WO2025035133A1 - Bmp-7 antibody compositions & methods for treating cancer - Google Patents

Bmp-7 antibody compositions & methods for treating cancer Download PDF

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Publication number
WO2025035133A1
WO2025035133A1 PCT/US2024/041808 US2024041808W WO2025035133A1 WO 2025035133 A1 WO2025035133 A1 WO 2025035133A1 US 2024041808 W US2024041808 W US 2024041808W WO 2025035133 A1 WO2025035133 A1 WO 2025035133A1
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seq
identity
cdr
bmp
amino acid
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James Welsh
Maria Angelica CORTEZ
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • cancer e.g., solid tumors such as non-small cell lung cancer and triple- negative breast cancer
  • polypeptides e.g., antibodies
  • the BMP-7-binding proteins for example BMP-7-neutralizing antibodies
  • described herein are unique in that they can specifically bind BMP-7 to overcome resistance to immunotherapies including anti-PD1, anti-PDL1, anti-TIM3, anti-LAG3, and anti-CTLA4.
  • the methods and compositions described herein provide a novel way to treat patients with resistance to immunotherapies.
  • the BMP-7-binding proteins for example BMP-7-neutralizing antibodies, described herein are further unique in that they can improve the efficacy of cancer therapies such as CAR-T cell therapies.
  • the methods and compositions described herein provide a novel way to treat patients with immunotherapies.
  • methods and compositions for treating a subject with cancer comprising providing a therapy comprising a BMP-7-binding protein to the subject, where the subject has been diagnosed with or is suspected of having cancer.
  • the disclosed methods comprise providing the therapy to a subject who was previously treated for cancer and was determined to be resistant to the previous treatment.
  • the previous cancer treatment comprised an immunotherapy.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • VL light chain variable region
  • SEQ ID NO:14 a heavy chain variable region having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14
  • a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16.
  • the VL has at least 85% identity to SEQ ID NO:14 and the VH has at least 85% identity to SEQ ID NO:16. In some aspects, the VL has at least 90% identity to SEQ ID NO:14 and the V H has at least 90% identity to SEQ ID NO:16. In some aspects, the V L has at least 95% identity to SEQ ID NO:14 and the V H has at least 95% identity to SEQ ID NO:16. In some aspects, the VL comprises SEQ ID NO:14 and the VH comprises SEQ ID NO:16.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • VL light chain variable region
  • SEQ ID NO:34 a heavy chain variable region having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:34
  • a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:40.
  • the VL has at least 85% identity to SEQ ID NO:34 and the VH has at least 85% identity to SEQ ID NO:40. In some aspects, the V L has at least 90% identity to SEQ ID NO:34 and the V H has at least 90% identity to SEQ ID NO:40. In some aspects, the V L has at least 95% identity to SEQ ID NO:34 and the VH has at least 95% identity to SEQ ID NO:40. In some aspects, the VL comprises SEQ ID NO:34 and the VH comprises SEQ ID NO:40.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • VL light chain variable region
  • SEQ ID NO:36 a heavy chain variable region having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:36
  • a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:42.
  • the VL has at least 85% identity to SEQ ID NO:36 and the VH has at least 85% identity to SEQ ID NO:42. In some aspects, the VL has at least 90% identity to SEQ ID NO:36 and the V H has at least 90% identity to SEQ ID NO:42. In some aspects, the V L has at least 95% identity to SEQ ID NO:36 and the VH has at least 95% identity to SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:36 and the VH comprises SEQ ID NO:42.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • VL light chain variable region
  • SEQ ID NO:38 a heavy chain variable region having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:38
  • a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:42.
  • the V L has at least 85% identity to SEQ ID NO:38 and the V H has at least 85% identity to SEQ ID NO:42. In some aspects, the VL has at least 90% identity to SEQ ID NO:38 and the VH has at least 90% identity to SEQ ID NO:42. In some aspects, the VL has at least 95% identity to SEQ ID NO:38 and the V H has at least 95% identity to SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:38 and the VH comprises SEQ ID NO:42.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • VL light chain variable region
  • SEQ ID NO:79 a heavy chain variable region having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:79
  • a heavy chain variable region (V H ) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:81.
  • the V L has at least 85% identity to SEQ ID NO:79 and the V H has at least 85% identity to SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:79 and the VH has at least 90% identity to SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:79 and the V H has at least 95% identity to SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:79 and the VH comprises SEQ ID NO:81.
  • the disclosed antigen-binding proteins, antibodies, antibody-like molecules, and fragments thereof are bone morphogenetic protein (BMP)-7-binding proteins (i.e., are capable of binding to BMP-7).
  • BMP bone morphogenetic protein
  • disclosed herein are monoclonal anti- BMP-7 antibodies.
  • disclosed herein are chimeric anti-BMP-7 antibodies.
  • disclosed herein are humanized anti-BMP-7 antibodies.
  • Antigen-binding proteins described herein may be used in treating one or more conditions associated with expression or activity of a BMP-7 protein such as, for example, cancer.
  • BMP- 7-binding proteins are be used in treating one or more BMP-7-associated conditions with reduced risk of toxicity and improved efficacy as compared to previously disclosed BMP-7- binding proteins.
  • Aspects include compositions comprising one or more antigen-binding proteins (e.g., BMP-7-binding proteins).
  • Aspects include an antigen-binding protein comprising one or more regions (e.g., heavy chain variable region, light chain variable region, etc.).
  • Aspects include monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody-like molecules.
  • Aspects also include nucleic acid molecules encoding for one or more antigen- binding proteins or portions thereof. Aspects include recombinant, transformed, or modified
  • compositions contemplated herein can comprise 1, 2, 3, 4, 5, or more of the following components: an antigen-binding protein, a nucleic acid, a vector, a cell, a polypeptide, an oligonucleotide, a complementarity determining region, a light chain variable region, a heavy chain variable region, a light chain constant region, a heavy chain constant region, and complementarity determining regions. Any one or more of these components may be excluded from the disclosed compositions.
  • aspects of the disclosure include methods and compositions for treating a subject having cancer, methods for diagnosing a subject with cancer, methods for prognosing a subject with cancer, methods and compositions for sensitizing a subject with cancer to immunotherapy, methods for identifying a subject with cancer as a candidate for a combination therapy, and methods and compositions for treating a subject having solid tumor cancer.
  • Methods of the disclosure can include 1, 2, 3, 4, 5, 6, or more of the following steps: providing a BMP-7 binding protein therapy to a subject, providing an immunotherapy to a subject, providing both a BMP-7 binding protein therapy and an immunotherapy to a subject, providing an alternative therapy to a subject, determining a subject to have cancer, providing two or more types of cancer therapy to a subject, identifying a subject as having had resistance to a previous cancer treatment, testing a subject for resistance to previous cancer treatments, and identifying a subject as being a candidate for a BMP-7 binding protein therapy or a double therapy comprising a BMP-7 binding protein therapy and an immunotherapy. Certain aspects of the disclosure may exclude one or more of the preceding elements and/or steps.
  • aspects also include methods of generating an antigen-binding protein, methods of producing an antigen-binding protein, methods of expressing an antigen-binding protein, methods of antigen-binding proteins, methods of detecting BMP-7, methods of treating one or more conditions, methods of purifying BMP-7, methods of treating cancer, and methods of eliminating one or more cells expressing BMP-7.
  • the steps and aspects discussed in this disclosure are contemplated as part of any of these methods.
  • the methods contemplated herein can comprise or exclude 1, 2, 3, 4, 5, or more of the following steps: providing an antigen-binding protein, providing a nucleic acid to a cell, subjecting a cell to conditions sufficient to express a nucleic acid, providing an additional therapeutic, covalently attaching a therapeutic to an antigen-binding protein, non-covalently attaching a therapeutic to an antigen-binding protein, expressing a vector in a cell, and providing a pharmaceutical composition to a subject. Any one or more of these steps may be excluded from the disclosed methods.
  • the disclosure relates to a bone morphogenetic protein-7 (BMP-7)- binding protein comprising: a light chain variable region (VL) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a heavy chain variable region (VH) comprising three complementarity
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a V L comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a V H comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a V L comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and
  • the disclosure relates to a BMP-7-binding protein, wherein the V L comprises a CDR-L1 having at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the disclosure relates to a BMP-7-binding protein, wherein the V L comprises a CDR-L1 having at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 95% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the VL comprises a CDR-L1 having at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least
  • the disclosure relates to a BMP-7-binding protein, wherein the V L comprises a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the V L comprises a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and
  • the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and
  • the disclosure relates to a BMP-7-binding protein, wherein the V H comprises a CDR-H1 having at least 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein, wherein the V H comprises a CDR-H1 having at least 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the VH and the VL are on the same polypeptide. In some aspects, the VH and the VL are on different polypeptides.
  • the BMP-7-binding protein has an affinity for BMP-7. In some aspects, the BMP-7-binding protein has an affinity for BMP-7 of between 0.001 and 1000 nM. In some aspects, the BMP-7-binding protein has an affinity for BMP-7 of between 0.01 and 100 nM. In some aspects, the BMP-7-binding protein has an association constant for a BMP-7 protein of between 0.1 and 50 nM. In some aspects, the BMP-7-binding protein has an association constant for a BMP-7 protein of between 1 and 20 nM.
  • the BMP- 7-binding protein has an association constant for a BMP-7 protein of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7.
  • the BMP-7-binding protein has an affinity for BPM-7 of about 10 nM. [0033] In some aspects, the BMP-7-binding protein specifically binds to human BMP-7. In some aspects, the BMP-7-binding protein is an antibody, an antibody-like molecule, or an antigen-binding fragment thereof. In some aspects, the BMP-7-binding protein is an antibody, a nanobody, a minibody, an scFv fragment, or a Fab fragment.
  • the BMP-7- binding protein is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, or a polyclonal antibody. In some aspects, the BMP-7-binding protein is a monoclonal antibody. In some aspects, the BMP-7-binding protein is a murine antibody. In some aspects, the BMP-7- binding protein is a chimeric antibody. In some aspects, the BMP-7-binding protein is a humanized antibody. In some aspects, the BMP-7-binding protein is a human antibody.
  • nucleic acid encoding for a polypeptide or fragment thereof that specifically binds BMP-7.
  • nucleic acid encodes both the heavy chain variable region and the light chain variable region of the BMP-7-binding protein.
  • nucleic acid encodes the light chain variable region of the BMP-7-binding protein.
  • nucleic acid encodes the heavy chain variable region of the BMP-7- binding protein.
  • the nucleic acid encoding the light chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78.
  • the nucleic acid encoding the light chain variable region comprises a nucleotide sequence having at least 85%
  • the nucleic acid encoding the light chain variable region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. In some aspects, the nucleic acid encoding the light chain variable region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78.
  • the nucleic acid encoding the light chain variable region comprises SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78.
  • the nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80.
  • the nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80.
  • the nucleic acid encoding the heavy chain variable region comprises SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:13; and a heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 85% identity to SEQ ID NO:13, and a heavy chain variable region comprising a nucleotide sequence having at least 85% identity to SEQ ID NO:15. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 90% identity to SEQ ID NO:13, and a heavy chain variable region comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:15. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence
  • the nucleic acid encodes a light chain variable region comprising SEQ ID NO:13, and a heavy chain variable region comprising SEQ ID NO:15.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:33; and a heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:39.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 85% identity to SEQ ID NO:33, and a heavy chain variable region comprising a nucleotide sequence having at least 85% identity to SEQ ID NO:39. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 90% identity to SEQ ID NO:33, and a heavy chain variable region comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:39.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 95% identity to SEQ ID NO:33, and a heavy chain variable region comprising a nucleotide sequence having at least 95% identity to SEQ ID NO:39. In some aspects, the nucleic acid encodes a light chain variable region comprising SEQ ID NO:33, and a heavy chain variable region comprising SEQ ID NO:39.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:35; and a heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:41.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 85% identity to SEQ ID NO:35, and a heavy chain variable region comprising a nucleotide sequence having at least 85% identity to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 90% identity to SEQ ID NO:35, and a heavy chain variable region
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 95% identity to SEQ ID NO:35, and a heavy chain variable region comprising a nucleotide sequence having at least 95% identity to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising SEQ ID NO:35, and a heavy chain variable region comprising SEQ ID NO:41.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:37; and a heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:41.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 85% identity to SEQ ID NO:37, and a heavy chain variable region comprising a nucleotide sequence having at least 85% identity to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 90% identity to SEQ ID NO:37, and a heavy chain variable region comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:41.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 95% identity to SEQ ID NO:37, and a heavy chain variable region comprising a nucleotide sequence having at least 95% identity to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising SEQ ID NO:37, and a heavy chain variable region comprising SEQ ID NO:41.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:78; and a heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:80.
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 85% identity to SEQ ID NO:78, and a heavy chain variable region comprising a nucleotide sequence having at least 85% identity to SEQ
  • the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 90% identity to SEQ ID NO:78, and a heavy chain variable region comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:80. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 95% identity to SEQ ID NO:78, and a heavy chain variable region comprising a nucleotide sequence having at least 95% identity to SEQ ID NO:80. In some aspects, the nucleic acid encodes a light chain variable region comprising SEQ ID NO:78, and a heavy chain variable region comprising SEQ ID NO:80.
  • nucleic acid(s) comprised in the vector is operably linked to an expression control sequence.
  • Certain aspects are directed to a cell comprising a polypeptide that specifically binds BMP-7 (the BMP-7-binding protein), a nucleic acid, and/or a vector of the present disclosure.
  • the cell is an immune cell.
  • the cell comprises the BMP-7-binding protein and can secrete the BMP-7-binding protein outside the cell.
  • the cell is a B cell.
  • the cell comprises the BMP-7-binding protein, and the BMP-7-binding protein is attached to a surface of the cell.
  • the BMP-7- binding protein is a chimeric antigen receptor.
  • the BMP-7-binding protein is a T cell receptor.
  • the cell is a T cell.
  • Certain aspects are directed to a composition comprising a BMP-7-binding protein, such as a BMP-7-binding protein described herein, and one or more additional therapeutics.
  • the one or more additional therapeutics comprise one or more immunotherapies.
  • the one or more immunotherapies comprise a cytotoxic T-lymphocyte- associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.
  • CTLA4 cytotoxic T-lymphocyte-associated protein 4
  • PD1 programmed cell death protein 1
  • PDL-1 programmed death-ligand 1
  • LAG3 lymphocyte activation gene-3
  • TIM-3 T cell immunoglobulin and mucin domain 3
  • the one or more immunotherapies comprise a CAR-T cell therapy.
  • the CAR- T cell therapy comprises EGFR-targeted CAR-T cells.
  • compositions comprising a BMP-7-binding protein, such as a BMP-7-binding protein described herein, a nucleic acid encoding for the BMP-7- binding protein, a vector comprising the nucleic acid, or a cell comprising the BMP-7-binding protein, the nucleic acid, or the vector; and a pharmaceutically acceptable excipient.
  • the composition further comprises an additional therapeutic.
  • the additional therapeutic comprises a biotherapeutic, a chemotherapeutic, an immunotherapeutic,
  • the immunotherapeutic comprises a cytotoxic T-lymphocyte- associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.
  • CTLA4 inhibitor is an anti-CTLA4 antibody.
  • the PD1 inhibitor is an anti- PD1 antibody.
  • the PDL-1 inhibitor is an anti-PDL-1 antibody.
  • the LAG3 inhibitor is an anti-LAG3 antibody.
  • the TIM-3 inhibitor is an anti-TIM-3 antibody.
  • the one or more immunotherapies comprise a CAR-T cell therapy.
  • the CAR-T cell therapy comprises EGFR-targeted CAR-T cells.
  • the CAR-T cell therapy comprises CAR-T cells genetically modified to have disrupted or reduced expression of one or more Bone Morphogenetic Protein (BMP) receptors (e.g., Bone Morphogenetic Protein Receptor Type 1A (BMPR1A), Bone Morphogenetic Protein Receptor Type 1B (BMPR1B), Activin A receptor type I (ACVR1), Activin receptor type 2A (ACVR2A), Activin receptor type 2B (ACVR2B), Bone morphogenetic protein receptor type 2 (BMPR2), or any combination thereof).
  • BMP Bone Morphogenetic Protein
  • compositions comprising a polypeptide that specifically binds BMP-7 which in some aspects comprises an additional therapeutic agent, in the manufacture of a medicament for the treatment or prevention of cancer. Certain aspects are directed to use of the composition comprising the BMP-7-binding protein in the manufacture of a medicament for the treatment or prevention of cancer.
  • a further aspect is directed to a polypeptide that specifically binds BMP-7 prepared by a method described herein. Some aspects are directed to a method for generating a BMP-7- binding protein, such as a BMP-7-binding protein described herein, by culturing a cell described herein under conditions sufficient to express a BMP-7-binding protein disclosed herein in the cell.
  • Some aspects are directed to a method for generating a BMP-7-binding protein, such as a BMP-7-binding protein described herein, comprising (a) providing to a cell a nucleic acid encoding for the BMP-7-binding protein; and (b) subjecting the cell to conditions sufficient to express the nucleic acid in the cell.
  • a method for treating cancer in a subject comprising providing to the subject a therapeutically effective amount of a BMP-7-binding protein or a composition, such as a pharmaceutical composition, comprising a BMP-7-binding
  • 202424720.3 15 protein such as a BMP-7-binding protein described herein; a nucleic acid encoding for the BMP-7-binding protein; an expression vector comprising the nucleic acid; or a cell comprising the BMP-7-binding protein, the nucleic acid, or the vector.
  • the method further comprises, prior to administering to the subject the therapeutically effective amount of a BMP- 7-binding protein or a composition, such as a pharmaceutical composition, comprising a BMP- 7-binding protein, such as a BMP-7-binding protein described herein; the nucleic acid encoding for the BMP-7-binding protein; the expression vector comprising the nucleic acid; or the cell comprising the BMP-7-binding protein, the nucleic acid, or the vector, diagnosing the subject with cancer.
  • the subject was previously treated for the cancer with a cancer therapy.
  • the cancer therapy comprises one or more immunotherapies.
  • the one or more immunotherapies comprise a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.
  • CTLA4 cytotoxic T-lymphocyte-associated protein 4
  • PD1 programmed cell death protein 1
  • PDL-1 programmed death-ligand 1
  • LAG3 lymphocyte activation gene-3
  • TIM-3 T cell immunoglobulin and mucin domain 3
  • the subject was determined to be resistant to the cancer therapy.
  • the cancer is a solid tumor.
  • the cancer is lung cancer, gastric cancer, brain cancer, colon cancer, melanoma, or breast cancer.
  • the cancer is lung cancer.
  • the lung cancer is non-small cell lung cancer.
  • the cancer is breast cancer. In some aspects, the breast cancer is triple-negative breast cancer. [0053] In some aspects, the cancer cells overexpress BMP-7 compared to non-cancerous cells. In some aspects, the cancer is pancreatic cancer, osteosarcoma, prostate cancer, colorectal cancer, malignant melanoma, breast cancer, ovarian cancer, esophageal cancer, gastric cancer, or renal cell cancer. In some aspects, the cancer is pancreatic cancer. [0054] In some aspects, the method further comprises providing to the subject one or more additional therapies. In some aspects, the one or more additional therapies are radiotherapy, chemotherapy, or immunotherapy.
  • a method of increasing the efficacy of a cancer therapy administered to a subject for treatment of cancer comprising administering to the subject a therapeutically effective amount of the cancer therapy and a BMP-7-binding protein or a composition, such as a pharmaceutical composition, comprising a BMP-7-binding protein, such as a BMP-7-binding protein described herein; a nucleic acid encoding for the BMP-7-binding protein; an expression vector comprising the nucleic acid; or
  • the cancer therapy and the a BMP-7-binding protein or a composition such as a pharmaceutical composition, comprising a BMP-7-binding protein, such as a BMP-7-binding protein described herein; a nucleic acid encoding for the BMP-7-binding protein; an expression vector comprising the nucleic acid; or a cell comprising the BMP-7-binding protein, the nucleic acid, or the vector, are comprised in the same composition.
  • the cancer therapy and a BMP-7-binding protein or a composition such as a pharmaceutical composition, comprising a BMP-7-binding protein, such as a BMP-7-binding protein described herein; a nucleic acid encoding for the BMP-7-binding protein; an expression vector comprising the nucleic acid; or a cell comprising the BMP-7-binding protein, the nucleic acid, or the vector, are comprised in the different compositions.
  • the cancer therapy comprises an immunotherapy.
  • the immunotherapy comprises a CAR-T cell therapy.
  • the immunotherapy comprises EGFR-targeted CAR-T cells.
  • the cancer cells overexpress BMP-7 compared to non-cancerous cells.
  • the cancer is pancreatic cancer, osteosarcoma, prostate cancer, colorectal cancer, malignant melanoma, breast cancer, ovarian cancer, esophageal cancer, gastric cancer, or renal cell cancer. In some aspects, the cancer is pancreatic cancer.
  • Some aspects are directed to a monoclonal antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a V H comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof comprising: a V L comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a V H comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%,
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the V L comprises a CDR-L1 having at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the V L comprises a CDR-L1 having at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the V L comprises a CDR-L1 having at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the V L comprises a CDR-L1 having at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 95% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the VL comprises a CDR-L1 having at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the V H comprises a CDR-H1 having at least, at most, exactly, or
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the VH comprises a CDR-H1 having at least 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75,
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the V H comprises a CDR-H1 having at least 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the V H comprises a CDR-H1 having at least 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the V H comprises a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the V H comprises a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, S
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14 and the VH comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16.
  • the VL has at least 85% identity to SEQ ID NO:14 and the VH has at least 85% identity to SEQ ID NO:16. In some aspects, the V L has at least 90% identity to SEQ ID NO:14 and the V H has at least 90% identity to SEQ ID NO:16. In some aspects, the V L has at least 95% identity to SEQ ID NO:14 and the VH has at least 95% identity to SEQ ID NO:16. In some aspects, the VL comprises SEQ ID NO:14 and the VH comprises SEQ ID NO:16. In some aspects, the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7).
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the V L comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:34 and the V H comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:40.
  • the VL has at least 85% identity to SEQ ID NO:34 and the V H has at least 85% identity to SEQ ID NO:40. In some aspects, the VL has at least 90% identity to SEQ ID NO:34 and the VH has at least 90% identity to SEQ ID NO:40. In some aspects, the VL has at least 95% identity to SEQ ID NO:34 and the V H has at least 95% identity to SEQ ID NO:40. In some aspects, the V L comprises SEQ ID NO:34 and the VH comprises SEQ ID NO:40. In some aspects, the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7).
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value
  • SEQ ID NO:36 and the V H comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:42.
  • the V L has at least 85% identity to SEQ ID NO:36 and the VH has at least 85% identity to SEQ ID NO:42.
  • the VL has at least 90% identity to SEQ ID NO:36 and the VH has at least 90% identity to SEQ ID NO:42.
  • the V L has at least 95% identity to SEQ ID NO:36 and the VH has at least 95% identity to SEQ ID NO:42.
  • the VL comprises SEQ ID NO:36 and the VH comprises SEQ ID NO:42.
  • the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7).
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:38 and the VH comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:42.
  • the VL has at least 85% identity to SEQ ID NO:38 and the V H has at least 85% identity to SEQ ID NO:42. In some aspects, the V L has at least 90% identity to SEQ ID NO:38 and the V H has at least 90% identity to SEQ ID NO:42. In some aspects, the VL has at least 95% identity to SEQ ID NO:38 and the VH has at least 95% identity to SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:38 and the V H comprises SEQ ID NO:42. In some aspects, the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7).
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:79 and the V H comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:81.
  • the V L has at least 85%
  • the VL has at least 90% identity to SEQ ID NO:79 and the VH has at least 90% identity to SEQ ID NO:81.
  • the VL has at least 95% identity to SEQ ID NO:79 and the V H has at least 95% identity to SEQ ID NO:81.
  • the V L comprises SEQ ID NO:79 and the VH comprises SEQ ID NO:81.
  • the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7).
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a V H comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12.
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a V H comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32.
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24; and a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30.
  • the V L comprises SEQ ID NO:14.
  • the V H comprises SEQ ID NO:16.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and a V H comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65.
  • the VL comprises SEQ ID NO:34.
  • the VH comprises SEQ ID NO:40.
  • the V L comprises SEQ ID NO:36.
  • the V H comprises SEQ ID NO:42.
  • the V L comprises SEQ ID NO:38. In some aspects, the V H comprises SEQ ID NO:42. [0079] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77. In some aspects, the V L comprises SEQ ID NO:79. In some aspects, the V H comprises SEQ ID NO:81.
  • the terms “about” and “approximately” and “substantially” are used according to their plain and ordinary meaning in the area of cell and molecular biology to indicate a deviation of ⁇ 10% of the value(s) to which it is attached. Therefore, in any disclosed aspect, the terms may be substituted with “within [a percentage] of” what is specified. In one non-limiting aspect, the percentage includes 0.1, 0.5, 1, 5, and 10 percent.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • “and/or” operates as an inclusive or.
  • compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification.
  • the term “consisting essentially of” refers to those elements required for a given aspect. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that aspect of the disclosure.
  • the term “consisting essentially of” includes the active ingredients recited, excludes any other active ingredients, but does not exclude any pharmaceutical excipients or other components that are not therapeutically active.
  • the term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the aspect.
  • any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “use of” any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.
  • any method or composition described herein can be implemented with respect to any other method or composition described herein and that different aspects may be combined.
  • Reference throughout this specification to “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” or “a further aspect” or combinations thereof means that a particular feature, structure, or characteristic described in connection with the aspect is included in at least one aspect of the present disclosure.
  • FIG.1 shows titration of an antibody generated to specifically target human BMP- 7 (BMP-7 mAb #5).
  • FIG. 2 shows functional validation of BMP-7 mAb in macrophages treated with BMP-7 protein for 48 hours.
  • FIGs.3A-3B show tumor growth and survival analysis of mice 129 Sv (Females) with 344SQR tumors.
  • Mouse survival rates were analyzed by the Kaplan–Meier method and compared with log-rank tests.
  • FIGs. 4A-4B show tumor growth and survival analysis of mice 129 Sv (Males) with 344SQR tumors.
  • FIG.4B Mouse survival rates were analyzed by the Kaplan–Meier method and compared with log-rank tests.
  • FIG. 5 shows functional validation of BMP-7 mAb antibodies in macrophages treated with BMP-7 antibody for 48 hours.
  • FIG.6 shows representative images of immunohistochemical stains of CD8 T cells (CD8 – brown dots) and M2 macrophages (CD163 – M2 macrophage marker) in formalin- fixed paraffin-embedded tissue sections from 344SQR tumors treated with BMP-7 mAb plus anti-PD1.
  • FIG.9 shows sensor-grams of chimeric antibody binding to human BMP-7.
  • FIGs. 10A-10B show alignment of sequenced humanized BMP-7 monoclonal antibody VH (FIG.10A) and VL chains (FIG.10B). [0101] FIGs.
  • FIG. 11A-11B show alignment of selected, sequenced humanized BMP-7 monoclonal antibody VH (FIG.11A) and VL chains (FIG.11B).
  • FIG. 12 shows 99% purification of selected humanized BMP-7 monoclonal antibodies by SDS-PAGE. Loading order: Marker (M); AHF17901, reduced (1); AHF17906, reduced (2); AHF17909, reduced (3); AHF17901, non-reduced (4); AHF17906, non-reduced (5); AHF17909, non-reduced (6); human IgG.
  • FIG.13 shows sensor-grams of chimeric BMP-7 monoclonal antibody binding to human BMP-7. [0104] FIG.
  • FIG. 14 shows BMP-7 gene expression in Capan-2 pancreatic cancer cells, GSU gastic cancer cells, MCF-7 breast cancer cells, and MDA-MB-231 breast cancer cells. BMP- 7 gene expression was measured using quantitative PCR using primers specific for BMP-7 gene.
  • FIG.15 shows apoptosis of Capan-2 pancreatic cancer cells by CAR-T cells after treatment with an anti-BMP-7 antibody. Apoptosis was measured using the Caspase 3/7 green, fluorescent dye (Essen Bioscience) analyzed by the INCUCYTE® Live-Cell Analysis System. A higher Y-axis value indicates increased apoptosis and killing capability of the CAR-T cells.
  • FIG. 15 shows apoptosis of Capan-2 pancreatic cancer cells by CAR-T cells after treatment with an anti-BMP-7 antibody. Apoptosis was measured using the Caspase 3/7 green, fluorescent dye (Essen Bioscience) analyzed by the INCUCYTE® Live-Cell Analysis System. A higher
  • FIG. 16 shows that addition of BMP7 protein blocks the effect of apoptosis of Capan-2 pancreatic cancer cells by CAR-T cells after treatment with an anti-BMP-7 antibody.
  • Apoptosis was measured using the Caspase 3/7 green, fluorescent dye (Essen Bioscience) analyzed by the INCUCYTE® Live-Cell Analysis System. A higher Y-axis value indicates increased apoptosis and killing capability of the CAR-T cells.
  • FIG. 17 shows that addition of BMP7 protein blocks the effect of apoptosis of Capan-2 pancreatic cancer cells by CAR-T cells after treatment with an anti-BMP-7 antibody.
  • Apoptosis was measured using the Caspase 3/7 green, fluorescent dye (Essen Bioscience) analyzed by the INCUCYTE® Live-Cell Analysis System. A higher Y-axis value indicates increased apoptosis and killing capability of the CAR-T cells.
  • FIG. 18 shows predicted target-potential vs. off-target risk for different gRNAs targeting the BMPR1B gene locus, with unique gRNAs predicted to have high target specificity highlighted in light grey.
  • FIG. 19 shows predicted target-potential vs. off-target risk for different gRNAs targeting the BMPR1A gene locus, with unique gRNAs predicted to have high target specificity highlighted in light grey. [0110] FIG.
  • FIG. 20 is a plasmid map of a lentiviral vector (VB200329-1029jwu) encoding a hCas9:T2A:Puro construct and two gRNAs (#29757 and #29702) targeting the hBMPR1B gene locus.
  • FIG. 21 is a plasmid map of a lentiviral vector (VB200329-1005ytw) encoding a hCas9 endonuclease and two gRNAs (#29757 and #29702) targeting the hBMPR1B gene locus.
  • FIG. 21 is a plasmid map of a lentiviral vector (VB200329-1005ytw) encoding a hCas9 endonuclease and two gRNAs (#29757 and #29702) targeting the hBMPR1B gene locus.
  • FIG. 22 is a plasmid map of a lentiviral vector (VB200328-7336rqg) encoding a hCas9:T2A:Puro construct and two gRNAs (#14262 and #14258) targeting the hBMPR1A gene locus.
  • FIG. 23 is a plasmid map of a lentiviral vector (VB200328-7298grx) encoding a hCas9 endonuclease and two gRNAs (#14262 and #14258) targeting the hBMPR1A gene locus.
  • VB200328-7298grx encoding a hCas9 endonuclease and two gRNAs (#14262 and #14258) targeting the hBMPR1A gene locus.
  • the present disclosure is based, at least in part, on the surprising discovery that cancer resistant to immunotherapy is responsive to a therapy comprising a BMP-7 binding protein. Further, administering a BMP-7 binding protein therapy was surprisingly found to re- sensitize tumors to immunotherapeutics. As disclosed herein, administration of a BMP-7 binding protein therapy, when associated with cancer, for example, a solid tumor cancer, relieves immunosuppression and enables efficacy of immunotherapy.
  • the BMP-7 binding protein therapy when associated with cancer, for example, a solid tumor cancer, relieves immunosuppression and enables efficacy of immunotherapy.
  • ⁇ can interact with functional BMP-7 protein secreted by cancer cells, for example, solid tumor cells, to neutralize BMP-7 protein and initiate downstream signaling events associated with a reduction in pro-inflammatory signaling and re-sensitization of tumors to immunotherapeutics.
  • cancer cells for example, solid tumor cells
  • BMP-7 binding protein therapy to a subject having or suspected or having cancer.
  • the cancer is a solid tumor cancer.
  • the subject has or has previously had resistance to immunotherapy.
  • Further aspects disclose methods for stratifying cancer patients based on a history of resistance to immunotherapy.
  • aspects are directed to methods for identifying a subject as being a candidate for a BMP-7 binding protein therapy by identifying the subject as having or having previously had resistance to immunotherapy.
  • a novel BMP-7 antibody and the use of such antibody for therapeutic applications such as treatment of resistance to immunotherapies in solid tumors.
  • Antibodies of the present disclosure and derivatives thereof may therefore be useful in the treatment of various conditions including, for example, cancer.
  • antibodies of the present disclosure are useful in the treatment of cancers which overexpress BMP-7, for example, solid tumor cancers such as lung cancer, including non-small cell lung cancer and small-cell lung cancer, breast cancer, including triple negative breast cancer, brain cancer, colon cancer, melanoma, and gastric cancer.
  • solid tumor cancers such as lung cancer, including non-small cell lung cancer and small-cell lung cancer
  • breast cancer including triple negative breast cancer
  • brain cancer colon cancer
  • melanoma gastric cancer.
  • “lower,” “lowered,” “reduce,” “reduced,” “reduction,” “decrease,” “decreased,” “inhibit,” “inhibited,” or “inhibition” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%
  • the terms “increased,” “increase,” “enhanced,” “enhance,” “activated,” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, “increased,” “increase,” “enhanced,” “enhance,” “activated,” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a
  • nucleic acid is a molecule comprising nucleic acid components and refers to DNA or RNA molecules.
  • nucleic acid may be used interchangeably with the term “polynucleotide.”
  • a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone.
  • Nucleic acids may also encompass modified nucleic acid molecules, such as base-modified, sugar-modified, backbone-modified, etc. DNA or RNA molecules.
  • Nucleic acids may exist in a variety of forms such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding polypeptides, e.g., antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof; polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide; anti-sense nucleic acids for inhibiting expression of a polynucleotide; mRNA; saRNA; and complementary sequences of the foregoing described
  • the nucleic acids can be single-stranded or double-stranded and can comprise RNA and/or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).
  • Nucleic acids may be single-stranded or double-stranded and may comprise RNA and/or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).
  • a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy.
  • heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.
  • polynucleotide refers to a nucleic acid molecule that may be recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (i.e., nucleic acids 100 residues or less in length) and recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or a combination thereof. Additional coding or non- coding sequences may, but need not, be present within a polynucleotide.
  • a “gene,” “polynucleotide,” “coding region,” “sequence,” “segment,” “fragment,” or “transgene” which “encodes” a particular protein is a nucleic acid molecule which is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
  • a nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide.
  • the coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5’ (amino) terminus (N-terminus) and a translation stop codon at the 3’ (carboxy) terminus (C-terminus).
  • a gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences.
  • a transcription termination sequence will
  • a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.
  • expression refers to the generation of any gene product from the nucleic acid sequence.
  • a gene product may be a transcript.
  • a gene product may be a polypeptide.
  • expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • engineered refers to the aspect of having been manipulated by the hand of man.
  • a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.
  • DNA means a nucleic acid molecule comprising nucleotides such as deoxy-adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate and deoxy-cytidine-monophosphate monomers which are composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, and polymerize by a characteristic backbone structure.
  • the backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, e.g., deoxyribose, of a first and a phosphate moiety of a second, adjacent monomer.
  • DNA sequence The specific order of the monomers, e.g., the order of the bases linked to the sugar/phosphate-backbone, is called the DNA sequence.
  • DNA may be single stranded or double stranded. In the double stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g., by A/T-base-pairing and G/C-base-pairing.
  • DNA may contain all, or a majority of, deoxyribonucleotide residues.
  • deoxyribonucleotide means a nucleotide lacking a hydroxyl group at the 2′ position of a ⁇ -D-ribofuranosyl group.
  • DNA may encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, DNA that is recombinantly produced, and modified DNA.
  • RNA means a nucleic acid molecule comprising nucleotides such as adenosine-monophosphate, uridine-monophosphate, guanosine- monophosphate and cytidine-monophosphate monomers which are connected to each other along a so-called backbone.
  • the backbone is formed by phosphodiester bonds between the sugar, e.g., ribose, of a first and a phosphate moiety of a second, adjacent monomer.
  • RNA may be obtainable by transcription of a DNA-sequence, e.g., inside a cell.
  • RNA messenger-RNA
  • Processing of the premature RNA comprises various posttranscriptional modifications such as splicing, 5′ capping, polyadenylation, and/or export from the nucleus or the mitochondria.
  • Mature messenger RNA is processed and provides the nucleotide sequence that may be translated into an amino acid sequence of a peptide or protein.
  • a mature mRNA may comprise a 5′ cap, a 5′ UTR, an open reading frame, a 3′ UTR and a poly-A tail sequence.
  • RNA may contain all, or a majority of, ribonucleotide residues.
  • ribonucleotide means a nucleotide with a hydroxyl group at the 2′ position of a ⁇ -D-ribofuranosyl group.
  • RNA may be messenger RNA (mRNA) that relates to an RNA transcript which encodes a peptide or protein.
  • mRNA generally contains a 5′ untranslated region (5′ UTR), a polypeptide coding region, and a 3′ untranslated region (3′ UTR).
  • RNA may encompass double stranded RNA, antisense RNA, single stranded RNA, isolated RNA, synthetic RNA, RNA that is recombinantly produced, and modified RNA (modRNA).
  • modified RNA modified RNA
  • the terms “protein,” “polypeptide,” or “peptide” are used herein as synonyms and refer to a polymer of amino acid monomers, e.g., a molecule comprising at least two amino acid residues.
  • Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • Polypeptides may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer.
  • a protein comprises one or more peptides or polypeptides and may be folded into a 3-dimensional form, which may be required for the protein to exert its biological function.
  • wild type or WT or “native” refer to the endogenous version of a molecule that occurs naturally in an organism.
  • wild type versions of a protein or polypeptide are employed, however, in other aspects of the disclosure, a modified protein or polypeptide is employed. The terms described above may be used interchangeably.
  • a “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide having a chemical structure, particularly an amino acid sequence, that is altered with respect to the wild type protein or polypeptide.
  • a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild type activity or function in other respects, such as immunogenicity.
  • a protein is specifically mentioned herein, it is in general a reference to a native (wild type) or recombinant (modified) protein.
  • the protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, produced by solid-phase peptide synthesis (SPPS) (i.e., an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution), liquid phase peptide synthesis (i.e., sequential addition of monomer building blocks in a liquid phase), a combination of solid and liquid phase peptide synthesis, or other in vitro methods.
  • SPPS solid-phase peptide synthesis
  • Assembling nucleic acids by a ligase may also be used to promote intermolecular ligation of the 5 ⁇ and 3′ ends of polynucleotide chains through the formation of a phosphodiester bond.
  • the term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.
  • fragment with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, e.g., a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus.
  • a fragment shortened at the C- terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3′-end of the open reading frame.
  • a fragment shortened at the N-terminus is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5′-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation.
  • a fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90%, or at least 99% of the amino acid residues from an amino acid sequence.
  • a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least, at most, exactly, or between (inclusive or
  • a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 70% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived.
  • a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 80% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 85% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived.
  • a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 90% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 95% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived.
  • a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 97% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 99% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived.
  • variant refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity.
  • a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule.
  • any biological or chemical reference molecule has certain characteristic structural elements.
  • a variant by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule.
  • a variant polypeptide or nucleic acid may differ from a
  • one or more particular amino acid residues are changed using, for example, a site- directed mutagenesis protocol.
  • one or more randomly selected residues are changed using, for example, a random mutagenesis protocol.
  • a mutant polypeptide can be expressed and screened for a desired property.
  • Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues.
  • one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes.
  • variants can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.
  • variant polypeptides encoded by nucleic acids of the disclosure may contain amino acid changes that confer any of a number of desirable properties. Variant polypeptides can be made using routine mutagenesis techniques and assayed as appropriate to determine whether they possess the desired property.
  • a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.
  • a reference polypeptide or nucleic acid has one or more biological activities.
  • a variant polypeptide or nucleic acid shares one or more of the biological
  • a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some aspects, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. [0141] In some aspects, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions.
  • amino acids may be substituted for other amino acids in a protein or polypeptide sequence inserted, or deleted, as compared to the reference, with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen- binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines its functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties.
  • Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants.
  • the variant polypeptide or nucleic acid sequence has at least one modification compared to the reference polypeptide or nucleic acid sequence, e.g., from 1 to about 50 modifications.
  • a variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type.
  • the variant polypeptide or nucleic acid sequence has from 1 to about 50 modifications compared to the reference polypeptide or nucleic acid sequence.
  • the variant polypeptide or nucleic acid sequence has from 1 to about 40 modifications compared to the reference polypeptide or nucleic acid sequence.
  • the variant polypeptide or nucleic acid sequence has from 1 to about 30 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has from 1 to about 20 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has from 1 to about 10 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has from 1 to about 5 modifications compared to the reference polypeptide or nucleic acid sequence. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or
  • a variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5’ or 3’ nucleic acid sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5’ or 3’ portions of the coding region.
  • the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.
  • Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted.
  • a stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.
  • Insertional mutants typically involve the addition of amino acid residues at a non- terminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties.
  • substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties.
  • “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class.
  • Conservative replacements also “conservative substitutions” or “conservative amino acid substitutions” are those that take place within a family of amino acids that possess similar biochemical properties, including charge, hydrophobicity, and size. Genetically encoded amino acids are generally divided into
  • 202424720.3 38 families based on the chemical nature of the side chain, e.g., acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine).
  • a conservative replacement may comprise replacement of an amino acid in one family for an amino acid in the same family (e.g., replacement of a lysine with an arginine, replacement of an aspartate for a glutamate, etc.).
  • amino acid similarity may be determined using a Blocks Substitution Matrix (BLOSUM), such as BLOSUM62 (Henikoff S and Henikoff JG, Proc. Natl. Acad. Sci. U.S.A 89(22): 10915-9 (1992)).
  • BLOSUM Blocks Substitution Matrix
  • a conservative replacement may be a substitution of amino acids having a non-negative value on a BLOSUM62 matrix. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative.
  • Standard ELISA, Surface Plasmon Resonance (SPR), or other antibody binding assays can be performed by one skilled in the art to make a quantitative comparison of antigen binging affinity between the unmodified antibody and any polypeptide derivatives with conservative substitutions generated through any of several methods available to one skilled in the art.
  • Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties. [0149]
  • substitutions may be “non-conservative” (also “nonconservative”). In some aspects, a non-conservative substitution affects a function or activity of the polypeptide.
  • a non-conservative substitution does not affect a function or activity of the polypeptide.
  • Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
  • Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.
  • a reference polypeptide or nucleic acid is a “wild type” or “WT” or “native” sequence found in nature, including allelic variations.
  • a wild type polypeptide or nucleic acid sequence has a sequence that has not been intentionally modified.
  • variants of an amino acid sequence comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants.
  • variants of a nucleotide sequence comprise nucleotide insertion variants, nucleotide addition variants, nucleotide deletion variants and/or nucleotide substitution variants.
  • variants includes all mutants, splice
  • variants include, in particular, fragments of an amino acid or nucleic acid sequence.
  • a fragment or variant of an amino acid sequence may be a “functional fragment” or “functional variant.”
  • the term “functional fragment” or “functional variant” of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, e.g., it is functionally equivalent.
  • one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived.
  • the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence.
  • amino acid sequence “derived from” a designated amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence.
  • amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to that particular sequence or a fragment thereof.
  • Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof.
  • the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.
  • Changes may be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antigen or antibody or antibody derivative) that it encodes. Mutations may be introduced using any technique known in the art. In one aspect, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. In some aspects, however it is made, a mutant polypeptide may be expressed and screened for a desired property.
  • a polypeptide e.g., an antigen or antibody or antibody derivative
  • Mutations may be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one may make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues.
  • one or more mutations may be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. For example, the mutation may quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.
  • “Sequence similarity” indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.
  • Sequence identity between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.
  • the terms “% identical,” “% identity,” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison,” in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads.
  • Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100. [0157] In some aspects, the degree of similarity or identity is given for a region that is at least, at most, exactly, or between (inclusive or exclusive) any two of about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the entire length of the reference
  • the degree of identity is given for at least, at most, exactly, or between any two of about 100, about 120, about 140, about 160, about 180, or about 200 nucleotides, in some aspects, continuous nucleotides. In some aspects, the degree of similarity or identity is given for the entire length of the reference sequence.
  • Homologous amino acid sequences may exhibit at least, at most, exactly, or between (inclusive or exclusive) any two of 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity of the amino acid residues.
  • a vector refers to a nucleic acid molecule, such as an artificial nucleic acid molecule.
  • a vector may be used to incorporate a nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame.
  • Vectors include, but are not limited to, storage vectors, expression vectors, cloning vectors, and transfer vectors.
  • a vector may be an RNA vector or a DNA vector.
  • the vector is a DNA molecule. In some aspects, the vector is a plasmid vector. In some aspects, the vector is a viral vector.
  • an expression vector will contain a desired coding sequence and appropriate other sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired fragment (typically a DNA fragment) and may lack functional sequences needed for expression of the desired fragment(s).
  • Treating” or “treatment,” as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest, and includes: (i) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; (ii) inhibiting the disease or condition, i.e., arresting its development; (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition.
  • the terms “disease” and “condition” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore
  • Prevent or “prevention,” as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder, or condition has been delayed for a predefined period of time.
  • risk of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition.
  • risk is expressed as a percentage.
  • risk is at least, at most, exactly, or between (inclusive or exclusive) any two of from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%.
  • risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples.
  • a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event.
  • a reference sample or group of reference samples are from individuals comparable to a particular individual.
  • risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and/or condition.
  • risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes.
  • An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public.
  • an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some aspects, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some aspects, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some aspects, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition. [0164] As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers approved by the
  • compositions may be immunogenic compositions.
  • active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation. II.
  • BMP-7 Bone morphogenic protein 7
  • OP-1 osteogenic protein-1
  • TGFB transforming growth factor beta
  • BMPs can act as either tumor suppressors or oncogenes depending on the cellular context and tumor type. Secreted BMP-7 impinges on effector T-cell functions, favoring the generation of immunosuppressive cells, and precluding response to immunotherapy.
  • BMP-7 is upregulated in mouse and human tumors resistant to anti-PD1 therapy, and BMP-7 levels are higher not only in blood from mice bearing resistant tumors but also in pretreatment blood from patients exhibiting disease progression while on anti-PD1 and radiotherapy.
  • BMP-7 has also been reported in a wide range of human cancers and is associated with metastasis and poor prognosis.
  • BMP-7 overexpression is associated with lymph node involvement and an indicator of bone metastasis.
  • BMPs regulate immune cell responses and are immunosuppressive in cancer. For example, BMPs have been shown to regulate activation, growth, and cytokine secretion in macrophages and to promote PDL1 and PDL2 upregulation in dendritic cells.
  • BMP-7 Treatment with BMP-7 in vitro and in vivo significantly enhanced monocyte polarization into M2 macrophages.
  • BMP ligands bind to and form heteromeric complexes with two types of serine/threonine kinase receptors on the cell surface, which then activate the “small mothers against decapentaplegic” (SMAD) proteins 1 and 5 in cells.
  • SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways.
  • SMAD proteins can be phosphorylated and activated by the BMP
  • BMP-7 Phosphorylated SMAD proteins form oligomeric complexes, which are important for their function in the transcription regulation. Specifically, binding of BMP to its receptor leads to the phosphorylation of intracellular SMADs, which then bind to co-SMAD4 and translocate into the nucleus to regulate gene expression.
  • Secreted BMP-7 promotes resistance to immunotherapy by repressing macrophage- mediated inflammatory responses and Th1-associated cytokines in the tumor microenvironment. BMP-7 downregulates MAPK14 and MAPK14-regulated cytokines and chemokines including IL1A, IL1B, TNF, and CCL5 via SMAD1 activation.
  • BMP-7 decreases CD4 + T-cell activation by downregulating IFNG and IL2 expression via SMAD1/MAPK14 signaling.
  • BMP-7 can specifically downregulate MAPK14 at the mRNA and protein levels.
  • MAPK14 can act as a tumor suppressor by regulating cell cycle progression and induction of apoptosis or as an oncogene by promoting invasion, inflammation, and angiogenesis.
  • MAPK14 is a member of the p38 MAPK family. It is downregulated not only in tumors but also in TILs from anti-PD1-resistant tumors.
  • MAPK14 is the critical isoform in inflammatory responses and is involved in the expression of proinflammatory mediators in macrophages such as IL1B, TNF, and IL12 as well as CCL5, COX-2, IL8, IL6, IL3, IL2, and IL1, all of which contain AU-rich elements in their 3′-untranslated regions to which MAPK14 binds.
  • MAPK14 participates in the regulation of IFNG expression and its mRNA stabilization in immune cells.
  • MAPK14 is known to be regulated by BMP-7 via SMAD activation.
  • BMP-7 can either promote or inhibit MAPK14 activation depending on the cellular context and BMP-7 dose.
  • MAPK14-regulated inflammatory cytokines IL1A, IL1B, and TNF are downregulated in tumor-infiltrating leukocytes (TILs) collected from 344SQR tumors treated with anti-PD1 versus 344SQP.
  • TILs tumor-infiltrating leukocytes
  • Cytokines and chemokines regulated by MAPK14 including IL1A, IL1B, TNF, CCL5, IFNG, and IL2 are also downregulated in blood from mice bearing 344SQR tumors compared with parental tumors.
  • BMP-7 regulates MAPK14 expression not only in tumors resistant to anti-PD1 but also in TILs in the tumor microenvironment, and BMP-7 also regulates expression of proinflammatory cytokines and chemokines in TILs via MAPK14 regulation.
  • BMP-7 Similar to results observed in tumors, BMP-7 regulates MAPK14 via SMAD1 activation in immune cells. Previous studies in macrophages isolated from a vivo model of
  • BMP-7 significantly reduced the number of proinflammatory macrophages and decreased MAPK14 activation while increasing SMAD1/5/8 phosphorylation in macrophages.
  • Other studies have shown that BMP-7 promotes M2 polarization in human and mouse macrophages in vitro and in vivo models.
  • MAPK14 signaling promotes not only M2 monocytes polarization into M1-type cells in response to lipopolysaccharides but also is central in the activation of proinflammatory gene transcription.
  • MAPK14 is activated by lipopolysaccharide and Toll-like receptor-4, which subsequently activates proinflammatory cytokines, including IL1 and TNF.
  • Murine macrophages co-cultured with 344SQR cells have lower expression of MAPK14, IL1A, IL1B, TNF, and CCL5 compared with cells co-cultured with 344SQP cells. That these findings depend on BMP-7 has been confirmed by co-culturing macrophages with BMP-7-knockdown 344SQR cells, which results in higher expression levels of MAPK14, IL1A, IL1B, TNF, and CCL5 compared with control cells.
  • BMP-7 suppresses the proinflammatory cytokine expression regulated by MAPK14 in macrophages.
  • P38 signaling is known to be activated in T cells stimulated via TCR signaling and reduced in anergic T cells.
  • MAPK14 also participates in the regulation of IFNG expression in CD4+ T cells and promotes the 3′-untranslated region stabilization of IFNG mRNA in NK cells. Further, the inhibition of MAPK14 in Th1 cells differentiated in vitro blocked the IFNG expression induced by IL12/IL18 and CD3/CD28 stimulation. Previous studies showed that treating cells with SB203580, a specific inhibitor of MAPK14, suppressed the transcriptional activation of the IL2 promoter in T lymphocytes.
  • SMAD regulatory pathways regulate different aspects of immune activation and immune suppression in T cells.
  • TGFB promotes the differentiation of CD4+ T cells into suppressive FOXP3+ T regulatory cells via SMAD activation.
  • BMP-7 regulates MAPK14 expression via SMAD1 signaling not only in tumors and macrophages but also in CD4+ T cells.
  • Activated CD4+ T cells incubated with BMP-7 had lower MAPK14, IFNG, and IL2 expression compared with untreated cells and cells treated with BMP-7 plus follistatin, a natural inhibitor of BMP-7.
  • other studies have also correlated IL2 activation with MAPK14 signaling in T cells.
  • BMP-7 decreases IFNG and IL2 expression in CD4+ T cells via SMAD1/MAPK14 signaling.
  • BMP-7-binding proteins relate to BMP-7-binding proteins.
  • the terms “polypeptide that specifically binds BMP-7” and “BMP-7-binding protein” are used interchangeably herein.
  • the BMP-7-binding protein specifically binds to human BMP-7.
  • the BMP-7-binding protein is an antibody, an antibody-like molecule, or an antigen-binding fragment thereof.
  • the BMP-7-binding protein is an antibody, a nanobody, a minibody, an scFv fragment, or a Fab fragment.
  • the BMP-7-binding protein is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, or a polyclonal antibody. In some aspects, the BMP-7-binding protein is a monoclonal antibody. In some aspects, the BMP-7-binding protein is a murine antibody. In some aspects, the BMP-7-binding protein is a chimeric antibody. In some aspects, the BMP-7- binding protein is a humanized antibody.
  • the disclosed BMP-7-binding proteins include antibodies comprising a light chain variable region (VL) having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and a heavy chain variable region (VH) having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • VL light chain variable region having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%
  • the V L has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the VH has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81
  • the VL has at least 90% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the VH has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the V H has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81
  • the VL comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the V H comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having 85%, 86%, 87%,
  • the VL has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the V H has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81
  • the VL has at least 90% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the V H has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the V L has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the VH has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81
  • the V L comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the VH comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7).
  • Some aspects are directed to an antibody comprising a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • Some aspects are directed to an antibody comprising a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6. Some aspects are directed to an antibody comprising a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24. Some aspects are directed to an antibody comprising a V L comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57.
  • Some aspects are directed to an antibody comprising a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71. [0180] Some aspects are directed to an antibody comprising a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-
  • H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • Some aspects are directed to an antibody comprising a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12.
  • Some aspects are directed to an antibody comprising a V H comprising a CDR- H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32.
  • Some aspects are directed to an antibody comprising a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30. Some aspects are directed to an antibody comprising a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65. Some aspects are directed to an antibody comprising a V H comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24; and a V H comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30.
  • the VL comprises SEQ ID NO:14.
  • the VH comprises SEQ ID NO:16.
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65.
  • the VL comprises SEQ ID NO:34.
  • the VH comprises SEQ ID NO:40.
  • the VL comprises SEQ ID NO:36.
  • the VH comprises SEQ ID NO:42.
  • the V L comprises SEQ ID NO:38. In some aspects, the V H comprises SEQ ID NO:42. [0184] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71; and a V H comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77. In some aspects, the VL comprises SEQ ID NO:79. In some aspects, the VH comprises SEQ ID NO:81.
  • an antibody refers to an intact immunoglobulin of any class or isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen.
  • An isotype refers to the genetic variations or differences in the constant regions of the heavy and light chains of an antibody. In humans, there are five heavy chain isotypes: IgA, IgD, IgG, IgE, and IgM and two light chain isotypes: kappa and lambda. The IgG class is divided into four isotypes: IgG1, IgG2, IgG3 and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice.
  • antibody includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody derivative, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, a multi-specific antibody, a DARPin, or a derivative or fragment of each thereof. Also contemplated are antibodies having specificity for more than one antigen or target, including bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and other multispecific antibodies.
  • an “antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof.
  • antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule.
  • the terms “antibody” or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response
  • the antibody or antigen binding fragment specifically binds human BMP-7.
  • the anti-BMP-7 antibody or antigen binding fragment thereof is a neutralizing antibody or antigen-binding fragment thereof.
  • neutralizing in the context of a BMP-7 neutralizing antibody refers to an antibody that may do one or more of: interfere with the BMP-7 receptor/BMP-7 interaction; reduce the concentration of BMP-7 receptor/BMP-7 interacted species in a subject or a cell; prevent the BMP-7 receptor/BMP-7 interaction in a subject or a cell; and/or reduce the biological function of BMP-7, which may include, but is not limited to, one or more of: promoting tumorigenesis or tumor progression; promoting cell growth and metastases; promoting inflammation, cell differentiation, adhesion, tumorigenesis, migration, invasion, and angiogenesis; inducing phosphorylation and activation of SMAD proteins; or downregulating MAPK14.
  • the term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody.
  • An antigen may possess one or more epitopes that can interact with different antibodies.
  • epitope refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component.
  • Epitope determinants may include a plurality of chemical atoms or groups on an antigen surface, such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In some aspects, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation.
  • such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some aspects, at least some such chemical atoms or groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Generally, antibodies specific for a particular target antigen would recognize an epitope on the target antigen within a complex mixture.
  • epitope regions of a given polypeptide can be identified using many different epitope mapping techniques well known in the art, including: x-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, and hydrogen-deuterium exchange see, e.g., Epitope Mapping Protocols, (Johan Rockberg and
  • immunogenic sequence means a molecule that includes an amino acid sequence of at least one epitope such that the molecule can stimulate the production of antibodies in an appropriate host.
  • immunogenic composition means a composition that comprises at least one immunogenic molecule (e.g., an antigen or carbohydrate).
  • An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains, such as antibodies naturally occurring in camelids that may comprise only heavy chains.
  • Antibodies as disclosed herein may be derived solely from a single source or may be “chimeric,” that is, different portions of the antibody may be derived from two different antibodies.
  • variable regions may be derived from a rat or murine source, while the constant region is derived from a different animal source, such as a human.
  • the antibodies or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • antibody includes derivatives, variants, fragments, and muteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol.2013; 4: 302; 2013).
  • variant region refers to a portion of the antibody that gives the antibody its specificity for binding antigen.
  • variable region is typically located at the ends of the heavy and light chains.
  • Variable loops of ⁇ -strands, three each on the light (V L ) and heavy (V H ) chains are responsible for binding to the antigen. These loops are referred to as the “complementarity determining regions” (CDRs).
  • CDRs complementarity determining regions
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • constant region refers to a portion of the antibody that is identical in all antibodies of the same isotype. The constant region differs in antibodies of different isotypes.
  • the term “light chain” may describe a full-length light chain or fragments thereof.
  • a full-length light chain has a molecular weight of around 25,000 Daltons and includes a variable region domain (abbreviated herein as VL), and a constant region domain (abbreviated herein as CL).
  • VL variable region domain
  • CL constant region domain
  • V L fragment means a fragment of the light chain of a monoclonal antibody that
  • V L fragment includes all or part of the light chain variable region, including CDRs.
  • a V L fragment can further include light chain constant region sequences.
  • the variable region domain of the light chain is at the amino-terminus of the polypeptide.
  • the term “heavy chain” may describe a full-length heavy chain or fragments thereof.
  • a full-length heavy chain for human IgG1 has a molecular weight of around 50,000 Daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as C H 1, C H 2, and C H 3).
  • V H fragment means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the heavy chain variable region, including CDRs.
  • a VH fragment can further include heavy chain constant region sequences. The number of heavy chain constant region domains will depend on the isotype.
  • the isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE and is defined by the heavy chains present of which there are five classifications: mu ( ⁇ ), delta (d), gamma ( ⁇ ), alpha ( ⁇ ), or epsilon ( ⁇ ) chains, respectively.
  • Human IgG has several subtypes, including, IgG1, IgG2, IgG3, and IgG4. A.
  • Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies with specificity to two or more antigens. They may also be fragments (e.g., F(ab’)2, Fab’, Fab, Fv, and the like), including hybrid fragments.
  • An immunoglobulin also includes natural, synthetic, or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex.
  • the term antibody includes genetically engineered or otherwise modified forms of immunoglobulins.
  • the term “monomer” means an antibody containing only one immunoglobulin unit. Monomers are the basic functional units of antibodies.
  • the term “dimer” means an antibody containing two immunoglobulin units attached to one another via constant domains of the antibody heavy chains (the Fc, or fragment crystallizable, region).
  • the complex may be stabilized by a joining (J) chain protein.
  • the term “multimer” means an antibody containing more than two immunoglobulin units attached to one another via constant domains of the antibody heavy chains (the Fc region).
  • the complex may be stabilized by a joining (J) chain protein.
  • bivalent antibody means an antibody that comprises two antigen- binding sites. The two binding sites may have the same antigen specificities, or they may be bi-specific, meaning the two antigen-binding sites have different antigen specificities.
  • Bispecific antibodies are a class of antibodies that have paratopes (i.e., antigen- binding sites) for two or more distinct epitopes.
  • Bispecific antibodies can be biparatopic, wherein a bispecific antibody may specifically recognize a different epitope from the same antigen.
  • Bispecific antibodies can be constructed from a pair of different single domain antibodies termed “nanobodies.” Single domain antibodies may be sourced and modified from cartilaginous fish and camelids. Nanobodies can be joined together by a linker using techniques typical to a person skilled in the art; such methods for selection and joining of nanobodies are described in PCT Publication No. WO2015044386A1, No.
  • Bispecific antibodies can be constructed as: a whole IgG, Fab’2, Fab’PEG, a diabody, or alternatively as a single chain variable fragment (scFv). Diabodies and scFvs can be constructed without an Fc region, using only variable domains. Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab’ fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp.
  • the antigen-binding domain may be multispecific or heterospecific by multimerizing with VH and VL region pairs that bind a different antigen.
  • the antibody may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component.
  • aspects may include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof that are directed to epitopes and to other targets, such as Fc receptors on effector cells.
  • Multispecific antibodies can be used and directly linked via a short flexible polypeptide chain, using routine methods known in the art.
  • One such example is diabodies that are bivalent, bispecific antibodies with two antigen-binding sites in which the V H and V L domains are expressed on a single polypeptide chain and utilize a linker that is too short to allow for pairing between domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain creating two antigen binding sites.
  • the linker functionality is applicable for aspects of triabodies, tetrabodies, and higher order antibody multimers.
  • the linker functionality is applicable for aspects of triabodies, tetrabodies, and higher order antibody multimers.
  • Hollinger et al. Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001), each of which is incorporated herein by reference in their entirety).
  • the part of the Fv fragment of an antibody molecule that binds with high specificity to the epitope of the antigen is referred to herein as the “paratope.”
  • the paratope consists of the amino acid residues that contact the epitope of an antigen to facilitate antigen recognition.
  • Each of the two Fv fragments of an antibody is composed of the two variable domains, V H and VL, in dimerized configuration.
  • the primary structure of each of the variable domains includes three hypervariable loops separated by, and flanked by, framework regions (FRs).
  • the hypervariable loops are the regions of highest primary sequences variability among the antibody molecules from any mammal.
  • hypervariable loop is sometimes used interchangeably with the term “complementarity determining region” (CDR).
  • CDR complementarity determining region
  • the length of the hypervariable loops (or CDRs) varies between antibody molecules.
  • the framework regions of all antibody molecules from a given mammal have high primary sequence similarity/consensus.
  • the consensus of framework regions can be used by one skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) which are interspersed among the framework regions.
  • the hypervariable loops are given identifying names which distinguish their position within the polypeptide, and on which domain they occur.
  • CDRs in the VL domain are identified as L1, L2, and L3, with L1 occurring at the most distal end and L3 occurring closest to the C L domain.
  • the CDRs may also be given the names CDR-L1, CDR-L2, and CDR-L3.
  • the L3 (CDR-L3) is generally the region of highest variability among all antibody molecules produced by a given organism.
  • the CDRs are regions of the polypeptide chain arranged linearly in the primary structure and separated from each other by FRs.
  • the amino terminal (N-terminal) end of the V L chain is named FR1.
  • the region identified as FR2 occurs between L1 and L2 hypervariable loops.
  • FR3 occurs between L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain.
  • V H chain which includes three CDRs identified as H1, H2, and H3, or CDR-H1, CDR-H2 and CDR-H3.
  • Fv fragments Fv fragments
  • Several methods have been developed and can be used by one skilled in the art to identify the exact amino acids that constitute each of these regions. This can be done using any of a number of multiple sequence alignment methods and algorithms, which identify the conserved amino acid residues that make up the framework regions, therefore identifying the CDRs that may vary in length but are located between framework regions.
  • This population of Fab expressing phage are then allowed to interact with the antigen which has been immobilized or may be expressed in by a different exogenous expression system. Non-binding Fab fragments are washed away, thereby leaving only the specific binding Fab fragments attached to the antigen.
  • the binding Fab fragments can be readily isolated and the genes which encode them determined. This approach can also be used for smaller regions of the Fab fragment including Fv fragments or specific V H and VL domains as appropriate.
  • affinity matured antibodies are enhanced with one or more modifications in one or more CDRs thereof (and/or one or more FRs thereof) that result in an improvement in the affinity of the antibody for a target antigen as compared to a parent antibody that does not possess those alteration(s).
  • Certain affinity matured antibodies will have nanomolar or picomolar affinities for the target antigen.
  • Affinity matured antibodies are produced by procedures known in the art, e.g., Marks et al., Bio/Technology 10:779 (1992) describes affinity maturation by VH and VL domain shuffling, random mutagenesis of CDR and/or framework residues employed in phage display is described by Rajpal et al., PNAS.24: 8466-8471 (2005) and Thie et al., Methods Mol Biol.525:309-22 (2009) in conjugation with
  • Chimeric immunoglobulins are the products of fused genes derived from different species (the various domains of the antibodies’ heavy and light chains are coded for by DNA from more than one species; see, e.g., U.S. Pat. No. 4,816,567); “humanized” antibodies generally have the FRs from human immunoglobulins and one or more CDRs are from a non- human source (e.g., murine).
  • humanized antibody or “humanized immunoglobulin” refers to a human/non-human chimeric antibody that contains a minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a variable region of the recipient are replaced by residues from a variable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity and capacity.
  • Humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody.
  • the humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, a non-human antibody containing one or more amino acids in a framework region, a constant region or a CDR, that have been substituted with a correspondingly positioned amino acid from a human antibody.
  • Fc immunoglobulin constant region
  • humanized antibodies are expected to produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody.
  • the humanized antibodies may have conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions.
  • Conservative substitutions groupings include: glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine and asparagine- glutamine.
  • Humanization or engineering of antibodies can be performed using any known method such as, but not limited to, those described in U.S. Pat. Nos.
  • Minimizing the antibody polypeptide sequence from the non-human species can optimize chimeric antibody function and reduces immunogenicity.
  • Specific amino acid residues of the non-human antibody are modified to be homologous to corresponding residues in a human antibody.
  • One example is the “CDR-grafted” antibody, in which an antibody comprises one or more CDRs from a particular species or belonging to a specific antibody class
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody to further refine performance.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin.
  • Intrabodies are intracellularly localized immunoglobulins that bind to intracellular antigens as opposed to secreted antibodies, which bind antigens in the extracellular space.
  • Antibodies also include “linear antibodies.” The procedure for making linear antibodies is known in the art and described in Zapata et al., 1995. Briefly, these antibodies comprise a pair of tandem Ed segments (V H -C H 1-V H -C H 1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
  • polyclonal antibody or “polyclonal antibody composition” as used herein refer to a preparation of antibodies that are derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope. Thus, polyclonal antibody preparations typically include different antibodies against different determinants (epitopes).
  • a monoclonal antibody or “mAb” refers to an antibody obtained from a population of substantially homogeneous antibodies from an exclusive parental cell, e.g., the population is identical except for naturally occurring mutations that may be present in minor amounts.
  • Each monoclonal antibody is directed against a single antigenic determinant (epitope). Monoclonal antibodies are highly specific, as each monoclonal antibody is directed against a single determinant on the antigen.
  • the term functional antibody fragment includes antigen-binding fragments of an antibody that retain the ability to specifically bind to an antigen. These fragments are constituted of various arrangements of the variable region heavy chain (VH) and/or light chain (V L ) and can include constant region heavy chain 1 (C H 1) and light chain (C L ).
  • aspects of antigen binding fragments and the modifications thereof may include: (i) the Fab fragment type constituted with the VL, VH, CL, and CH1 domains; (ii) the Fd fragment type constituted with the V H and C H 1 domains; (iii) the Fv fragment type constituted with the V H and V L domains; (iv) the single domain fragment type, dAb, (Holt et al. Trends Biotechnol. 21(11):484-90 (2003)) constituted with a single VH or VL domain; (v) isolated complementarity determining region (CDR) regions.
  • CDR complementarity determining region
  • Antigen-binding fragments also include fragments of an antibody that retain exactly, at least, or at most 1, 2, or 3 CDRs from a light chain variable region. Fusions of CDR- containing sequences to an Fc region (or a CH2 or CH3 region thereof) are included within the scope of this definition including, for example, scFv fused, directly or indirectly, to an Fc region are included herein.
  • the term Fab fragment means a monovalent antigen-binding fragment of an antibody containing the variable (V L and V H ) and the constant (C L and C H 1) domains.
  • the term Fab’ fragment means a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab’ fragment includes the VL, VH, CL and CH1
  • F(ab’)2 fragment means a bivalent antigen-binding fragment of a monoclonal antibody comprising two Fab’ fragments linked by a disulfide bridge at the hinge region.
  • An F(ab’)2 fragment includes, for example, all or part of the two V H and V L domains and can further include all or part of the two C L and C H 1 domains.
  • Fd fragment means a fragment of the heavy chain of a monoclonal antibody, which includes all or part of the VH, including the CDRs.
  • An Fd fragment can further include C H 1 region sequences.
  • Fv fragment means a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of the VL and VH, and absent of the CL and CH1 domains.
  • the V L and V H include, for example, the CDRs.
  • Single-chain antibodies are Fv molecules in which the V L and V H regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding fragment. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are herein incorporated by reference.
  • (scFv)2 means bivalent or bispecific sFv polypeptide chains that include oligomerization domains at their C-termini, separated from the sFv by a hinge region.
  • the oligomerization domain comprises self-associating ⁇ -helices, e.g., leucine zippers, which can be further stabilized by additional disulfide bonds.
  • (scFv)2 fragments are also known as “miniantibodies” or “minibodies.”
  • a single domain antibody is an antigen-binding fragment containing only a V H or the V L domain. In some instances, two or more V H regions are covalently joined with a peptide linker to create a bivalent domain antibody.
  • Fc region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C H 3 domains.
  • Fc polypeptide as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing a hinge region that promotes dimerization are included. 3. Cell Surface Receptors
  • Antigen-binding proteins of the present disclosure may be expressed on the surface of a cell.
  • antigen-binding proteins are cell surface receptors comprising antigen binding domains (e.g., BMP-7-binding domains) disclosed herein.
  • described herein are cell surface receptors comprising a BMP-7-binding domain and one or more additional components or domains. Examples of cell surface receptors of the present disclosure include chimeric antigen receptor (CARs) and T-cell receptors (TCRs).
  • a BMP-7-specific cell surface receptor may comprise one or more of an antigen binding domain, a signal peptide, an extracellular spacer, a transmembrane domain, a cytoplasmic region, and a linker.
  • a cell surface receptor of the present disclosure comprises an antigen binding domain having or comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with one or more of SEQ ID NOs:1-16 and 19-81.
  • Cells expressing a BMP-7-specific cell surface receptor may be useful in treating one or more BMP-7-associated conditions, as described elsewhere herein. C.
  • Antigen-binding peptide scaffolds such as CDRs, are used to generate protein- binding molecules in accordance with the aspects.
  • a person skilled in the art can determine the type of protein scaffold on which to graft at least one of the CDRs. It is known that scaffolds, optimally, must meet a number of criteria such as: good phylogenetic conservation; known three-dimensional structure; small size; few or no post-transcriptional modifications; and/or be easy to produce, express, and purify. Skerra, J Mol Recognit, 13:167- 87 (2000).
  • the protein scaffolds can be sourced from but are not limited to fibronectin type III FN3 domain (known as “monobodies”), fibronectin type III domain 10, lipocalin, anticalin, Z- domain of protein A of Staphylococcus aureus, thioredoxin A or proteins with a repeated motif such as the “ankyrin repeat”, the “armadillo repeat”, the “leucine-rich repeat” and the “tetratricopeptide repeat”.
  • Such proteins are described in US Patent Publication Nos. 2010/0285564, 2006/0058510, 2006/0088908, 2005/0106660, and PCT Publication No. WO2006/056464, each of which are specifically incorporated herein by reference in their entirety.
  • Scaffolds derived from toxins from scorpions, insects, plants, mollusks, etc., and the protein inhibiters of neuronal nitric oxide synthase (PIN) may also be used.
  • binding agent refers to a molecule that binds to an antigen.
  • Non-limiting examples include antibodies, antigen-binding fragments, scFv, Fab, Fab’, F(ab’)2, single chain antibodies, peptides, peptide fragments and proteins.
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • Immunologically reactive means that the selective binding agent or antibody of interest will bind with antigens present in a biological sample.
  • immuno complex refers the combination formed when an antibody or selective binding agent binds to an epitope on an antigen.
  • affinity refers the strength with which an antibody or selective binding agent binds an epitope. In antibody binding reactions, this is expressed as the affinity constant (K a or k a sometimes referred to as the association constant) for any given antibody or selective binding agent. Affinity is measured as a comparison of the binding strength of the antibody to its antigen relative to the binding strength of the antibody to an unrelated amino acid sequence.
  • k on is the rate of antibody and antigen association per unit time and is related to the concentration of the bound antigen- antibody complex at equilibrium.
  • the units used for measuring the KD are mol/L (molarity, or M), or concentration.
  • examples of some experimental methods that can be used to determine the KD value are enzyme-linked immunosorbent assays (ELISA), isothermal titration calorimetry (ITC), fluorescence anisotropy, surface plasmon resonance (SPR), and affinity capillary electrophoresis (ACE).
  • Antibodies deemed useful in certain aspects may have an equilibrium dissociation constant of at least, at most, exactly, or between (inclusive or exclusive) any two of about 10- 6, 10 -7 , 10 -8 , 10 -9 , 10 -10 M, 10 -11 M, 10 -12 M, or any range derivable therein. These values are reported for antibodies discussed herein and the same assay may be used to evaluate the binding properties of such antibodies. An antibody of the disclosure is said to “specifically bind” its target antigen when the dissociation constant (KD) is about 10 -8 M.
  • KD dissociation constant
  • the antibody specifically binds antigen with “high affinity” when the K D is about 5 ⁇ 10 -9 M, and with “very high affinity” when the K D is about 5 ⁇ 10 -12 M.
  • the epitope of an antigen is the specific region of the antigen for which an antibody has binding affinity. In the case of protein or polypeptide antigens, the epitope is the specific residues (or specified amino acids or protein segment) that the antibody binds with high affinity. An antibody does not necessarily contact every residue within the protein. Nor does every single amino acid substitution or deletion within a protein necessarily affect binding affinity.
  • epitopes and “antigenic determinant” are used interchangeably to refer to the site on an antigen to which B and/or T cell receptors respond or recognize.
  • Polypeptide epitopes can be formed from both contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a polypeptide.
  • an epitope includes at least 3, for example 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, in a unique spatial conformation.
  • One approach involves testing a collection of overlapping peptides of about 15 amino acids spanning the full sequence of the protein and differing in increments of a small number of amino acids (e.g., 3 to 30 amino acids).
  • the peptides are immobilized in separate wells of a microtiter dish. Immobilization can be accomplished, for example, by biotinylating one terminus of the peptides. This process may affect the antibody affinity for the epitope, therefore different samples of the same peptide can be biotinylated at the N and C terminus and immobilized in separate wells for the purposes of comparison. This is useful for identifying end-specific antibodies.
  • additional peptides can be included terminating at a particular amino acid of interest. This approach is useful for identifying end-specific antibodies to internal fragments. An antibody or antigen-binding fragment is screened for binding to each
  • the epitope is defined as a segment of amino acids that is common to all peptides to which the antibody shows high affinity binding.
  • the antibodies of this disclosure may be modified to yield variant antibodies.
  • Variant antibodies are substantially identical to the antibody polypeptide sequences, or fragments thereof, and still bind the epitopes of the present disclosure.
  • Polypeptide sequences are “substantially identical” when optimally aligned using such programs as Clustal Omega, IGBLAST, GAP, or BESTFIT using default gap weights, they share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity or any range therein.
  • variable region of the antibodies of the present disclosure can be modified by mutating amino acid residues within the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in
  • 202424720.3 64 vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions.
  • Framework modifications can be made to the antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to the corresponding germline sequence.
  • the antibodies of the disclosure may be engineered to include modifications within the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • Such modifications include, but are not limited to, alterations of the number of cysteine residues in the hinge region to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody (U.S. Pat. No. 5,677,425) and amino acid mutations in the Fc hinge region to decrease the biological half-life of the antibody (U.S. Pat. No.6,165,745).
  • Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. Certain preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases.
  • computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function.
  • Standard methods to identify protein sequences that fold into a known three-dimensional structure are available to those skilled in the art; Dill and McCallum., Science 338:1042-1046 (2012).
  • Several algorithms for predicting protein structures and the gene sequences that encode these have been developed, and many of these algorithms can be found at the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov/guide/proteins/) and at the Bioinformatics Resource Portal (on the World Wide Web at expasy.org/proteomics).
  • the antigen-binding domain may be multi-specific or multivalent by multimerizing the antigen-binding domain with V H and V L region pairs that bind either the same antigen (multi-valent) or a different antigen (multi-specific).
  • V H and V L region pairs that bind either the same antigen (multi-valent) or a different antigen (multi-specific).
  • the antibodies of the disclosure may be enzymatically or chemically modified to produce further derivatives of the antibodies and antigen binding fragments that are described herein.
  • the term “antibody derivative” can include post-translational modification to the linear polypeptide sequence of the antibody or fragment.
  • the derivatized antibody or fragment thereof may comprise any molecule or substance that imparts a desired property to the antibody or fragment.
  • the derivatized antibody can comprise, for example, a chemical post-translational modification, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead), a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antibody for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses).
  • a detectable (or labeling) moiety e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead
  • a detectable moiety e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule,
  • an antibody or fragment thereof can be covalently attached to a molecule or substance, such as a labeling moiety or a therapeutic moiety; covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
  • An antibody or fragment thereof can be non-covalently attached to a molecule or substance, such as a labeling moiety or a therapeutic moiety.
  • an antibody or an antigen-binding fragment can be chemically conjugated to, or expressed as, a fusion protein with other proteins.
  • polypeptides may be chemically modified by conjugating or fusing the polypeptide to serum protein, such as human serum albumin, to increase half-life of the resulting molecule.
  • the polypeptides may be conjugated to a diagnostic agent and used diagnostically, for example, to monitor the development or progression of a disease and determine the efficacy of a given treatment regimen.
  • the polypeptides may also be conjugated to a therapeutic agent to provide a therapy in combination with the therapeutic effect of the polypeptide.
  • disclosed are antibodies and antibody-like molecules that are linked to at least one agent to form an antibody conjugate or payload. To increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety.
  • Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
  • Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity.
  • Non-limiting examples of effector molecules include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides and the like.
  • a reporter molecule is defined as any moiety that may be detected
  • reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands. 1.
  • the antigen-binding protein can have or lack one or more post-translational modifications such as myristoylation, palmitoylation, isoprenylation or prenylation, farnesylation, geranylgeranylation, glypiation, acylation, acetylation, formylation, alkylation, methylation, amide bond formation, amidation at C-terminus, arginylation, polyglutamylation, polyglycylation, butyrylation, glycosylation, glycation, polysialylation, malonylation, hydroxylation, iodination, phosphorylation, adenylylation, propionylation, S- glutathionylation, S-nitrosylation, S-sulfenylation (aka S-sulphenylation), succinylation, sulfation, biotinylation, pegylation, SUMOylation, ubiquitination,
  • the antigen-binding protein can have reduced or increased amounts of one or more post-translational modifications as compared to the same antigen- binding protein expressed in the cell that is native to the encoded gene.
  • the reduction or increase may be by at least or at most 25, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500% or more (or any range derivable therein).
  • contemplated are glycosylation variants of antibodies, wherein the number and/or type of glycosylation site(s) has been altered compared to the amino acid sequences of the parent polypeptide.
  • Glycosylation of the polypeptides can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861, incorporated herein by reference).
  • Antibody protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native antibody.
  • An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline.
  • the substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain.
  • substitutions that eliminate or alter this sequence will prevent addition of an N-linked carbohydrate chain present in the native polypeptide.
  • the glycosylation can be reduced by the deletion of an Asn or by substituting the Asn
  • Additional antibody variants include cysteine variants, wherein one or more cysteine residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g., serine). Cysteine variants are useful, inter alia, when antibodies must be refolded into a biologically active conformation. Cysteine variants may have fewer cysteine residues than the native antibody and typically have an even number to minimize interactions resulting from unpaired cysteines.
  • the polypeptides can be pegylated to increase biological half-life by reacting the polypeptide with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the polypeptide.
  • PEG polyethylene glycol
  • Polypeptide pegylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • Methods for pegylating proteins are known in the art and can be applied to the polypeptides of the disclosure to obtain PEGylated derivatives of antibodies. See, e.g., EP 0154316 and EP 0401384, incorporated herein by reference.
  • the antibody is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant.
  • TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyrrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols, and polyvinyl alcohols.
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins. 2.
  • antibody conjugates are those conjugates in which the antibody is linked to a detectable label.
  • Detectable labels are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to be detected, and/or further quantified if desired.
  • the term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like.
  • GFP green fluorescent protein
  • the label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • the labels can be suitable for small scale detection or more suitable for
  • the label may be simply detected, or it may be quantified.
  • a response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property.
  • detectable labels include, but not limited to, radioactive isotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like.
  • labels are, but not limited to, horseradish peroxidase (HRP), fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, nicotinamide adenine dinucleotide phosphate (NADPH), and ⁇ - or ß-galactosidase.
  • HRP horseradish peroxidase
  • FITC fluorescein isothiocyanate
  • rhodamine dansyl
  • umbelliferone umbelliferone
  • DMAE dimethyl acridinium ester
  • Texas red Texas red
  • luminol nicotinamide adenine dinucleotide phosphate
  • NADPH nicotinamide adenine dinucleotide phosphate
  • NADPH ⁇ - or ß-galactosidas
  • the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.
  • luminescent labels that produce signals include but are not limited to bioluminescence and chemiluminescence.
  • Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P.
  • luminescent probes include, but are not limited to, aequorin and luciferases.
  • suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl- coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red.
  • suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.).
  • the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker.
  • Suitable functional groups including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
  • the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
  • Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker. Suitable binding pairs for use in indirectly
  • linking the fluorescent label to the intermediate include, but are not limited to, antigens/antibodies, e.g., rhodamine/anti-rhodamine, biotin/avidin and biotin/streptavidin.
  • Antibodies may also be coupled to low molecular weight haptens to increase the sensitivity of the antibody in an assay. The haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See, Harlow and Lane (1988) supra.
  • Antibody conjugates also include those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme to generate a colored product upon contact with a chromogenic substrate.
  • suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase.
  • Preferred secondary binding ligands are biotin and/or avidin and streptavidin compounds. The uses of such labels are well known to those of skill in the art and are described, for example, in U.S.
  • RNA molecules may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light.
  • Additional suitable conjugated molecules include ribonuclease (RNase), DNase I, an antisense oligonucleotide, an inhibitory RNA molecule such as a siRNA molecule, an immunostimulatory nucleic acid, aptamers, ribozymes, triplex forming molecules, and external guide sequences (e.g., guide RNAs).
  • Aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets, and can bind small molecules, such as ATP (U.S. Pat. No. 5,631,146) and theophiline (U.S. Pat. No.5,580,737), as well as large molecules, such as reverse transcriptase (U.S. Pat. No.5,786,462) and thrombin (U.S. Pat. No.5,543,293).
  • Ribozymes are nucleic acid molecules that can catalyze a chemical reaction, either intramolecularly or intermolecularly.
  • Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage.
  • Triplex forming function nucleic acid molecules can interact with double-stranded or single-stranded nucleic acid by forming a triplex, in which three strands of DNA form a complex dependent on both Watson-Crick and Hoogsteen base- pairing. Triplex molecules can bind target regions with high affinity and specificity.
  • the functional nucleic acid molecules may act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules may possess a de novo activity independent of any other molecules.
  • the antibodies of the disclosure or antigen-binding regions thereof can also be linked to another functional molecule such as another antibody or ligand for a receptor to generate a bi-specific or multi-specific molecule that binds to at least two or more different binding sites or target molecules.
  • Linking of the antibody to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, can be done, for example, by chemical coupling, genetic fusion, or noncovalent association.
  • Multi- specific molecules can further include a third binding specificity, in addition to the first and second target epitope.
  • the antibodies or fragments thereof of the present disclosure may be linked to a moiety that is toxic to a cell to which the antibody is bound to form “depleting” antibodies.
  • the antibodies of the disclosure may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • the antibodies also can be bound to many different carriers. Thus, this disclosure also provides compositions containing the antibodies and another substance, active or inert.
  • Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite.
  • the nature of the carrier can be either soluble or insoluble for purposes of the disclosure. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation.
  • contemplated are immunoconjugates comprising an antibody or antigen-binding fragment thereof conjugated (e.g., covalently attached) to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g
  • the immunoconjugate can be provided in the form of a genetic fusion protein.
  • an antibody may be conjugated to various therapeutic substances to target the cell surface antigen.
  • conjugated agents include, but are not limited to, metal chelate complexes, drugs, toxins, and other effector molecules, such as cytokines,
  • ADC antibody drug conjugates
  • the ADC may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form antibody- L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody.
  • ADC may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug.
  • a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis.
  • the length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
  • Examples of ADC known to a person skilled in the art are pro-drugs useful for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605-614 (1999); Niculescu- Duvaz and Springer, Adv. Drg. Del. Rev.
  • ADCs include covalent or aggregative conjugates of antibodies, or antigen-binding fragments thereof, with other proteins or peptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an antibody polypeptide.
  • the conjugated peptide may be a heterologous signal (or leader) polypeptide, e.g., the yeast alpha-factor leader, or a peptide such as an epitope tag (e.g., V5-His).
  • Antibody-containing fusion proteins may comprise peptides added to facilitate purification or identification of the antibody (e.g., poly-His).
  • polypeptide also can be linked to the FLAG® (Sigma-Aldrich, St. Louis, Mo.) peptide as described in Hopp et al., Bio/Technology 6:1204 (1988), and U.S. Pat. No.5,011,912.
  • the conjugated agents can be linked to the antibody directly or indirectly, using any of a large number of available methods. Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety (Amon et al., 1985; Hellstrom et al., 1987; Thorpe, 1985; Baldwin et al., 1985; Thorpe et al., 1982).
  • Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro- 3-6-diphenylglycouril-3 attached to the antibody (U.S. Patent Nos.4,472,509 and 4,938,948, each incorporated herein by reference).
  • DTPA diethylenetriaminepentaacetic acid anhydride
  • ethylenetriaminetetraacetic acid N-chloro-p-toluenesulfonamide
  • tetrachloro- 3-6-diphenylglycouril-3 attached to the antibody
  • Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
  • Conjugates may also be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyridyld
  • derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site, are contemplated.
  • Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No.5,196,066, incorporated herein by reference).
  • Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region has also been disclosed in the literature (O’Shannessy et al., J. Immunol. Methods 99(2):153-61 (1987)).
  • Bi-specific and multi-specific molecules can be prepared using methods known in the art. For example, each binding unit of the hi-specific molecule can be generated separately and then conjugated to one another.
  • the binding molecules are proteins or peptides
  • a variety of coupling or cross-linking agents can be used for covalent conjugation.
  • cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5’-dithiobis(2-nitroberizoic acid) (DTNB), o-phenylenedimaleimide (oRDM), N-
  • succinimidyl-3-(2-pyridyldithio)propionate SPDP
  • sulfo-SMCC sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohaxane-I-carboxylate
  • the size of a protein or polypeptide may comprise, but is not limited to, at least, at most, exactly, or between (inclusive or exclusive) any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
  • polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).
  • domain refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.
  • polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids (e.g., amino acid substitutions) or be at least, at most, exactly, or between (inclusive or exclusive) any two of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any de
  • the protein or polypeptide may comprise amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 (or any derivable range
  • the protein or polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 (or any derivable range therein) con
  • the polypeptide or protein may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107
  • polypeptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 of any of SEQ ID NOs:
  • Nucleotide as well as protein, polypeptide, and peptide sequences for various genes have been previously disclosed and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s GENBANK® and GENPEPT® databases (on the World Wide Web at ncbi.nlm.nih.gov) and The Universal Protein Resource (UNIPROT®; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art. [0282] It is contemplated that in compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide per ml.
  • the concentration of polypeptide in a composition can be at least, at most, exactly, or between (inclusive or exclusive) any two of about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein).
  • Amino acid sequences from 12 light chain variable region CDRs from the BMP-7- binding proteins (e.g., antibodies) of the present disclosure are provided in SEQ ID NOs:2, 4,
  • CDR-L1 SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67
  • CDR-L2 SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69
  • CDR-L3 SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • amino acid sequence for a first full light chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:14 as follows: [0286] DIVMTQSPSSMYASLGERVTITCKASQDITGYLGWFQQKPGKSPKTLIYRA DRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO:14).
  • amino acid sequence for a second full light chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:34 as follows: [0288] DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYY TSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDYSSPPTFGGGTKLEIK (SEQ ID NO:34).
  • amino acid sequence for a third full light chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:36 as follows: [0290] DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGQAPKLLIYY TSNRFTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQDYSSPPTFGGGTKLEIK (SEQ ID NO:36).
  • amino acid sequence for a fourth full light chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:38 as follows: [0292] DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGQAPKLLIYY TSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQDYSSPPTFGGGTKLEIK (SEQ ID NO:38).
  • SEQ ID NO:79 The amino acid sequence for a fifth full light chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:79 as follows: [0294] SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYY TSNRFTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPPTFGGGTKLEIK (SEQ ID NO:79).
  • SEQ ID NO:16 The amino acid sequence for a first full heavy chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:16 as follows: [0296] SGPELKKPGETVKISCKASGYTFTDSSMHWVKQAPGKGLKWMGWINTET GEPTYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCSRDYDWFAYWGQGTL VTVSA (SEQ ID NO:16).
  • SEQ ID NO:40 The amino acid sequence for a second full heavy chain variable region of a BMP- 7-binding protein disclosed herein is provided in SEQ ID NO:40 as follows: [0298] QVQLVQSGSELKKPGASVKVSCKASGYTFTDSSMHWVRQAPGQGLKWM GWINTETGEPTYVDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYDWFAY WGQGTLVTVSS (SEQ ID NO:40).
  • amino acid sequence for a third full heavy chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:42 as follows: [0300] QVQLVQSGSELKKPGASVKVSCKASGYTFTDSSMHWVRQAPGQGLEWM GWINTETGEPTYVDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYDWFAY WGQGTLVTVSS (SEQ ID NO:42).
  • amino acid sequence for a fourth full heavy chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:81 as follows: [0302] IQLVQSGPELKKPGETVKISCKASGYTFTDSSMHWVKQAPGKGLKWMG WINTETGEPTYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCSRDYDWFAY WGQGTLVTVSA (SEQ ID NO:81). [0303] In some aspects, the VH and the VL are on the same polypeptide. In some aspects, the V H and the V L are on different polypeptides.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • V L light chain variable region
  • antigen-binding proteins e.g., antibodies, antibody- like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • VH heavy chain variable region
  • the V L has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79. In some aspects, the VH has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the V L has at least 90% identity
  • the VH has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79.
  • the V H has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the VL comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79.
  • the V H comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79; and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 8
  • the VL has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79; and the V H has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the VL has at least 90% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79; and the VH has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79; and the VH has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the V L comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79; and the VH comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • antigen-binding proteins e.g., antibodies, antibody- like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%
  • VL light chain variable region
  • VH heavy chain variable region
  • the VL has at least 85% identity to SEQ ID NO:14
  • the VH has at least 85% identity to SEQ ID NO:16.
  • the VL has at least 90% identity to SEQ ID NO:14, and the V H has at least 90% identity to SEQ ID NO:16. In some aspects, the VL has at least 95% identity to SEQ ID NO:14, and the VH has at least 95% identity to SEQ ID NO:16. In some aspects, the VL comprises SEQ ID NO:14, and the VH comprises SEQ ID NO:16.
  • antigen-binding proteins e.g., antibodies, antibody- like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a light chain variable region (V L ) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:34; and a heavy chain variable region (V H ) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
  • V L light chain variable region having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%
  • the V L has at least 85% identity to SEQ ID NO:34, and the V H has at least 85% identity to SEQ ID NO:40. In some aspects, the V L has at least 90% identity to SEQ ID NO:34, and the VH has at least 90% identity to SEQ ID NO:40. In some aspects, the VL has at least 95% identity to SEQ ID NO:34, and the VH has at least 95% identity to SEQ ID NO:40. In some aspects, the V L comprises SEQ ID NO:34, and the V H comprises SEQ ID NO:40.
  • antigen-binding proteins e.g., antibodies, antibody- like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:36; and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
  • the V L has at least 85% identity to SEQ ID NO:36, and the VH has at least 85% identity to SEQ ID NO:42.
  • the VL has at least 90% identity to SEQ ID NO:36, and the VH has at least 90% identity to SEQ ID NO:42.
  • the V L has at least 95% identity to SEQ ID NO:36, and the V H has at least 95% identity to SEQ ID NO:42.
  • the VL comprises SEQ ID NO:36, and the VH comprises SEQ ID NO:42.
  • antigen-binding proteins e.g., antibodies, antibody- like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a light chain variable region (V L ) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:38; and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
  • the VL has at least 85% identity to SEQ ID NO:38, and the V H has at least 85% identity to SEQ ID NO:42. In some aspects, the V L has at least 90% identity to SEQ ID NO:38, and the VH has at least 90% identity to SEQ ID NO:42. In some aspects, the VL has at least 95% identity to SEQ ID NO:38, and the VH has at least 95% identity to SEQ ID NO:42. In some aspects, the V L comprises SEQ ID NO:38, and the V H comprises SEQ ID NO:42.
  • RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:44.
  • amino acid sequence for a full heavy chain constant region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:46 as follows: [0313] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
  • the CH and the CL are on the same polypeptide.
  • the C H and the C L are on different polypeptides.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a light chain constant region (CL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:44.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen- binding fragment thereof comprising a heavy chain constant region (C H ) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:46.
  • the C L has at least 85% identity to SEQ ID NO:44. In some aspects, the CH has at least 85% identity to SEQ ID NO:46. In some aspects, the CL has at least 90% identity to SEQ ID NO:44. In some aspects, the CH has at least 90% identity to SEQ ID NO:46. In some aspects, the C L has at least 95% identity to SEQ ID NO:44. In some aspects, the C H has at least 95% identity to SEQ ID NO:46. In some aspects, the C L comprises SEQ ID NO:44. In some aspects, the CH comprises SEQ ID NO:46.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a light chain constant region (CL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:44; and a heavy chain constant region (CH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
  • CH heavy chain constant region
  • the CL has at least 85% identity to SEQ ID NO:44; and the CH has at least 85% identity to SEQ ID NO:46. In some aspects, the CL has at least 90% identity to SEQ ID NO:44; and the C H has at least 90% identity to SEQ ID NO:46.
  • the C L has at least 95% identity to SEQ ID NO:44; and the C H has at least 95% identity to SEQ ID NO:46.
  • the CL comprises SEQ ID NO:44; and the CH comprises SEQ ID NO:46.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a light chain variable region (V L ) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; and a light chain constant region (C L ) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%
  • V L light chain variable region having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region (V H ) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:40 or SEQ ID NO:42; and a heavy chain constant region (C H ) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%,
  • V H heavy chain variable region having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 8
  • the VL has at least 85% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; and the C L has at least 85% identity to SEQ ID NO:44.
  • the VH has at least 85% identity to SEQ ID NO:40 or SEQ ID NO:42; and the CH has at least 85% identity to SEQ ID NO:46.
  • the VL has at least 90% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; and the C L has at least 90% identity to SEQ ID NO:44.
  • the VH has at least 90% identity to SEQ ID NO:40 or SEQ ID NO:42; and the CH has at least 90% identity to SEQ ID NO:46.
  • the V L has at least 95% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; and the C L has at least 95% identity to SEQ ID NO:44.
  • the V H has at least 95% identity to SEQ ID NO:40 or SEQ ID NO:42; and the CH has at least 95% identity to SEQ ID NO:46.
  • the VL comprises SEQ ID NO:34, SEQ ID NO:36, or SEQ ID
  • V H comprises SEQ ID NO:40 or SEQ ID NO:42; and the CH comprises SEQ ID NO:46.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38
  • the VL has at least 85% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; the C L has at least 85% identity to SEQ ID NO:44; the V H has at least 85% identity to SEQ ID NO:40 or SEQ ID NO:42; and the C H has at least 85% identity to SEQ ID NO:46.
  • the VL has at least 90% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; the CL has at least 90% identity to SEQ ID NO:44; the VH has at least 90% identity to SEQ ID NO:40 or SEQ ID NO:42; and the C H has at least 90% identity to SEQ ID NO:46.
  • the VL has at least 95% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; the CL has at least 95% identity to SEQ ID NO:44; the VH has at least 95% identity to SEQ ID NO:40 or SEQ ID NO:42; and the C H has at least 95% identity to SEQ ID NO:46.
  • the VL comprises SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; the CL comprises SEQ ID NO:44; the VH comprises SEQ ID NO:40 or SEQ ID NO:42; and the C H comprises SEQ ID NO:46.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • a monoclonal antibody or antigen-binding fragment thereof comprising a light chain variable region (V L ) having at least, at most, exactly, or
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:81.
  • VH heavy chain variable region
  • the VL has at least 85% identity to SEQ ID NO:79. In some aspects, the V H has at least 85% identity to SEQ ID NO:81. In some aspects, the V L has at least 90% identity to SEQ ID NO:79. In some aspects, the VH has at least 90% identity to SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:79. In some aspects, the VH has at least 95% identity to SEQ ID NO:81. In some aspects, the V L comprises SEQ ID NO:79. In some aspects, the V H comprises SEQ ID NO:81.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:79; and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the VL has at least 85% identity to SEQ ID NO:79; and the VH has at least 85% identity to SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:79; and the V H has at least 90% identity to SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:79; and the VH has at least 95% identity to SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:79; and the V H comprises SEQ ID NO:81.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a CDR-L1 having at least, at most, exactly, or between (inclusive or
  • antigen-binding proteins e.g., antibodies, antibody- like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • BMP-7 bone morphogenetic protein-7
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 95% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:2; a CDR-L2 having 85% sequence identity to SEQ ID NO:4; and a CDR-L3 having 85% sequence identity to SEQ ID NO:6. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:2; a CDR-L2 having 95% sequence identity to SEQ ID NO:4; and a CDR-L3 having 95% sequence identity to SEQ ID NO:6. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:2; a CDR-L2 comprising SEQ ID NO:4; and a CDR-L3 comprising SEQ ID NO:6.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:20; a CDR-L2 having 85% sequence identity to SEQ ID NO:22; and a CDR-L3 having 85% sequence identity to SEQ ID NO:24. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:20; a CDR-L2 having 90% sequence identity to SEQ ID NO:22; and a CDR-L3 having 90% sequence identity to SEQ ID NO:24.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:20; a CDR-L2 having 95% sequence identity to SEQ ID NO:22; and a CDR-L3 having 95% sequence identity to SEQ ID NO:24. Some aspects are directed to antigen-binding proteins comprising a CDR- L1 comprising SEQ ID NO:20; a CDR-L2 comprising SEQ ID NO:22; and a CDR-L3 comprising SEQ ID NO:24.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:50; a CDR-L2 having 85% sequence identity to SEQ ID NO:53; and a CDR-L3 having 85% sequence identity to SEQ ID NO:57. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:50; a CDR-L2 having 90% sequence identity to SEQ ID NO:53; and a CDR-L3 having 90% sequence identity to SEQ ID NO:57.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:50; a CDR-L2 having 95% sequence identity to SEQ ID NO:53; and a CDR-L3 having 95% sequence identity to SEQ ID NO:57. Some aspects are directed to antigen-binding proteins comprising a CDR- L1 comprising SEQ ID NO:50; a CDR-L2 comprising SEQ ID NO:53; and a CDR-L3 comprising SEQ ID NO:57.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:67; a CDR-L2 having 85% sequence identity to SEQ ID NO:69; and a CDR-L3 having 85% sequence identity to SEQ ID NO:71. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:67; a CDR-L2 having 90% sequence identity to SEQ ID NO:69; and a CDR-L3
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:67; a CDR-L2 having 95% sequence identity to SEQ ID NO:69; and a CDR-L3 having 95% sequence identity to SEQ ID NO:71. Some aspects are directed to antigen-binding proteins comprising a CDR- L1 comprising SEQ ID NO:67; a CDR-L2 comprising SEQ ID NO:69; and a CDR-L3 comprising SEQ ID NO:71.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a CDR-H1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR- H3 having 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • aspects are directed to antigen-binding proteins comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:8; a CDR-H2 having 85% sequence identity to SEQ ID NO:10; and a CDR-H3 having 85% sequence identity to SEQ ID NO:12. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:8; a CDR-H2 having 90% sequence identity to SEQ ID NO:10; and a CDR-H3 having 90% sequence identity to SEQ ID NO:12.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:8; a CDR-H2 having 95% sequence identity to SEQ ID NO:10; and a CDR-H3 having 95% sequence identity to SEQ ID NO:12. Some aspects are directed to antigen-binding proteins comprising a CDR- H1 comprising SEQ ID NO:8; a CDR-H2 having 85% comprising SEQ ID NO:10; and a CDR- H3 comprising SEQ ID NO:12.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:26; a CDR-H2 having 85% sequence identity to SEQ ID NO:28; and a CDR-H3 having 85% sequence identity to SEQ ID NO:30. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:26; a CDR-H2 having 90% sequence identity to SEQ ID NO:28; and a CDR-H3 having 90% sequence identity to SEQ ID NO:30.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:26; a CDR-H2 having 95% sequence identity to SEQ ID NO:28; and a CDR-H3 having 95% sequence identity to SEQ ID NO:30. Some aspects are directed to antigen-binding proteins comprising a CDR- H1 comprising SEQ ID NO:26; a CDR-H2 comprising SEQ ID NO:28; and a CDR-H3 comprising SEQ ID NO:30.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:26; a CDR-H2 having 85% sequence identity to SEQ ID NO:28; and a CDR-H3 having 85% sequence identity to SEQ ID NO:32. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:26; a CDR-H2 having 90% sequence identity to SEQ ID NO:28; and a CDR-H3 having 90% sequence identity to SEQ ID NO:32.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:26; a CDR-H2 having 95% sequence identity to SEQ ID NO:28; and a CDR-H3 having 95% sequence identity
  • Some aspects are directed to antigen-binding proteins comprising a CDR- H1 comprising SEQ ID NO:26; a CDR-H2 comprising SEQ ID NO:28; and a CDR-H3 comprising SEQ ID NO:32. [0332] Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:59; a CDR-H2 having 85% sequence identity to SEQ ID NO:62; and a CDR-H3 having 85% sequence identity to SEQ ID NO:65.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:59; a CDR-H2 having 90% sequence identity to SEQ ID NO:62; and a CDR-H3 having 90% sequence identity to SEQ ID NO:65. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:59; a CDR-H2 having 95% sequence identity to SEQ ID NO:62; and a CDR-H3 having 95% sequence identity to SEQ ID NO:65.
  • Some aspects are directed to antigen-binding proteins comprising a CDR- H1 comprising SEQ ID NO:59; a CDR-H2 comprising SEQ ID NO:62; and a CDR-H3 comprising SEQ ID NO:65. [0333] Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:73; a CDR-H2 having 85% sequence identity to SEQ ID NO:75; and a CDR-H3 having 85% sequence identity to SEQ ID NO:77.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:73; a CDR-H2 having 90% sequence identity to SEQ ID NO:75; and a CDR-H3 having 90% sequence identity to SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:73; a CDR-H2 having 95% sequence identity to SEQ ID NO:75; and a CDR-H3 having 95% sequence identity to SEQ ID NO:77.
  • antigen-binding proteins comprising a CDR- H1 comprising SEQ ID NO:73; a CDR-H2 comprising SEQ ID NO:75; and a CDR-H3 comprising SEQ ID NO:77.
  • antigen-binding proteins e.g., antibodies, antibody-like molecules, or fragments thereof
  • BMP-7 bone morphogenetic protein-7
  • monoclonal antibody or antigen-binding fragment thereof comprising a CDR-L1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:
  • SEQ ID NO:4 SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a CDR-H1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:26,
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a CDR-H1 having 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a CDR-H1 having 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 95% sequence
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:2; a CDR-L2 having 85% sequence identity to SEQ ID NO:4; and a CDR-L3 having 85% sequence identity to SEQ ID NO:6; and a CDR-H1 having 85% sequence identity to SEQ ID NO:8; a CDR-H2 having 85% sequence identity to SEQ ID NO:10; and a CDR-H3 having 85% sequence identity to SEQ ID NO:12.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:2; a CDR-L2 having 90% sequence identity to SEQ ID NO:4; and a CDR-L3 having 90% sequence identity to SEQ ID NO:6; and a CDR-H1 having 90% sequence identity to SEQ ID NO:8; a CDR-H2 having 90% sequence identity to SEQ ID NO:10; and a CDR-H3 having 90% sequence identity to SEQ ID NO:12.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:2; a CDR-L2 having 95% sequence identity to SEQ ID NO:4; and a CDR-L3 having 95% sequence identity to SEQ ID NO:6; and a CDR-H1 having 95% sequence identity to SEQ ID NO:8; a CDR-H2 having 95% sequence identity to SEQ ID NO:10; and a CDR-H3 having 95% sequence identity to SEQ ID NO:12.
  • Some aspects are directed to antigen-binding proteins comprising a CDR- L1 comprising SEQ ID NO:2; a CDR-L2 comprising SEQ ID NO:4; and a CDR-L3 comprising SEQ ID NO:6; and a CDR-H1 comprising SEQ ID NO:8; a CDR-H2 comprising SEQ ID NO:10; and a CDR-H3 comprising SEQ ID NO:12.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:20; a CDR-L2 having 85% sequence identity to SEQ ID NO:22; and a CDR-L3 having 85% sequence identity to SEQ ID NO:24; and a CDR-H1 having
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:20; a CDR-L2 having 90% sequence identity to SEQ ID NO:22; and a CDR-L3 having 90% sequence identity to SEQ ID NO:24; and a CDR-H1 having 90% sequence identity to SEQ ID NO:26; a CDR-H2 having 90% sequence identity to SEQ ID NO:28; and a CDR- H3 having 90% sequence identity to SEQ ID NO:30.
  • Some aspects are directed to antigen- binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:20; a CDR-L2 having 95% sequence identity to SEQ ID NO:22; and a CDR-L3 having 95% sequence identity to SEQ ID NO:24; and a CDR-H1 having 95% sequence identity to SEQ ID NO:26; a CDR-H2 having 95% sequence identity to SEQ ID NO:28; and a CDR-H3 having 95% sequence identity to SEQ ID NO:30.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:20; a CDR-L2 comprising SEQ ID NO:22; and a CDR-L3 comprising SEQ ID NO:24; and a CDR-H1 comprising SEQ ID NO:26; a CDR-H2 comprising SEQ ID NO:28; and a CDR-H3 comprising SEQ ID NO:30.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:20; a CDR-L2 having 85% sequence identity to SEQ ID NO:22; and a CDR-L3 having 85% sequence identity to SEQ ID NO:24; and a CDR-H1 having 85% sequence identity to SEQ ID NO:26; a CDR-H2 having 85% sequence identity to SEQ ID NO:28; and a CDR-H3 having 85% sequence identity to SEQ ID NO:32.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:20; a CDR-L2 having 90% sequence identity to SEQ ID NO:22; and a CDR-L3 having 90% sequence identity to SEQ ID NO:24; and a CDR-H1 having 90% sequence identity to SEQ ID NO:26; a CDR-H2 having 90% sequence identity to SEQ ID NO:28; and a CDR- H3 having 90% sequence identity to SEQ ID NO:32.
  • Some aspects are directed to antigen- binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:20; a CDR-L2 having 95% sequence identity to SEQ ID NO:22; and a CDR-L3 having 95% sequence identity to SEQ ID NO:24; and a CDR-H1 having 95% sequence identity to SEQ ID NO:26; a CDR-H2 having 95% sequence identity to SEQ ID NO:28; and a CDR-H3 having 95% sequence identity to SEQ ID NO:32.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:20; a CDR-L2 comprising SEQ ID NO:22; and a CDR-L3 comprising SEQ ID NO:24; and a CDR-H1 comprising SEQ ID NO:26; a CDR-H2 comprising SEQ ID NO:28; and a CDR-H3 comprising SEQ ID NO:32.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:50; a CDR-L2 having 85% sequence identity to SEQ ID NO:53; and a CDR-L3 having 85% sequence identity to SEQ ID NO:57; and a CDR-H1 having 85% sequence identity to SEQ ID NO:59; a CDR-H2 having 85% sequence identity to SEQ ID NO:62; and a CDR-H3 having 85% sequence identity to SEQ ID NO:65.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:50; a CDR-L2 having 90% sequence identity to SEQ ID NO:53; and a CDR-L3 having 90% sequence identity to SEQ ID NO:57; and a CDR-H1 having 90% sequence identity to SEQ ID NO:59; a CDR-H2 having 90% sequence identity to SEQ ID NO:62; and a CDR- H3 having 90% sequence identity to SEQ ID NO:65.
  • Some aspects are directed to antigen- binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:50; a CDR-L2 having 95% sequence identity to SEQ ID NO:53; and a CDR-L3 having 95% sequence identity to SEQ ID NO:57; and a CDR-H1 having 95% sequence identity to SEQ ID NO:59; a CDR-H2 having 95% sequence identity to SEQ ID NO:62; and a CDR-H3 having 95% sequence identity to SEQ ID NO:65.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:50; a CDR-L2 comprising SEQ ID NO:53; and a CDR-L3 comprising SEQ ID NO:57; and a CDR-H1 comprising SEQ ID NO:59; a CDR-H2 comprising SEQ ID NO:62; and a CDR-H3 comprising SEQ ID NO:65.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:67; a CDR-L2 having 85% sequence identity to SEQ ID NO:69; and a CDR-L3 having 85% sequence identity to SEQ ID NO:71; and a CDR-H1 having 85% sequence identity to SEQ ID NO:73; a CDR-H2 having 85% sequence identity to SEQ ID NO:75; and a CDR-H3 having 85% sequence identity to SEQ ID NO:77.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:67; a CDR-L2 having 90% sequence identity to SEQ ID NO:69; and a CDR-L3 having 90% sequence identity to SEQ ID NO:71; and a CDR-H1 having 90% sequence identity to SEQ ID NO:73; a CDR-H2 having 90% sequence identity to SEQ ID NO:75; and a CDR- H3 having 90% sequence identity to SEQ ID NO:77.
  • Some aspects are directed to antigen- binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:67; a CDR-L2 having 95% sequence identity to SEQ ID NO:69; and a CDR-L3 having 95% sequence identity to SEQ ID NO:71; and a CDR-H1 having 95% sequence identity to SEQ ID NO:73; a CDR-H2 having 95% sequence identity to SEQ ID NO:75; and a CDR-H3 having 95% sequence identity to SEQ ID NO:77.
  • Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:67; a CDR-L2 comprising SEQ ID
  • the disclosure relates to a bone morphogenetic protein-7 (BMP-7)- binding protein comprising: a light chain variable region (V L ) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71.
  • V L light chain variable region
  • CDRs complementarity determining regions
  • the disclosure relates to a bone morphogenetic protein-7 (BMP-7)-binding protein comprising: a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a bone morphogenetic protein-7 (BMP-7)- binding protein comprising: a light chain variable region (V L ) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a heavy chain variable region (V H ) comprising three complementarity determining regions (CDR
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71.
  • the disclosure relates to a BMP-7-binding protein comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71.
  • the disclosure relates to a BMP-7-binding protein comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71.
  • the disclosure relates to a BMP-7-binding protein comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71.
  • the disclosure relates to a BMP-7-binding protein comprising: a V H comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein comprising: a V L comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and V H comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50,
  • SEQ ID NO:53 SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a V H comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69,
  • the disclosure relates to a BMP-7-binding protein, wherein the V L comprises a CDR-L1 having at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the disclosure relates to a BMP-7-binding protein, wherein the V L comprises a CDR-L1 having at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 95% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID
  • the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and
  • the disclosure relates to a BMP-7-binding protein, wherein the V H comprises a CDR-H1 having at least 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein, wherein the V H comprises a CDR-H1 having at least 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID
  • a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75
  • a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • Some aspects are directed to a monoclonal (e.g., humanized) antibody or antigen- binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71.
  • a monoclonal (e.g., humanized) antibody or antigen- binding fragment thereof comprising: a VL comprising three CDR
  • Some aspects are directed to a monoclonal (e.g., humanized) antibody or antigen- binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • Some aspects are directed to a monoclonal (e.g., humanized) antibody or antigen- binding fragment thereof comprising: a V L comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most,
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a V H comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a V L comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71.
  • a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a V L comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, S
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a V L comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71.
  • a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a V L comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:2, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:2, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:2, SEQ
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a V L comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a V H comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a V H comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12.
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the V L comprises a CDR-L1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:4, SEQ ID NO:22,
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the VL comprises a CDR-L1 having at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the V L comprises a CDR-L1 having at least 95%
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • the VL comprises a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the V H comprises a CDR-H1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:10, SEQ ID NO:28, S
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, S
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the VH comprises a CDR-H1 having at least 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the VH comprises a CDR-H1 having at least 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, S
  • the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the VH comprises a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or any range derivable therein, with SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or any range derivable therein, with SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a V H comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:2, a CDR- L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:6; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:12.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:2, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:6; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:12.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:2, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:6; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:12.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR- L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:2, a CDR- L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:6; and a V H comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:32.
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:2, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:6; and a V H comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:32.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:2, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:6; and a V H comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:32.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR- L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a V H comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:20, a CDR- L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:22, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:24; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:26, a CDR-H2 comprising an amino acid sequence having at
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:20, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:22, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:24; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:26, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:28, and a CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:30.
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:20, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:22, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:24; and a V H comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:26, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:28, and a CDR-H3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:30.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24; and a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30.
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:50, a CDR- L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:53, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:57; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:59, a CDR-H2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:62, and a CDR-H3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:65.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:50, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:53, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:57; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:59, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:62, and a
  • CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:65.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:50, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:53, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:57; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:59, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:62, and a CDR-H3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:65.
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and a V H comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:67, a CDR- L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:71; and a V H comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:75, and a CDR-H3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:77.
  • Some aspects are directed to an antibody comprising: a V L comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:71; and a V H comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:75, and a CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:77.
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:71; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:75, and a CDR-H3 comprising an amino acid sequence having
  • Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77.
  • the antibody VL comprises SEQ ID NO:14.
  • the antibody VH comprises SEQ ID NO:16.
  • nucleic Acids [0392] Some aspects are directed to a nucleic acid encoding for a BMP-7-binding protein. Some aspects are directed to a nucleic acid encoding for a polypeptide or fragment thereof that specifically binds BMP-7. [0393] In some aspects, the nucleic acid encodes the heavy chain variable region and the light chain variable region of the BMP-7-binding protein. In some aspects, the nucleic acid encodes the light chain variable region of the BMP-7-binding protein. In some aspects, the nucleic acid encodes the heavy chain variable region of the BMP-7-binding protein. In some aspects, the nucleic acid encoding the light chain variable region comprises SEQ ID NO:13.
  • nucleic acid encoding the heavy chain variable region comprises SEQ ID NO:15.
  • a nucleic acid encoding for a BMP-7-binding protein comprises SEQ ID NO:13 and SEQ ID NO:15.
  • polynucleotide variants having substantial identity to the sequences disclosed herein, e.g., those comprising at least, at most, exactly, or between (inclusive or exclusive) any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters).
  • the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 80%, at least 85%, at least 90%, or at least 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.
  • the nucleic acid segments regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.
  • the nucleic acids can be any combination of the nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.
  • the nucleic acids can be any combination of the nucleic acid sequences, such as promoters, poly
  • 202424720.3 112 length can be, for example, at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and/or can comprise one or more additional sequences, for example, regulatory sequences, and/or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.
  • a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post- translational modification, or for therapeutic benefits such as targeting or efficacy.
  • a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide. 1.
  • Nucleotide sequences from 12 light chain variable region CDRs from the BMP-7- binding proteins (e.g., antibodies) of the present disclosure are provided in SEQ ID NOs:1, 3, 5, 19, 21, 23, 47-49, 51, 52, 54-56, 66, 68, and 70 as follows and in Table 3: CDR-L1 (SEQ ID NO:1, SEQ ID NO:19, SEQ ID NOs:47-49, or SEQ ID NO:66), CDR-L2 (SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:68), CDR-L3 (SEQ ID NO:5, SEQ ID NO:23, SEQ ID NOs:54-56, or SEQ ID NO: 70).
  • CDR-L1 SEQ ID NO:1, SEQ ID NO:19, SEQ ID NOs:47-49, or SEQ ID NO:66
  • CDR-L2 SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO
  • Nucleotide sequences from 13 heavy chain variable region CDRs from the BMP-7- binding proteins (e.g., antibodies) of the present disclosure are provided in SEQ ID NOs:7, 9, 11, 25, 27, 29, 31, 58, 60, 61, 63, 64, 72, 74, and 76 as follows and in Table 4: CDR-H1 (SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:58, or SEQ ID NO:72), CDR-H2 (SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:74), and CDR-H3 (SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO:76).
  • nucleotide sequence encoding for a first full light chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:13 as follows: [0399] GATATTGTGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAG AGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTACTGGCTATTTAGGCTG
  • nucleotide sequence encoding for a second full light chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:33 as follows: [0401] GATATCCAGATGACCCAGTCTCCATCTAGCCTGAGCGCCAGCGTCGGA GATAGAGTGACCATCACCTGTAAAGCTTCTCAGAGCGTGTCCAACGACGTGGCCT GGTATCAGCAAAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTACACCAGCA ACCGGTTTACAGGCGTGCCTAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACT TCACCCTGACCATCAGCTCCCTGCAGCCTGAGGACTTCGCCACATACTACTGCCA GCAGGACTACAGCAGCCCCCCCACATTCGGCGGCGGAACAAAGCTGGAAATCAA G (SEQ ID NO:33).
  • SEQ ID NO:35 The nucleotide sequence encoding for a third full light chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:35 as follows: [0403] GACATCCAGATGACCCAGAGCCCCAGCTCCCTGAGCGCCTCTGTCGGC GACAGAGTGACCATCACCTGTAAAGCCAGCCAGTCCGTGTCCAACGACGTGGCC TGGTATCAGCAAAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTACACCAGC AACCGGTTCACCGGCGTGCCTGATAGATTCAGCGGCTCTGGCAGCGGCACAGAT TTTACACTGACCATCAGCAGCCTGCAGCCTGAGGACTTCGCTACATACTACTGCC AGCAGGACTACAGCTCTCCACCTACCTTCGGCGGAGGAACAAAGCTGGAAATCA AG (SEQ ID NO:35).
  • nucleotide sequence encoding for a fourth full light chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:37 as follows: [0405] GATATCCAGATGACCCAGTCCCCATCTAGCCTGAGCGCCTCTGTCGGC GACAGAGTGACCATCACCTGTAAAGCCAGCCAGAGCGTGTCCAACGACGTGGCC TGGTATCAGCAAAAGCCTGGCCAGGCCCCTAAGCTGCTGATCTACTACACCAGC AACCGGTTCACCGGCGTGCCTAGCAGATTCAGCGGCAGCGGATCTGGCACAGAT TTTACACTGACCATCAGCTCCCTGCAGCCTGAGGACTTCGCTACATACTTCTGCC AGCAGGACTACAGCAGCCCCCCCACCTTCGGCGGCGGAACAAAGCTGGAAATCA AG (SEQ ID NO:37). [0406] The nucleotide sequence encoding for a fifth full light chain variable region of a BMP-7-binding protein disclosed herein is
  • nucleotide sequence encoding for a first full heavy chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:15 as follows: [0409] AGTCAGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCC TGCAAGGCTTCTGGTTATACCTTCACAGACTCTTCAATGCACTGGGTGAAGCAGG CTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGC CAACATATGTAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGC CAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATA TTTCTGTTCTAGAGATTACGACTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTC ACTGTCTCCGCAG (SEQ ID NO:15).
  • nucleotide sequence encoding for a second full heavy chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:39 as follows: [0411] CAAGTGCAGCTGGTTCAGAGCGGCAGCGAGCTGAAAAAGCCCGGCGC TTCTGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCGACAGCAGCATG CACTGGGTCAGACAGGCCCCTGGACAGGGCCTGAAATGGATGGGCTGGATCAAC ACCGAGACAGGCGAACCTACATACGTGGACGACTTCAAGGGCAGATTCGTGTTC AGCCTGGATACCAGCGTGTCCACCGCCTACCTGCAGATCAGCAGCCTGAAGGCT GAAGATACAGCCGTGTACTTTTGTGCCCGGGACTACGACTGGTTCGCCTATTGGG GCCAGGGAACCCTGGTGACCGTGTCTTCT (SEQ ID NO:39).
  • nucleotide sequence encoding for a third full heavy chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:41 as follows: [0413] CAAGTGCAGCTGGTCCAGAGCGGCAGCGAGCTGAAAAAGCCCGGAGC TTCTGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCGACAGCAGCATG CACTGGGTGCGGCAGGCCCCTGGCCAGGGCCTGGAATGGATGGGCTGGATCAAC ACCGAGACAGGCGAGCCTACCTACGTGGACGACTTCAAGGGCAGATTCGTGTTC AGCCTGGACACCAGCGTGTCCACCGCCTACCTGCAGATCAGCAGCCTGAAGGCT GAAGATACAGCCGTGTACTTTTGTGCCAGAGATTACGACTGGTTCGCCTATTGGG GCCAGGGAACACTGGTTACCGTGTCTTCT (SEQ ID NO:41).
  • nucleotide sequence encoding for a fourth full heavy chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:80 as follows: [0415] CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGA GACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTCTTCAATG CACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAAC ACTGAGACTGGTGAGCCAACATATGTAGATGACTTCAAGGGACGGTTTGCCTTCT CTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGA GGACACGGCTACATATTTCTGTTCTAGAGATTACGACTGGTTTGCTTACTGGGGC CAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:80).
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78.
  • the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78.
  • the nucleic acid comprises SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78.
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80.
  • the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80.
  • the nucleic acid comprises SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. [0418] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%,
  • the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:13 and a nucleotide sequence having at least 85% identity to SEQ ID NO:15. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:13 and a nucleotide sequence having at least 90% identity to SEQ ID NO:15. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:13 and a nucleotide sequence having at least 95% identity to SEQ ID NO:15.
  • the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:13 and a nucleotide sequence comprising SEQ ID NO:15. In some aspects, the nucleic acid comprises SEQ ID NO:13 and SEQ ID NO:15.
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:33 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:39.
  • the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:33 and a nucleotide sequence having at least 85% identity to SEQ ID NO:39. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:33 and a nucleotide sequence having at least 90% identity to SEQ ID NO:39. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:33 and a nucleotide sequence having at least 95% identity to SEQ ID NO:39.
  • the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:33 and a nucleotide sequence comprising SEQ ID NO:39. In some aspects, the nucleic acid comprises SEQ ID NO:33 and SEQ ID NO:39.
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:35 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein,
  • the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:35 and a nucleotide sequence having at least 85% identity to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:35 and a nucleotide sequence having at least 90% identity to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:35 and a nucleotide sequence having at least 95% identity to SEQ ID NO:41.
  • the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:35 and a nucleotide sequence comprising SEQ ID NO:41. In some aspects, the nucleic acid comprises SEQ ID NO:35 and SEQ ID NO:41.
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:37 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:41.
  • the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:37 and a nucleotide sequence having at least 85% identity to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:37 and a nucleotide sequence having at least 90% identity to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:37 and a nucleotide sequence having at least 95% identity to SEQ ID NO:41.
  • the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:37 and a nucleotide sequence comprising SEQ ID NO:41. In some aspects, the nucleic acid comprises SEQ ID NO:37 and SEQ ID NO:41.
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:78 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:80.
  • the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:78 and a nucleotide sequence having at least 85% identity to SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:78 and a nucleotide sequence having at least 90% identity
  • the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:78 and a nucleotide sequence having at least 95% identity to SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:78 and a nucleotide sequence comprising SEQ ID NO:80. In some aspects, the nucleic acid comprises SEQ ID NO:78 and SEQ ID NO:80.
  • nucleotide sequence encoding for a first full light chain constant region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:43 as follows: [0424] AGGACAGTGGCCGCCCCAAGCGTGTTCATCTTTCCCCCTTCCGACGAG CAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTC GGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCC AGGAGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCA CCCTGACACTGAGCAAGGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAG TCACCCATCAGGGGCTGTCATCACCCGTCACTAAGTCATTCAATCGCGGAGAATG C (SEQ ID NO:43).
  • nucleotide sequence encoding for a first full heavy chain constant region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:45 as follows: [0426] GCCAGCACCAAAGGCCCAAGCGTATTCCCACTGGCCCCTAGCTCTAAA AGCACCAGCGGCGGAACCGCAGCCCTGGGATGTCTGGTCAAGGACTACTTCCCC GAGCCTGTGACCGTGTCTTGGAACAGCGGTGCTCTCACCAGCGGAGTGCACACCT TCCCTGCTGTGCTTCAGTCCTCCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGT GCCCTCCAGCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC AGCAATACCAAGGTGGATAAAAAAAGGTCGAGCCTAAGTCCTGCGACAAGACCCAC ACATGCCCTCCTTGCCCCGCCCCTGAGCTGCTGGGCGGCCCCTCCGTGTTTCTGTT CCCCTAAACCAAAGGATACACTGATGATCTGTT CCCCT
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:43 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:45.
  • the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:43 and a nucleotide sequence having at least 85% identity to SEQ ID NO:45. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:43 and a nucleotide sequence having at least 90% identity to SEQ ID NO:45. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:43 and a nucleotide sequence having at least 95% identity to SEQ ID NO:45.
  • the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:43 and a nucleotide sequence comprising SEQ ID NO:45. In some aspects, the nucleic acid comprises SEQ ID NO:43 and SEQ ID NO:45.
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80; a nucleotide sequence having at least, at most,
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range
  • SEQ ID NO:13 a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:15; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:43; and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:33; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:39,; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 95%, 95%, 95%
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:35; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:41; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 96%, 97%,
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:37; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:41; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:78; and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:80.
  • Antibody Production A. Antibody Production
  • Methods for preparing and characterizing antibodies for use in diagnostic and detection assays, for purification, and for use as therapeutics are well known in the art as disclosed in, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745, each incorporated herein by reference (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
  • antibodies may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab’)2 fragments, Fab fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof which bind to the antigen in question.
  • polypeptides, peptides, and proteins may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab’)2 fragments, Fab fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof which bind to the antigen in question.
  • polypeptides, peptides, and proteins may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, chimeric antibodies, such as humanized antibodies, altered antibodies, F(a
  • the antibodies can be isolated from any suitable biological source, e.g., murine, rat, rabbit, goat, camelid, sheep, or canine.
  • a polyclonal antibody is prepared by immunizing an animal with an antigen or a portion thereof and collecting antisera from that immunized animal. The antigen may be altered compared to an antigen sequence found in nature. A variant or altered antigenic peptide or polypeptide can be employed to generate antibodies.
  • Inocula are typically prepared by dispersing the antigenic composition in a physiologically tolerable diluent to form an aqueous composition.
  • Antisera is subsequently collected by methods known in the arts, and the serum may be used as-is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • affinity chromatography Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • Methods of making monoclonal antibodies are also well known in the art (e.g., U.S. Patent 4,196,265, herein incorporated by reference in its entirety for all purposes).
  • this technique involves immunizing a suitable animal with a selected immunogenic composition, e.g., a purified or partially purified protein, polypeptide, peptide, or domain. Resulting antibody-producing B-cells from the immunized animal, or all dissociated splenocytes, are then induced to fuse with cells from an immortalized cell line to form hybridomas.
  • a selected immunogenic composition e.g., a purified or partially purified protein, polypeptide, peptide, or domain.
  • Resulting antibody-producing B-cells from the immunized animal, or all dissociated splenocytes are then induced to fuse with cells from an immortalized cell line to form hybridomas.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing and have high fusion efficiency and enzyme deficiencies that render then incapable of growing in certain selective media that support the growth of only the desired fused cells (hybridomas).
  • fusion partners can be hypoxanthine/aminopterin/thymidine (HAT)-sensitive.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • selection of hybridomas can be performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. Fusion procedures for making hybridomas, immunization protocols, and techniques for isolation of immunized splenocytes for fusion are known in the art.
  • a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8,653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MIA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 313, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O) or the like, or heteromyelomas, fusion products thereof, or any cell or fusion cell derived there from, or any other suitable cell line as known in the art, with antibody producing cells, such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph
  • Antibody producing cells can also be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest. Any other suitable host cell can also be used for expressing-heterologous or endogenous nucleic acid encoding an antibody, specified fragment or variant thereof, of the present disclosure.
  • the fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods.
  • SLAM lymphocyte antibody method
  • Monoclonal antibodies may be further purified using filtration, centrifugation, and various chromatographic methods such as high-performance liquid chromatography (HPLC). Monoclonal antibodies may be further screened or optimized for properties relating to specificity, avidity, half-life, immunogenicity, binding association, binding disassociation, or
  • monoclonal antibodies may have alterations in the amino acid sequence of CDRs, including insertions, deletions, or substitutions with a conserved or non-conserved amino acid.
  • Chimeric, humanized, or primatized antibodies of the present disclosure can be prepared based on the sequence of a reference monoclonal antibody prepared using standard molecular biology techniques.
  • DNA encoding the heavy and light chain immunoglobulins can be obtained from the hybridoma of interest and engineered to contain non-reference (e.g., human) immunoglobulin sequences using standard molecular biology techniques.
  • the murine variable regions can be linked to human constant regions using methods known in the art (U.S. Pat. No. 4,816,567).
  • the murine CDR regions can be inserted into a human framework using methods known in the art (U.S. Pat. No.5,225,539 and U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,762 and 6,180,370).
  • the murine CDR regions can be inserted into a primate framework using methods known in the art (WO 93/02108 and WO 99/55369).
  • Fully human antibody sequences are made in a transgenic mouse which has been engineered to express human heavy and light chain antibody genes. Multiple strains of such transgenic mice have been made which can produce different classes of antibodies. B cells from transgenic mice which are producing a desirable antibody can be fused to make hybridoma cell lines for continuous production of the desired antibody.
  • the antibodies of this disclosure can also be modified to create veneered antibodies.
  • Veneered antibodies are those in which the exterior amino acid residues of the antibody of one species are judiciously replaced or “veneered” with those of a second species so that the antibodies of the first species will not be immunogenic in the second species thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein is primarily dependent on the nature of its surface, the immunogenicity of an antibody could be reduced by replacing the exposed residues which differ from those usually found in another mammalian species antibodies. This judicious replacement of exterior residues should have
  • Non- limiting examples of the methods used to generate veneered antibodies include EP 519596; U.S. Pat. No.6,797,492; and described in Padlan et al., 1991.
  • Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, cDNA, or the like, display library; e.g., as available from various commercial vendors such as MorphoSys (Martinsreid/Planegg, Del.), BioInvent (Lund, Sweden), Affitech (Oslo, Norway) using methods known in the art.
  • a peptide or protein library e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, cDNA, or the like, display library
  • Such techniques include, but are not limited to, ribosome display (Wanes et al., 1997; Hanes et al., 1998); single cell antibody producing technologies (e,g., selected lymphocyte antibody method (“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al., 1987; Babcook et al., 1996); gel microdroplet and flow cytometry (Powell et al., 1990; Gray et al., 1995; Kenny et al., 1995); B-cell selection (Steenbakkers et al., 1994).
  • SLAM selected lymphocyte antibody method
  • the antibodies of this disclosure can be recovered and purified from recombinant cell cultures by known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be used for purification.
  • HPLC high performance liquid chromatography
  • the immunogenicity of a particular immunogen composition can be enhanced using non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants that may be
  • 127 used include, but are not limited to, interleukin-1 (IL-1), IL-2, IL-4, IL-7, IL-12, ⁇ -interferon (INF- ⁇ ), granulocyte-macrophage colony-stimulating factor (GMCSF), Bacillus Calmette- Guérin (BCG), aluminum hydroxide, muramyl dipeptide (MDP) compounds, muramyl tripeptide phosphatidyl ethanolamine (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-4 IL-7
  • IL-12 ⁇ -interferon
  • GMCSF granulocyte-macrophage colony-stimulating factor
  • BCG Bacillus Calmette- Guérin
  • aluminum hydroxide aluminum hydroxide
  • muramyl dipeptide (MDP) compounds muramyl tripeptide phosphatidyl ethanolamine
  • Exemplary adjuvants may include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants, and/or aluminum hydroxide adjuvant.
  • BRM biologic response modifiers
  • Cimetidine CIM; 1200 mg/d
  • CYP Cyclophosphamide
  • cytokines such as INF-ß, IL-2, or IL-12
  • genes encoding proteins involved in immune helper functions such as B7-1 (CD80) or B7-2 (CD86).
  • a phage-display system can be used to expand antibody molecule populations in vitro. Saiki, et al., Nature 324:163 (1986); Scharf et al., Science 233:1076 (1986); U.S. Pat. Nos. 4,683,195 and 4,683,202; Yang et al., J Mol Biol.254:392 (1995); Barbas, III et al., Methods: Comp. Meth Enzymol. (1995) 8:94; Barbas, III et al., Proc Natl Acad Sci USA 88:7978 (1991).
  • B. Antibody Fragments Production Antibody fragments that retain the ability to recognize the antigen of interest will also find use herein.
  • antibody fragments are known in the art that comprise antigen- binding sites capable of exhibiting immunological binding properties of an intact antibody molecule and can be subsequently modified by methods known in the arts.
  • Functional fragments including only the variable regions of the heavy and light chains, can also be produced using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as Fv. See, e.g., Inbar et al., Proc. Nat. Acad. Sci. USA 69:2659-2662 (1972); Hochman et al., Biochem.15:2706-2710 (1976); and Ehrlich et al., Biochem.19:4091-4096 (1980).
  • Single-chain variable fragments may be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (V L and V H ).
  • scFvs can form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et al., Prot. Eng. 10:423 (1997); Kort et al., Biomol. Eng. 18:95-108 (2001)).
  • V L - and V H -comprising polypeptides By combining different V L - and V H -comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al., Biomol. Eng.18:31-40 (2001)). Antigen-binding
  • VL and VH are coded for by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be made as a single chain polypeptide (known as single chain Fv (sFv or scFv); see e.g., Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988).
  • sFv or scFv single chain Fv
  • Design criteria include determining the appropriate length to span the distance between the C-terminus of one chain and the N-terminus of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not tend to coil or form secondary structures.
  • Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues and may include glutamic acid and lysine residues inserted to enhance solubility.
  • Antigen-binding fragments are screened for utility in the same manner as intact antibodies. Such fragments include those obtained by N-terminal and/or C-terminal deletions, where the remaining amino acid sequence is substantially identical to the corresponding positions in the naturally occurring sequence deduced, for example, from a full-length cDNA sequence.
  • non-peptide compounds having properties analogous to those of a template peptide. These types of non-peptide compounds are termed “peptide mimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al., J. Med. Chem.30:1229 (1987).
  • peptide mimetics or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al., J. Med. Chem.30:1229 (1987).
  • ABSiPs antibody like binding peptidomimetics
  • peptidomimetics of the disclosure are proteins that are structurally similar to an antibody displaying a desired biological activity, such as the ability to bind a protein, but have one or more peptide linkages optionally replaced by a linkage selected from: —CH 2 NH—, —CH 2 S—, —CH 2 —CH 2 —, —CH-CH— (cis and trans), —COCH2—, —CH(OH)CH2—, and —CH2SO— by methods well known in the art.
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type may be used to generate more stable
  • constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • a phage display library can be used to improve the immunological binding affinity of Fab molecules using known techniques. See, e.g., Figini et al., J. Mol. Biol. 239:68 (1994).
  • the coding sequences for the heavy and light chain portions of the Fab molecules selected from the phage display library can be isolated or synthesized and cloned into any suitable vector or replicon for expression. Any suitable expression system can be used.
  • V. Obtaining Antibodies there are nucleic acid molecules encoding antibody or antibody- like polypeptides (e.g., heavy or light chain, variable domain only, or full-length). These may be generated by methods known in the art, e.g., isolated from B cells of mice that have been immunized and isolated, phage display, expressed in any suitable recombinant expression system and allowed to assemble to form antibody molecules.
  • the nucleic acid molecules may be used to express large quantities of recombinant antibodies or to produce chimeric antibodies, single chain antibodies, antigen-binding fragments, immunoadhesins, diabodies, bi-specific antibodies, mutated antibodies, and other antibody derivatives. If the nucleic acid molecules are derived from a non-human, non- transgenic animal, the nucleic acid molecules may be used for antibody humanization.
  • vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Expression vectors comprising the nucleic acid molecules may encode the heavy chain, light chain, or the antigen- binding portion thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, and/or probes thereof.
  • vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
  • DNA encoding partial or full-length light and heavy chains are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences.
  • expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences.
  • flanking sequences typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • a promoter one or more enhancer sequences
  • an origin of replication a transcriptional termination sequence
  • a complete intron sequence containing a donor and acceptor splice site a sequence encoding a leader sequence for polypeptide secretion
  • ribosome binding site a sequence encoding a leader sequence for polypeptide secretion
  • polyadenylation sequence a polylinker region for inserting the nucleic acid encoding the polypeptid
  • Prokaryote- and/or eukaryote-based systems can be employed to produce nucleic acid sequences, or their cognate polypeptides, proteins, and peptides.
  • Commercially and widely available systems include, but are not limited to bacterial, mammalian, yeast, and insect cell systems.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • Those skilled in the art can express a vector to produce a nucleic acid sequence or its cognate polypeptide using an appropriate expression system. 3.
  • nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue, or an organism, as described herein
  • a nucleic acid e.g., DNA, including viral and nonviral vectors
  • Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S.
  • Patent No.5,384,253, incorporated herein by reference by calcium phosphate precipitation; by using DEAE dextran followed by polyethylene glycol; by direct sonic loading; by liposome mediated transfection; by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium-mediated transformation (U.S.
  • Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.
  • Host Cells [0460] In another aspect, contemplated are the use of host cells into which a polypeptide, nucleic acid, or recombinant expression vector has been introduced. Antibodies and antibody- like molecules can be expressed in a variety of cell types. An expression construct encoding an antibody can be transfected into cells according to a variety of methods known in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • the antibody expression construct can be placed under control of a promoter that is linked to immune cell (e.g., T cell) activation. Control of antibody expression allows immune cells, such as tumor-targeting immune cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cells themselves and in surrounding endogenous immune cells.
  • immune cells such as tumor-targeting immune cells
  • One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector.
  • Host cells which may be used to express antibodies and other antigen-binding proteins of the present disclosure include, for example, murine myeloma cells (e.g., NS0 cells,
  • the cell is an immune cell.
  • the immune cell is a T cell.
  • the immune cell is a B cell.
  • a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts.
  • the cells disclosed herein can be used in methods to generate the BMP-7-binding proteins of the disclosure. The methods can comprise culturing the cell under conditions sufficient to express a nucleic acid encoding for a BMP-7-binding protein in the cell.
  • B. Isolation The nucleic acid molecule encoding either or both of the entire heavy and light chains of an antibody or the variable regions thereof may be obtained from any source that produces antibodies. Methods of isolating mRNA encoding an antibody are well known in the art. The sequences of human heavy and light chain constant region genes are also known in the art.
  • Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed in a cell into which they have been introduced and the antibody isolated.
  • BMP bone morphogenetic protein
  • the one or more BMP receptors may be selected from Bone Morphogenetic Protein Receptor Type 1A (BMPR1A), Bone Morphogenetic Protein Receptor Type 1B (BMPR1B), Activin A receptor type I (ACVR1), Activin receptor type 2A (ACVR2A), Activin receptor
  • BMPR1A Bone Morphogenetic Protein Receptor Type 1B
  • ACVR1 Activin A receptor type I
  • ACVR2A Activin receptor type 2A
  • the genetically modified cells may be prepared using any standard gene editing method, including, but not limited to CRISPR/Cas gene editing.
  • the genetically modified cells may be prepared by delivering a CRISPR associated (Cas) endonuclease and one or more gRNAs targeting a gene locus encoding a BMP receptor.
  • the genetically modified cells are prepared by delivering a Cas9 endonuclease and two gRNAs targeting a gene locus encoding a BMP receptor (e.g., a BMPR1A gene locus, a BMPR1B gene locus, a ACVR1 gene locus, an ACVR2A gene locus, a ACVR2B gene locus, or a BMPR2 gene locus).
  • a BMP receptor e.g., a BMPR1A gene locus, a BMPR1B gene locus, a ACVR1 gene locus, an ACVR2A gene locus, a ACVR2B gene locus, or a BMPR2 gene locus.
  • Exemplary gRNA spacer sequences targeting these gene loci are provided in Table 5 below.
  • Table 5 [0466]
  • the genetically modified cells may be an immune cell (e.g., a T cell).
  • the genetically modified cells may be further modified to express one or more of
  • the genetically engineered cells may be a CAR-T cell (e.g., a CAR-T cell targeting PD1/PDL1, TIM3, LAG3, and/or CTLA-4).
  • CAR-T cell e.g., a CAR-T cell targeting PD1/PDL1, TIM3, LAG3, and/or CTLA-4.
  • compositions comprising the genetically modified cells. These compositions may, in certain aspects, further comprise antibodies targeting PD1/PDL1 pathway, anti-TIM3 antibodies, anti-LAG3 antibodies, and/or anti-CTLA-4 antibodies.
  • Therapeutic Methods [0468] Aspects of the disclosure are directed to compositions of the disclosure and methods for therapeutic use. The compositions of the disclosure may be used for in vivo, in vitro, or ex vivo administration.
  • the route of administration of the composition may be, for example, intratumoral, intravenous, intramuscular, intraperitoneal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, through inhalation, or through a combination of two or more routes of administration.
  • the disclosed methods comprise administering a cancer therapy to a subject or patient.
  • the cancer therapy may be chosen based on the expression level measurements, alone or in combination with the clinical risk score calculated for the subject.
  • the cancer therapy comprises a local cancer therapy.
  • the cancer therapy excludes a systemic cancer therapy.
  • the cancer therapy excludes a local therapy.
  • the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy.
  • the cancer therapy comprises an immunotherapy, which may be a checkpoint inhibitor therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.
  • the term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer.
  • the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer is a Stage I cancer.
  • the cancer is a Stage II cancer.
  • the cancer is a Stage III cancer.
  • the cancer is a Stage IV cancer.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma;
  • the cancer is lung cancer.
  • the cancer is non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the cancer is breast cancer.
  • the cancer is triple negative breast cancer.
  • the cancer is a recurrent cancer.
  • the cancer is an immunotherapy-resistant cancer.
  • management regimen refers to a management plan that specifies the type of examination, screening, diagnosis, surveillance, care, and treatment (such as dosage, schedule and/or duration of a treatment) provided to a subject in need thereof (e.g., a subject diagnosed with cancer).
  • the selected treatment regimen can be an aggressive one which is expected to result in the best clinical outcome (e.g., complete cure of the disease) or a more moderate one which may relieve symptoms of the disease yet may result in incomplete cure of the disease.
  • the type of treatment can include a surgical intervention, administration of a therapeutic drug such as a BMP-7-binding protein, immunotherapy, an exposure to radiation therapy, and/or any combination thereof.
  • the dosage, schedule and duration of treatment can vary, depending on the severity of disease and the selected type of treatment, and those of skill in the art can adjust the type of treatment with the dosage, schedule, and duration of treatment.
  • Biomarkers like BMP-7 that can predict the efficacy of certain therapeutic regimen and can be used to identify patients who will receive benefit of a conventional single or combined modality therapy before treatment begins or to modify or design a future treatment plan after treatment. In the same way, those patients who do not receive much benefit from such conventional single or combined modality therapy and can offer them alternative treatment(s) may be identified.
  • a cancer treatment may include or exclude any of the cancer treatments described herein.
  • aspects of the disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein.
  • a radiotherapy such as ionizing radiation
  • ionizing radiation means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons).
  • ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.
  • the radiotherapy can comprise external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT).
  • the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy.
  • the internal radiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy.
  • the radiotherapy is administered to a primary tumor.
  • the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some aspects, the amount of ionizing radiation is 18 Gy and is administered in three doses.
  • the amount of ionizing radiation is at least, at most, exactly, or between (inclusive or exclusive) of about 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range
  • the ionizing radiation is administered in at least, at most, exactly, or between (inclusive or exclusive) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any derivable range therein).
  • the doses may be about 1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.
  • the amount of radiotherapy administered to a subject may be presented as a total dose of radiotherapy, which is then administered in fractionated doses.
  • the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each.
  • the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each.
  • the total dose of radiation is at least, at most, exactly, or between (inclusive or exclusive) of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99
  • the total dose is administered in fractionated doses of at least, at most, exactly, or between (inclusive or exclusive) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein).
  • At least, at most, exactly, or between (inclusive or exclusive) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day. In some aspects, at least, at most, exactly, or between (inclusive or exclusive) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.
  • the methods comprise administration of a cancer immunotherapy.
  • Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer.
  • Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor- associated antigens (TAAs); they are often proteins or other macromolecules (e.g., carbohydrates).
  • TAAs tumor- associated antigens
  • Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes, and cytokines.
  • checkpoint inhibitor therapy also “immune checkpoint blockade therapy”, “immune checkpoint therapy”, “ICT,” “checkpoint blockade immunotherapy,” or “CBI”
  • ICT immune checkpoint therapy
  • CBI checkpoint blockade immunotherapy
  • PD-1, PDL1, and PDL2 inhibitors can act in the tumor microenvironment where T cells encounter an infection or tumor.
  • Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues.
  • Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells.
  • PDL2 is expressed on macrophages and dendritic cells.
  • the main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response.
  • Inhibitors of the disclosure may block one or more functions of PD-1 and/or PDL1 activity.
  • Alternative names for “PD-1” include CD279 and SLEB2.
  • Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H.
  • PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.
  • the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners.
  • the PD-1 ligand binding partners are PDL1
  • a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners.
  • PDL1 binding partners are PD-1 and/or B7- 1.
  • a PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners.
  • a PDL2 binding partner is PD-1.
  • the inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference.
  • the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
  • the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab.
  • the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
  • the PDL1 inhibitor comprises AMP- 224.
  • Nivolumab also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006/121168.
  • Pembrolizumab also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335.
  • Pidilizumab also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611.
  • AMP-224 also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.
  • Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.
  • the immune checkpoint inhibitor is a PDL1 inhibitor such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof.
  • the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.
  • the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab.
  • the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the V H region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab.
  • the antibody competes for binding with and/or binds to the same epitope on PD-1, PDL1, or PDL2 as the above-
  • the antibody has at least, at most, exactly, or between (inclusive or exclusive) of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • CD152 cytotoxic T-lymphocyte-associated protein 4
  • CTLA-4 is found on the surface of T cells and acts as an “off” switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells.
  • CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells.
  • CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells.
  • CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal.
  • Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function.
  • the T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.
  • Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and/or B7-2 activity.
  • the inhibitor blocks the CTLA-4 and B7- 1 interaction.
  • the inhibitor blocks the CTLA-4 and B7-2 interaction.
  • the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
  • art recognized anti-CTLA-4 antibodies can be used.
  • the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No.6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein.
  • the teachings of each of the aforementioned publications are hereby incorporated by reference.
  • Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used.
  • a humanized CTLA-4 antibody is described in International Patent Application No. WO2001/014424, WO2000/037504, and U.S. Patent No.8,017,114; all incorporated herein by reference.
  • a further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and YERVOY®) or antigen binding fragments and variants thereof (see, e.g., WO 01/14424).
  • the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab.
  • the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the V L region of tremelimumab or ipilimumab.
  • the antibody competes for binding with and/or binds to the same epitope on PD-1, B7-1, or B7- 2 as the above- mentioned antibodies.
  • the antibody has at least, at most, exactly, or between (inclusive or exclusive) of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above- mentioned antibodies.
  • LAG3 Another immune checkpoint that can be targeted in the methods provided herein is the lymphocyte-activation gene 3 (LAG3), also known as CD223 and lymphocyte activating 3.
  • LAG3 lymphocyte-activation gene 3
  • the complete mRNA sequence of human LAG3 has the GENBANK® accession number NM_002286.
  • LAG3 is a member of the immunoglobulin superfamily that is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells.
  • LAG3’s main ligand is MHC class II, and it negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD-1 and has been reported to play a role in Treg suppressive function.
  • the immune checkpoint inhibitor is an anti-LAG3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • an anti-LAG3 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody
  • an antigen binding fragment thereof e.g., an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-LAG3 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
  • art recognized anti-LAG3 antibodies can be used.
  • the anti-LAG3 antibodies can include: GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017, or GSK2831781.
  • BMS-986016 also known as relatlimab
  • US 10,711,060 IMP-701, also known as LAG525
  • US 9,244,059 IMP731, also known as H5L7BW
  • US 10,344,089 25F7, also known as LAG3.1
  • WO 2016/028672 MK-4280, also known as 28G-10
  • WO 2017/019894 BAP050
  • Burova E. et al., J. ImmunoTherapy Cancer, 2016; 4(Supp.1):P195 (REGN3767)
  • Yu X., et al., mAbs, 2019; 11:6 (LBL-007) can be used in the methods disclosed herein.
  • anti-LAG-3 antibodies useful in the claimed invention can be found in, for example: WO 2016/028672, WO 2017/106129, WO 2017062888, WO 2009/044273, WO 2018/069500, WO 2016/126858, WO 2014/179664, WO 2016/200782, WO 2015/200119, WO 2017/019846, WO 2017/198741, WO 2017/220555, WO 2017/220569, WO 2018/071500, WO 2017/015560; WO 2017/025498, WO 2017/087589 , WO 2017/087901, WO 2018/083087, WO 2017/149143, WO 2017/219995, US 2017/0260271, WO 2017/086367, WO 2017/086419, WO 2018/034227, and WO 2014/140180.
  • the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the V H region of an anti-LAG3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-LAG3 antibody.
  • the antibody has at least, at most, exactly, or between (inclusive or exclusive) of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.
  • TIM-3 T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2) and CD366.
  • HAVCR2 hepatitis A virus cellular receptor 2
  • CD366 CD366.
  • the complete mRNA sequence of human TIM-3 has the GENBANK® accession number NM_032782.
  • TIM-3 is found on the surface IFN ⁇ -producing CD4+ Th1 and CD8+ Tc1 cells.
  • the extracellular region of TIM-3 consists of a membrane distal single variable immunoglobulin domain (IgV) and a glycosylated mucin domain of variable length located closer to the membrane.
  • IgV membrane distal single variable immunoglobulin domain
  • TIM-3 is an immune checkpoint and, together with other inhibitory receptors including PD-1 and LAG3, it mediates the T-cell exhaustion.
  • TIM-3 has also been shown as a CD4+ Th1-specific cell surface protein that
  • the immune checkpoint inhibitor is an anti-TIM-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-TIM-3 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-TIM-3 antibodies can be used.
  • anti-TIM-3 antibodies including: MBG453, TSR-022 (also known as cobolimab), and LY3321367 can be used in the methods disclosed herein.
  • anti-TIM-3 antibodies useful in the claimed invention can be found in, for example: US 9,605,070, US 8,841,418, US2015/0218274, and US 2016/0200815.
  • the teachings of each of the aforementioned publications are hereby incorporated by reference.
  • Antibodies that compete with any of these art-recognized antibodies for binding to TIM-3 also can be used.
  • the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-TIM-3 antibody.
  • the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the V H region of an anti-TIM-3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-TIM-3 antibody.
  • the antibody has at least, at most, exactly, or between (inclusive or exclusive) of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range or value therein) variable region amino acid sequence identity with the above-mentioned antibodies. 2.
  • CAR-T cell therapy Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy. [0503] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for
  • CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T cells.
  • the extracellular ligand recognition domain is usually a single-chain variable fragment (scFv).
  • scFv single-chain variable fragment
  • Example CAR-T therapies include Tisagenlecleucel (KYMRIAH) and Axicabtagene ciloleucel (YESCARTA®).
  • the CAR-T cell therapy comprises CAR-T cells genetically modified to have disrupted or reduced expression of one or more bone morphogenic protein (BMP) receptors.
  • BMP bone morphogenic protein
  • the one or more bone morphogenic protein (BMP) receptors may comprise Bone Morphogenetic Protein Receptor Type 1A (BMPR1A), Bone Morphogenetic Protein Receptor Type 1B (BMPR1B), Activin A receptor type I (ACVR1), Activin receptor type 2A (ACVR2A), Activin receptor type 2B (ACVR2B), Bone morphogenetic protein receptor type 2 (BMPR2), or any combination thereof.
  • the CAR-T cell therapy comprises CAR-T cells having disrupted or reduced expression of a BMPR1A receptor, a BMPR1B receptor or any combination thereof. 3.
  • the immunotherapy comprises an inhibitor of a co-stimulatory molecule.
  • the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof.
  • Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.
  • Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen.
  • Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting.
  • APCs antigen presenting cells
  • dendritic cells can also be activated in vivo by making tumor cells express GM- CSF.
  • dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells.
  • Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor.
  • Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets. 5.
  • Cytokine therapy [0511] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins. [0512] Interferons are produced by the immune system. They are usually involved in anti- viral response, but also have use for cancer.
  • Interleukins have an array of immune system effects.
  • IL-2 is an example interleukin cytokine therapy. 6.
  • Adoptive T-cell therapy is an example interleukin cytokine therapy.
  • Adoptive T cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically, they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens.
  • APCs antigen presenting cells
  • T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.
  • TILs tumor sample
  • D. Oncolytic virus [0516] In some aspects, the additional therapy comprises an oncolytic virus.
  • An oncolytic virus is a virus that preferentially infects and kills cancer cells.
  • the additional therapy comprises polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.
  • Neoantigens [0518] In some aspects, the additional therapy comprises neoantigen administration. Many tumors express mutations. These mutations potentially create new targetable antigens
  • the additional therapy comprises a chemotherapy.
  • chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thiouracil
  • cisplatin is a particularly suitable chemotherapeutic agent.
  • Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors.
  • Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection.
  • Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg/m 2 to about 20 mg/m 2 for 5 days every three weeks for a total of three courses being contemplated in certain aspects.
  • the amount of cisplatin delivered to the cell and/or subject in conjunction with the construct comprising an Egr-1 promoter operatively linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.
  • chemotherapeutic agents include antimicrotubule agents, e.g., paclitaxel (“TAXOL®”) and doxorubicin hydrochloride (“doxorubicin”).
  • TAXOL® paclitaxel
  • doxorubicin hydrochloride doxorubicin hydrochloride
  • the combination of an Egr-1 promoter/TNF ⁇ construct delivered via an adenoviral vector and doxorubicin was determined to be effective in overcoming resistance to chemotherapy and/or TNF- ⁇ , which suggests that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF- ⁇ .
  • Doxorubicin is absorbed poorly and is preferably administered intravenously.
  • appropriate intravenous doses for an adult include about 60 mg/m 2 to about 75 mg/m 2 at about 21-day intervals or about 25 mg/m 2 to about 30 mg/m 2 on each of 2 or 3 successive days repeated at about 3-week to about 4-week intervals or about 20 mg/m 2 once a week.
  • the lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.
  • Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure.
  • a nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and/or ifosfamide, melphalan (L-sarcolysin), and chlorambucil.
  • HN2 mechlorethamine
  • cyclophosphamide and/or ifosfamide melphalan
  • L-sarcolysin L-sarcolysin
  • chlorambucil chlorambucil.
  • Cyclophosphamide CYTOXAN®
  • NEOSTAR® is available from Adria
  • Adria is another suitable chemotherapeutic agent.
  • Suitable oral doses for adults include, for example, about 1 mg/kg/day to about 5 mg/kg/day
  • intravenous doses include, for example, initially about 40 mg/kg to about 50 mg/kg in divided doses over a period of about 2 days to about 5 days or about 10 mg/kg to about 15 mg/kg about every 7 days to about 10 days or about 3 mg/kg to about 5 mg/kg twice a week or about 1.5 mg/kg/day to about 3 mg/kg/day.
  • the intravenous route is preferred.
  • the drug also sometimes is administered intramuscularly, by infiltration or into body cavities.
  • chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode- oxyuridine; FudR).
  • 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg/m 2 . Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.
  • the amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable aspect, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered
  • the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000-fold less than the chemotherapeutic effective dose of the chemotherapeutic agent.
  • the chemotherapeutic agent may be administered in an amount that is about 20-fold less, about 500-fold less or even about 5000- fold less than the chemotherapeutic effective dose of the chemotherapeutic agent.
  • the chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc.
  • In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.
  • H. Surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present aspects, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
  • Other Agents It is contemplated that other agents may be used in combination with certain aspects of the present aspects to improve the therapeutic efficacy of treatment.
  • Additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase
  • cytostatic or differentiation agents can be used in combination with certain aspects of the present aspects to improve the anti-hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present aspects.
  • Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present aspects to improve the treatment efficacy.
  • compositions e.g., antigen-binding proteins
  • methods described herein may be administered to any patient having a condition in which targeting BMP-7 may have therapeutic benefit.
  • Conditions in which targeting BMP-7 may have a therapeutic benefit include, for example, a condition associated with activation of BMP receptors and a condition in which targeting BMP-7 may be used to specifically deliver a therapeutic.
  • Such conditions include, for example, cancer.
  • aspects of the present disclosure are directed to methods comprising treatment of a subject suffering from, or suspected of having, cancer.
  • the cancer is lung cancer, breast cancer, brain cancer, colon cancer, melanoma, or gastric cancer.
  • the cancer is lung cancer.
  • the lung cancer is non-small cell lung cancer.
  • the disclosed methods comprise treating a subject who currently has or has previously had resistance to immunotherapy.
  • a subject may be identified as having resistance to immunotherapy using tests and diagnostic methods known in the art.
  • the therapy provided herein may comprise administration of a therapeutic agent (e.g., a BMP-7-binding protein).
  • therapy provided herein comprises administration of a BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, a vector comprising the nucleic acid encoding for the BMP-7-binding protein, or a cell comprising the BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, or a vector comprising the nucleic acid encoding for the BMP-7-binding protein, and a pharmaceutically acceptable excipient.
  • the disclosed methods comprise treating a subject suffering from a cancer with a BMP-7-binding protein.
  • cancers associated with resistance to immunotherapy are surprisingly and unexpectedly sensitive to treatment with a BMP-7- binding protein. Further, administering a BMP-7-binding protein can decrease
  • the cancer is a cancer characterized as being resistant to immunotherapy (e.g., a solid tumor cancer).
  • the cancer is lung cancer.
  • the cancer is breast cancer.
  • the disclosed BMP-7-binding proteins include antibodies comprising a light chain variable region (VL) having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and a heavy chain variable region (VH) having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • VL light chain variable region having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%
  • the VL has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the VH has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81
  • the VL comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the VH comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the disclosed methods comprise treating a subject suffering from a cancer with a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the VL has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at
  • VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • VH has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the VL comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79
  • the VH comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81.
  • the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7).
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
  • an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57,
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6.
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24.
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71.
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a
  • VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32.
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30.
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VH comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77.
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12.
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR- H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32.
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24; and a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30.
  • the VL comprises SEQ ID NO:14.
  • the VH comprises SEQ ID NO:16.
  • the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65.
  • the VL comprises SEQ ID NO:34.
  • the VH comprises SEQ ID NO:40.
  • the VL comprises SEQ ID NO:36.
  • the VH comprises SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:38. In some aspects, the VH comprises SEQ ID NO:42. [0539] In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77.
  • the VL comprises SEQ ID NO:79. In some aspects, the VH comprises SEQ ID NO:81.
  • therapy provided herein comprises administration of a combination of therapeutic agents, such as a BMP-7-binding protein and an additional therapeutic agent, optionally comprised in a composition.
  • the additional therapeutic comprises a biotherapeutic, a chemotherapeutic, an immunotherapeutic, a toxin, an antisense oligonucleotide, a small inhibitory RNA (siRNA), an enzyme, a protein (e.g., a toxin, an enzyme, etc.), a viral vector, or a nanodrug.
  • the immunotherapeutic comprises a cytotoxic T-lymphocyte- associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.
  • CTL4 cytotoxic T-lymphocyte-associated protein 4
  • PD1 programmed cell death protein 1
  • PDL-1 programmed death-ligand 1
  • LAG3 lymphocyte activation gene-3
  • TIM-3 T cell immunoglobulin and mucin domain 3
  • therapy provided herein comprises administration of a combination of a BMP- 7-binding protein and a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor, optionally comprised in a composition.
  • CTLA4 cytotoxic T-lymphocyte-associated protein 4
  • PD1 programmed cell death protein 1
  • PDL-1 programmed death-ligand 1
  • LAG3 lymphocyte activation gene-3
  • TIM-3 T cell immunoglobulin and mucin domain 3
  • the immunotherapeutic comprises a CAR-T cell therapy.
  • the CAR-T cell therapy may be CAR-T cells targeted to one or more tumor markers or antigens that are present in or produced by cancer cells or other cells of the body in response to cancer or certain benign (noncancerous) conditions, where the cancer cells or other cells of the body also express BMP-7.
  • Non-limiting examples of tumor markers or antigens to which CAR-T cells may be targeted include: EBNA, CD123, HER1, HER2, CA-125, CA 19-9, CA 72-4, CA 15-3 ⁇ CA 27.29 ⁇ BCAA, CA-195, CA-242, CA-50, CA LX, MN-CA IX, TRAIL/DR4, CD2, CD5, CD7, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v6, CD47, CD56, CD68/P1, CD70, CD97, CD99, CD123, CD171, CD179, CD200, CD319 (CS1), HLA-G, carcinoembryonic antigen, alphafetoprotein, b-human chorionic gonadotropin, AKT, Her3, epithelial tumor antigen, ROR1, folate binding protein, folate receptor, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, HERV-
  • sequences for antigens are known in the art, for example, in the GENBANK® database: CD19 (Accession No. NG_007275.1), EBNA (Accession No. NG_002392.2), WT1 (Accession No. NG_009272.1), CD123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No. NG_007503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No.
  • therapy provided herein comprises administration of a BMP-7-binding protein and CAR-T cells targeted to one or more tumor markers or antigens, optionally comprised in a composition.
  • administration of a BMP-7-binding protein with CAR-T cells targeted to one or more tumor markers or antigens improves or increases killing by the CAR-T cells (i.e., apoptosis of cancer cells expressing the markers or antigens to which the CAR-T cells are targeted).
  • the cancer is pancreatic cancer
  • the CAR-T cells are targeted to EGFR
  • administration of a BMP-7-binding protein with EGFR-targeted CAR-T cells improves or increases killing by the CAR-T cells of the pancreatic cancer cells.
  • the CAR-T cells are genetically modified to have disrupted or reduced expression of one or more Bone Morphogenetic Protein (BMP) receptors (e.g., a Bone Morphogenetic Protein Receptor Type 1A (BMPR1A), Bone Morphogenetic Protein Receptor Type 1B (BMPR1B), Activin A receptor type I (ACVR1), Activin receptor type 2A (ACVR2A), Activin
  • BMP Bone Morphogenetic Protein
  • the subject was previously treated for the cancer with a cancer therapy.
  • the subject was determined to be resistant to the cancer therapy.
  • the cancer therapy comprises one or more immunotherapies.
  • the one or more immunotherapies comprise a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death- ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.
  • CTL4 cytotoxic T-lymphocyte-associated protein 4
  • PD1 programmed cell death protein 1
  • PDL-1 programmed death- ligand 1
  • LAG3 lymphocyte activation gene-3
  • TIM-3 T cell immunoglobulin and mucin domain 3
  • the CTLA4 inhibitor is an anti-CTLA4 antibody.
  • the PD1 inhibitor is an anti-PD1 antibody.
  • the PDL-1 inhibitor is an anti-PDL- 1 antibody.
  • the LAG3 inhibitor is an anti-LAG3 antibody.
  • the TIM-3 inhibitor is an anti-TIM-3 antibody.
  • the cancer is a solid tumor.
  • the cancer is lung cancer, gastric cancer, brain cancer, colon cancer, melanoma, or breast cancer.
  • the cancer is lung cancer.
  • the lung cancer is non-small cell lung cancer.
  • the cancer is breast cancer.
  • the breast cancer is triple-negative breast cancer.
  • the disclosed methods comprise identifying one or more subjects as being candidates for treatment with a BMP-7-binding protein, based on current or former resistance to treatment with an immunotherapy.
  • a method comprising identifying a subject having cancer as being a candidate for treatment with a BMP-7-binding protein by determining that the subject currently has or previously had resistance to treatment with an immunotherapy.
  • the disclosed methods comprise determining an optimal cancer treatment for a subject with resistance to treatment with an immunotherapy.
  • a subject is given multiple types of cancer therapy, for example a cancer immunotherapy and a chemotherapy.
  • pharmaceutical compositions comprising a BMP-7-binding protein.
  • the pharmaceutical compositions can further comprise one or more additional therapeutics, for example, an immunotherapeutic, including but not limited to the immunotherapeutics disclosed herein. VIII. Administration of Therapeutic Compositions
  • the therapy provided herein may comprise administration of a combination of therapeutic agents, such as a first cancer therapy (e.g., a BMP-7-binding protein therapy comprising a BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, a vector comprising the nucleic acid encoding for the BMP-7-binding protein, or a cell comprising the BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, or a vector comprising the nucleic acid encoding for the BMP-7-binding protein), and a second cancer therapy (e.g., an immunotherapy, for example, a checkpoint inhibitor therapy).
  • a first cancer therapy e.g., a BMP-7-binding protein therapy comprising a BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, a vector comprising the nucleic acid encoding for the BMP-7-binding protein
  • the therapies may be administered in any suitable manner known in the art.
  • the first and second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time or approximately the same time; also “simultaneously” or “substantially simultaneously”).
  • the first and second cancer treatments are administered in a separate composition.
  • the first and second cancer treatments are in the same composition.
  • the BMP-7-binding protein therapy and the immunotherapy are administered substantially simultaneously.
  • the BMP-7-binding protein therapy and the immunotherapy are administered substantially simultaneously.
  • the BMP-7-binding protein therapy and the immunotherapy are administered sequentially.
  • the BMP-7-binding protein therapy, the immunotherapy, and the chemotherapy are administered sequentially. In some aspects, the BMP-7-binding protein therapy is administered before administering the immunotherapy. In some aspects, the BMP- 7-binding protein therapy is administered after administering the immunotherapy. [0552] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed. [0553] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration.
  • the cancer therapy is administered intratumorally, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • the appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
  • An effective amount of therapeutic or prophylactic composition is determined based on the intended goal.
  • the treatments may include various “unit doses.”
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered, both according to number of treatments and unit dose, and the particular route and formulation is within the skill of determination of those in the clinical arts.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • a unit dose comprises a single administrable dose.
  • the BMP-7-binding protein therapy comprises a BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, a vector comprising the nucleic acid encoding for the BMP-7-binding protein, or a cell comprising the BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, or a vector comprising the nucleic acid encoding for the BMP-7-binding protein.
  • a single dose of the BMP-7-binding protein therapy is administered.
  • multiple doses of the BMP-7-binding protein are administered.
  • the BMP-7-binding protein is administered at a dose of between 1 mg/kg and 5000 mg/kg. In some aspects, the BMP-7-binding protein is administered at a dose that is at least, at most, exactly, or between (inclusive or exclusive) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
  • a single dose of the immunotherapy is administered. In some aspects, multiple doses of the immunotherapy are administered. In some aspects, the immunotherapy is administered at a dose of between 1 mg/kg and 100 mg/kg. In some aspects, the immunotherapy is administered at a dose of at least, at most, exactly, or between (inclusive or exclusive) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87
  • an effective dose is understood to refer to an amount necessary to achieve a particular effect.
  • doses include doses of at least, at most, exactly, or between (inclusive or exclusive) of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 ⁇ g/kg, mg/kg, ⁇ g/day, or mg/day or any range derivable therein.
  • such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
  • the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 ⁇ M to 150 ⁇ M.
  • the effective dose provides a blood level of about 4 ⁇ M to 100 ⁇ M.; or about 1 ⁇ M to 100 ⁇ M; or about 1 ⁇ M to 50 ⁇ M; or about 1 ⁇ M to 40 ⁇ M; or about 1 ⁇ M to 30 ⁇ M; or about 1 ⁇ M to 20 ⁇ M; or about 1 ⁇ M to 10 ⁇ M; or about 10 ⁇ M to 150 ⁇ M; or about 10 ⁇ M to 100 ⁇ M; or about 10 ⁇ M to 50 ⁇ M; or about 25 ⁇ M to 150 ⁇ M; or about 25 ⁇ M to 100 ⁇ M; or about 25 ⁇ M to 50 ⁇ M; or about 50 ⁇ M to 150 ⁇ M; or about 50 ⁇ M to 100 ⁇ M (or any range derivable therein).
  • the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: at least, at most, exactly, or between (inclusive or exclusive) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ⁇ M or any range de
  • the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent.
  • the blood levels discussed herein may refer to the unmetabolized therapeutic agent.
  • Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.
  • dosage units of ⁇ g/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of ⁇ g/ml or ⁇ M (blood levels), such as 4 ⁇ M to 100 ⁇ M. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein. [0561] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.
  • compositions are administered to a subject. Different aspects may involve administering an effective amount of a composition to a subject.
  • an antibody or antigen binding fragment capable of binding to BMP-7 may be administered to the subject to protect against or treat a condition (e.g., cancer).
  • a nucleic acid or an expression vector encoding one or more such antibodies or polypeptides or peptides may be given to a subject as a preventative treatment.
  • a cell comprising the BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, or a vector comprising the nucleic acid encoding for the BMP-7-binding protein may be administered to the subject to protect against or treat a condition (e.g., cancer). Additionally, such compositions can be administered in combination with an additional therapeutic agent (e.g., a chemotherapeutic, an immunotherapeutic, a biotherapeutic, etc.). Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.
  • the active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes.
  • such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and
  • the proteinaceous compositions may be formulated into a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • a pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [0568] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically
  • kits containing compositions of the disclosure or compositions to implement methods of the disclosure can be used to evaluate one or more biomarkers.
  • a kit contains, contains at least, at most, exactly, or between (inclusive or exclusive) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more therapeutic agents, e.g., a BMP-7-binding protein therapy comprising a BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, a vector comprising the nucleic acid encoding for the BMP-7-binding protein, or a cell comprising the BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, or a vector comprising the nucleic acid encoding for the BMP-7-binding protein, or an immunotherapy, or any value or range and combination derivable therein.
  • Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means. [0573] Individual components may also be provided in a kit in concentrated amounts; in some aspects, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as 1X, 2X, 5X, 10X, or 20X or more. [0574] Kits for using therapeutic agents of the disclosure for prognostic or diagnostic applications are included as part of the disclosure.
  • any such molecules corresponding to any biomarker identified herein which includes nucleic acid primers/primer sets and probes that are identical to or complementary to all or part of a biomarker, which may include noncoding sequences of the biomarker, as well as coding sequences of the biomarker.
  • negative and/or positive control nucleic acids, probes, and inhibitors are included in some kit aspects. Aspects of the disclosure include kits
  • the kit can further comprise reagents for labeling nucleic acids in the sample.
  • the kit may also include labeling reagents, including at least one of amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer.
  • any aspect of the disclosure involving specific therapeutic agents by name is contemplated also to cover aspects involving therapeutic agents whose sequences are at least, at most, exactly, or between (inclusive or exclusive) of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the specified nucleic acid. VII. Examples [0577] The following examples are included to demonstrate aspects of the disclosure.
  • Example 1 Monoclonal Antibody to BMP-7 to treat resistance to immunotherapies
  • Antibody generation Hybridoma cells were provided. Total RNA was extracted from the hybridoma clone cell pellet using RNA extraction protocol (Zymo Research). RT- PCR cDNA was created from the RNA by reverse-transcription with random primers.
  • VH and VL products were cloned into the Invitrogen sequencing vector pCR2.1-TOPO (SEQ ID NO:17 corresponding to VL in pCR.2.1-TOPO, SEQ ID NO:18 corresponding to V H in pCR.2.1-TOPO) and transformed into DH5a cells and screened by PCR for positive transformants. Selected colonies were picked and analyzed by
  • FIG.1 shows titration of an antibody generated to specifically target human BMP-7 (BMP-7 mAb 344-5-1, having a VL comprising SEQ ID NO:14 and a CDR-L1 comprising SEQ ID NO:2 or SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:4 or SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:6 or SEQ ID NO:24; and having a V H corresponding to SEQ ID NO:16 and a CDR-H1 comprising SEQ ID NO:8 or SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:10 or SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, or SEQ ID NO:32).
  • BMP-7 mAb 344-5-1 having a VL comprising SEQ ID NO:14 and a CDR-L1 comprising SEQ ID NO:2 or SEQ ID NO:
  • BMP-7 mAb 344-5-1 treatment produced the highest upregulation of p38 alpha, a marker for Th1 cytokines/chemokines (pro-inflammatory phenotype). Therefore, the specific inhibition of BMP7 by BMP-7 mAb 344-5-1 promotes release of cytokines and chemokines by M1-like macrophages to induce inflammation, which can lead to anti-tumor immune response.
  • BMP-7 mAb 344-5-1 produced the highest upregulation of p38 alpha, a marker for Th1 cytokines/chemokines (pro-inflammatory phenotype). Therefore, the specific inhibition of BMP7 by BMP-7 mAb 344-5-1 promotes release of cytokines and chemokines by M1-like macrophages to induce inflammation, which can lead to anti-tumor immune response.
  • Tumor growth and survival analysis of mice treated twice a week. All mice were bred and maintained in a specific pathogen-free mouse colony.
  • Tumor growth and survival analysis of mice treated three times a week. All mice were bred and maintained in a specific pathogen-free mouse colony. Tumors were established by subcutaneous injection of 344SQR or 4T1 cells (0.5 ⁇ 10 6 in 100 ⁇ L of sterile
  • mice 202424720.3 168 PBS
  • mice 202424720.3 168 PBS
  • BMP-7 inhibition by BMP-7 mAb 344-5-1 was found to re-sensitize tumors to anti-PD1 and extended mouse survival relative to the control group.
  • a mouse monoclonal antibody (mAb) antibody was humanized using a back mutation library method without sacrificing the binding affinity of the BMP-7344-5-1 parent mouse antibody. The following experiments were carried out: (1) confirmation of antigen-antibody interaction; (2) back mutation library design and construction; and (3) selection of humanized antibodies. [0584] Binding confirmation of chimeric antibody by SPR. A chimeric antibody was created from the BMP-7344-5-1 parent antibody and used for BMP-7 antibody humanization. The chimeric antibody has a VL corresponding to SEQ ID NO:79 and a CDR-L1 comprising
  • NC refers to a non-related FASEBA supernatant control and blank refers to 2xYT medium only. The concentration of 13 ⁇ g/ml antigen was selected for further FASEBA screening.
  • Table 8 [0590] Generation of deluxe humanized library. The humanized FASEBA screening library containing all back mutants in the inner core of antibody structure were designed as described in design report. The construction of the library was carried out following GenScript ProBio’s standard operating procedures (SOP), described below. [0591] The real size of the constructed FAB library was 7.0x10 7 cfu. Library in-frame rate and diversity were evaluated by DNA sequencing. A total of 47 clones were sequenced. The qualities are summarized in Tables 9-11. The sequence alignments are summarized in FIGs.
  • the AHF17901 antibody clone (mAb #1) had a binding OD450nm of 1.377 and an expression OD450nm of 2.183, and has a V L corresponding to SEQ ID NO:34 and a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and a VH corresponding to SEQ ID NO:40 and a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR- H3 comprising SEQ ID NO:65.
  • the AHF17906 antibody clone (mAb #2) had a binding OD450nm of 1.1 and an expression OD450nm of 2.099, and has a VL corresponding to SEQ ID NO:36 and a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53,
  • the AHF17909 antibody clone (mAb #3) had a binding OD450nm of 1.243 and an expression OD450nm of 2.168, and has a V L corresponding to SEQ ID NO:38 and a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR- L3 comprising SEQ ID NO:57; and a VH corresponding to SEQ ID NO:42 and a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65.
  • the heavy chain expression plasmid and the light chain expression plasmid was added into one tube and mixed them. Then transfection reagent was added into another tube and mixed fully, incubation for 5 min , then mixed the plasmids and transfection reagent, mixed fully, followed by incubation for 10 min at room temperature. The mixture was added into the Expi293F cells and incubated for 48-72 h. Supplement was added into the cells, and then the cells were put back into the shaker. The supernatant was collected for purification after 6 days of cell culture. The recombinant IgGs secreted to the medium were purified using protein A affinity chromatography. Protein A Magnetic Beads were removed from the refrigerator.
  • the supernatant was added to Protein A Magnetic Beads and incubated at room temperature for 2 h. After incubation, protein A Magnetic Beads was balanced with 10 CV (Column volume) binding buffer. Protein A Magnetic Beads was washed with elution buffer. Collect the elution buffer. The eluted antibody was neutralized by adding 1/10 volume of neutralizing buffer. After elution, Protein A Magnetic Beads was balanced with 10 CV (Column volume) binding buffer. The eluted and neutralized antibody was dialyzed in dialysis buffer at room temperature for 2 hours, and then dialyzed at 2-8 oC 16 hours after buffer changing.
  • Antibody was transferred from the dialysis bag into the tubes and filtered with 0.22 ⁇ m membrane filter. Antibody concentration was measured the by Nanodrop 2000 with extinction coefficient of 1.43. Binding confirmation was tested by Surface Plasmon Resonance (SPR) using Biacore 8K/T200.
  • SPR Surface Plasmon Resonance
  • the affinity of purified antibody binding to antigen was individually determined using a Surface Plasmon Resonance (SPR) biosensor, BIACORETM 8K (GE Healthcare). Antigen was immobilized on the sensor chip. Antibodies were used as the analytes. The data of dissociation (kd) and association (ka) rate constants were obtained using BIACORETM 8K evaluation software. The equilibrium dissociation constants (KD) were calculated from the ratio of kd over ka. [0599] The parameters for affinity measurement are shown in Table 18. Table 18 [0600] The antibody-antigen binding kinetics are summarized in Table 19, and sensor- grams are shown in FIG. 10. Real-time responses are shown as gray curves. Fitting of BIACORETM experimental data to 1:1 interaction model was shown as black curves.
  • Example 3 Humanized BMP-7 Antibody to Treat Resistance to Immunotherapies
  • mice 129 Sv
  • 344SQR primary tumors FIG.13A
  • secondary tumors FIG.13B
  • anti-PD1 10 mg/kg
  • chimeric human BMP-7 mAb clone 344-5-1 10 mg/kg
  • Example 4 – Humanized BMP-7 Antibody May Improve CAR-T Cell Killing [0606]
  • BMP-7 is overexpressed in many cancers, including pancreatic cancer, osteosarcoma, prostate cancer, colorectal cancer, malignant melanoma, breast cancer, ovarian cancer, esophageal cancer, gastric cancer, or renal cell cancer.
  • anti-BMP7 antibodies e.g., one or more BMP-7 antibodies disclosed herein
  • the addition of BMP-7 protein can block, or inhibit, the effect of anti-BMP7 antibodies on CAR-T cell (e.g., EGFR-CAR-T cells) killing.
  • CAR-T cell e.g., EGFR-CAR-T cells
  • BMP-7 is overexpressed in Capan-2 human pancreatic cancer cells (FIG. 14), which also overexpress epidermal growth factor receptor (EGFR).
  • FIG.15 the addition of humanized anti-BMP-7 antibody clone AHF17901 (Ab1) or humanized anti-BMP- 7 antibody clone AHF17906 (Ab2) increased the ability of EGFR-expressing CAR-T cells to effect apoptosis of Capan-2 cells.
  • BMP-7 protein blocks, or inhibits, the effect of humanized anti-BMP-7 antibody clone AHF17901 (Ab1) and humanized anti-BMP-7 antibody clone AHF17906 (Ab2) on the ability of EGFR- expressing CAR-T cells to effect apoptosis of Capan-2 cells.
  • CRISPR-edited CAR T cell to treat resistance to immunotherapies CRISPR-edited CAR T cells having reduced or disrupted expression of bone morphogenic receptors (BMP receptors) were generated using standard techniques. Specifically, lentiviral vectors encoding two gRNAs targeting a gene locus for a BMP receptor and a Cas nuclease were transfected into isolated human T-cells. The Cas nuclease effected a double stranded break at two target locations, allowing for removal of the intervening nucleic acid and successful disruption of the target gene.
  • BMP receptors bone morphogenic receptors
  • FIGs.20-23 depict plasmid maps for illustrative vectors targeting hBMPR1B using a hCas9:TCA:Puro construct (FIG.

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Abstract

Aspects of the disclosure relate to BMP-7-binding proteins. Aspects include methods for treating one or more conditions, for example cancer, using a BMP-7-binding protein. In some aspects, the disclosed methods and compositions involve one or more antibodies that are capable of binding BMP-7.

Description

BMP-7 ANTIBODY COMPOSITIONS & METHODS FOR TREATING CANCER CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of the U.S. provisional application number 63/518,669 filed August 10, 2023, the disclosure of which is herein incorporated by reference in its entirety. SEQUENCE LISTING [0002] This application contains a Sequence Listing that has been submitted via Patent Center in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, created on August 9, 2024, is named MDACP1370WO_SequenceListing.xml and is about 89,100 bytes in size. BACKGROUND OF THE DISCLOSURE Field of the Disclosure [0003] This disclosure relates generally to the fields of molecular biology, immunology, immunotherapy, and medicine. Background [0004] Immunotherapies revolutionized cancer treatment by harnessing the immune system to target cancer cells. However, most patients are resistant to immunotherapies, and the mechanisms underlying this resistance are still poorly understood. For example, although antibodies blocking PD1/PDL1 have led to impressive clinical responses in some patients with melanoma, lung cancer, or renal cell carcinoma, the objective response rates to single-agent anti-PD1 or -PDL1 therapies are only 15–25% in chemotherapy-refractory non-small cell lung cancer (NSCLC). That many patients either do not respond to or develop recurrence after immunotherapy indicates the presence of intrinsic or acquired resistance. Thus, there is a need in the art for methods and compositions for targeting the mechanisms underlying non-response to immunotherapies and for enhancing antitumor immune function to overcome treatment resistance.
202424720.3 1 SUMMARY [0005] Described herein, in some aspects, are methods and compositions for effectively treating subjects with cancer (e.g., solid tumors such as non-small cell lung cancer and triple- negative breast cancer) and for sensitizing a subject with cancer to immunotherapy using polypeptides (e.g., antibodies) targeting BMP-7. In certain aspects, the BMP-7-binding proteins, for example BMP-7-neutralizing antibodies, described herein are unique in that they can specifically bind BMP-7 to overcome resistance to immunotherapies including anti-PD1, anti-PDL1, anti-TIM3, anti-LAG3, and anti-CTLA4. Thus, the methods and compositions described herein provide a novel way to treat patients with resistance to immunotherapies. In certain aspects, the BMP-7-binding proteins, for example BMP-7-neutralizing antibodies, described herein are further unique in that they can improve the efficacy of cancer therapies such as CAR-T cell therapies. Thus, the methods and compositions described herein provide a novel way to treat patients with immunotherapies. [0006] Accordingly, provided herein, in some aspects, are methods and compositions for treating a subject with cancer comprising providing a therapy comprising a BMP-7-binding protein to the subject, where the subject has been diagnosed with or is suspected of having cancer. In some aspects, the disclosed methods comprise providing the therapy to a subject who was previously treated for cancer and was determined to be resistant to the previous treatment. In some aspects, the previous cancer treatment comprised an immunotherapy. [0007] Aspects of the present disclosure include, inter alia, antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16. In some aspects, the VL has at least 85% identity to SEQ ID NO:14 and the VH has at least 85% identity to SEQ ID NO:16. In some aspects, the VL has at least 90% identity to SEQ ID NO:14 and the VH has at least 90% identity to SEQ ID NO:16. In some aspects, the VL has at least 95% identity to SEQ ID NO:14 and the VH has at least 95% identity to SEQ ID NO:16. In some aspects, the VL comprises SEQ ID NO:14 and the VH comprises SEQ ID NO:16.
202424720.3 2 [0008] Aspects of the present disclosure include, inter alia, antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:34, and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:40. In some aspects, the VL has at least 85% identity to SEQ ID NO:34 and the VH has at least 85% identity to SEQ ID NO:40. In some aspects, the VL has at least 90% identity to SEQ ID NO:34 and the VH has at least 90% identity to SEQ ID NO:40. In some aspects, the VL has at least 95% identity to SEQ ID NO:34 and the VH has at least 95% identity to SEQ ID NO:40. In some aspects, the VL comprises SEQ ID NO:34 and the VH comprises SEQ ID NO:40. [0009] Aspects of the present disclosure include, inter alia, antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:36, and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:42. In some aspects, the VL has at least 85% identity to SEQ ID NO:36 and the VH has at least 85% identity to SEQ ID NO:42. In some aspects, the VL has at least 90% identity to SEQ ID NO:36 and the VH has at least 90% identity to SEQ ID NO:42. In some aspects, the VL has at least 95% identity to SEQ ID NO:36 and the VH has at least 95% identity to SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:36 and the VH comprises SEQ ID NO:42. [0010] Aspects of the present disclosure include, inter alia, antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:38, and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:42. In some
202424720.3 3 aspects, the VL has at least 85% identity to SEQ ID NO:38 and the VH has at least 85% identity to SEQ ID NO:42. In some aspects, the VL has at least 90% identity to SEQ ID NO:38 and the VH has at least 90% identity to SEQ ID NO:42. In some aspects, the VL has at least 95% identity to SEQ ID NO:38 and the VH has at least 95% identity to SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:38 and the VH comprises SEQ ID NO:42. [0011] Aspects of the present disclosure include, inter alia, antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:79, and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:81. In some aspects, the VL has at least 85% identity to SEQ ID NO:79 and the VH has at least 85% identity to SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:79 and the VH has at least 90% identity to SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:79 and the VH has at least 95% identity to SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:79 and the VH comprises SEQ ID NO:81. [0012] In some aspects, the disclosed antigen-binding proteins, antibodies, antibody-like molecules, and fragments thereof are bone morphogenetic protein (BMP)-7-binding proteins (i.e., are capable of binding to BMP-7). In some aspects, disclosed herein are monoclonal anti- BMP-7 antibodies. In some aspects, disclosed herein are chimeric anti-BMP-7 antibodies. In some aspects, disclosed herein are humanized anti-BMP-7 antibodies. Antigen-binding proteins described herein may be used in treating one or more conditions associated with expression or activity of a BMP-7 protein such as, for example, cancer. In some aspects, BMP- 7-binding proteins are be used in treating one or more BMP-7-associated conditions with reduced risk of toxicity and improved efficacy as compared to previously disclosed BMP-7- binding proteins. [0013] Aspects include compositions comprising one or more antigen-binding proteins (e.g., BMP-7-binding proteins). Aspects include an antigen-binding protein comprising one or more regions (e.g., heavy chain variable region, light chain variable region, etc.). Aspects include monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody-like molecules. Aspects also include nucleic acid molecules encoding for one or more antigen- binding proteins or portions thereof. Aspects include recombinant, transformed, or modified
202424720.3 4 cells, vectors, and/or expression cassettes comprising such nucleic acid molecules. In some aspects, the compositions contemplated herein can comprise 1, 2, 3, 4, 5, or more of the following components: an antigen-binding protein, a nucleic acid, a vector, a cell, a polypeptide, an oligonucleotide, a complementarity determining region, a light chain variable region, a heavy chain variable region, a light chain constant region, a heavy chain constant region, and complementarity determining regions. Any one or more of these components may be excluded from the disclosed compositions. [0014] Aspects of the disclosure include methods and compositions for treating a subject having cancer, methods for diagnosing a subject with cancer, methods for prognosing a subject with cancer, methods and compositions for sensitizing a subject with cancer to immunotherapy, methods for identifying a subject with cancer as a candidate for a combination therapy, and methods and compositions for treating a subject having solid tumor cancer. Methods of the disclosure can include 1, 2, 3, 4, 5, 6, or more of the following steps: providing a BMP-7 binding protein therapy to a subject, providing an immunotherapy to a subject, providing both a BMP-7 binding protein therapy and an immunotherapy to a subject, providing an alternative therapy to a subject, determining a subject to have cancer, providing two or more types of cancer therapy to a subject, identifying a subject as having had resistance to a previous cancer treatment, testing a subject for resistance to previous cancer treatments, and identifying a subject as being a candidate for a BMP-7 binding protein therapy or a double therapy comprising a BMP-7 binding protein therapy and an immunotherapy. Certain aspects of the disclosure may exclude one or more of the preceding elements and/or steps. [0015] Aspects also include methods of generating an antigen-binding protein, methods of producing an antigen-binding protein, methods of expressing an antigen-binding protein, methods of antigen-binding proteins, methods of detecting BMP-7, methods of treating one or more conditions, methods of purifying BMP-7, methods of treating cancer, and methods of eliminating one or more cells expressing BMP-7. The steps and aspects discussed in this disclosure are contemplated as part of any of these methods. In some aspects, the methods contemplated herein can comprise or exclude 1, 2, 3, 4, 5, or more of the following steps: providing an antigen-binding protein, providing a nucleic acid to a cell, subjecting a cell to conditions sufficient to express a nucleic acid, providing an additional therapeutic, covalently attaching a therapeutic to an antigen-binding protein, non-covalently attaching a therapeutic to an antigen-binding protein, expressing a vector in a cell, and providing a pharmaceutical composition to a subject. Any one or more of these steps may be excluded from the disclosed methods.
202424720.3 5 [0016] In some aspects, the disclosure relates to a bone morphogenetic protein-7 (BMP-7)- binding protein comprising: a light chain variable region (VL) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0017] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0018] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
202424720.3 6 [0019] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0020] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0021] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0022] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
202424720.3 7 [0023] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0024] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 95% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0025] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0026] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0027] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
202424720.3 8 [0028] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0029] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0030] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0031] In some aspects, the VH and the VL are on the same polypeptide. In some aspects, the VH and the VL are on different polypeptides. [0032] In some aspects, the BMP-7-binding protein has an affinity for BMP-7. In some aspects, the BMP-7-binding protein has an affinity for BMP-7 of between 0.001 and 1000 nM. In some aspects, the BMP-7-binding protein has an affinity for BMP-7 of between 0.01 and 100 nM. In some aspects, the BMP-7-binding protein has an association constant for a BMP-7 protein of between 0.1 and 50 nM. In some aspects, the BMP-7-binding protein has an association constant for a BMP-7 protein of between 1 and 20 nM. In some aspects, the BMP- 7-binding protein has an association constant for a BMP-7 protein of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0.19.5, 20.0, 20.5, 21, 21.5, 22, 22.5, 23, 23.5,
202424720.3 9 24, 24.5, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, or 1000 nM, or any range derivable therein. In some aspects, the BMP-7-binding protein has an affinity for BPM-7 of about 10 nM. [0033] In some aspects, the BMP-7-binding protein specifically binds to human BMP-7. In some aspects, the BMP-7-binding protein is an antibody, an antibody-like molecule, or an antigen-binding fragment thereof. In some aspects, the BMP-7-binding protein is an antibody, a nanobody, a minibody, an scFv fragment, or a Fab fragment. In some aspects, the BMP-7- binding protein is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, or a polyclonal antibody. In some aspects, the BMP-7-binding protein is a monoclonal antibody. In some aspects, the BMP-7-binding protein is a murine antibody. In some aspects, the BMP-7- binding protein is a chimeric antibody. In some aspects, the BMP-7-binding protein is a humanized antibody. In some aspects, the BMP-7-binding protein is a human antibody. [0034] Some aspects are directed to a nucleic acid encoding for a polypeptide or fragment thereof that specifically binds BMP-7. In some aspects, the nucleic acid encodes both the heavy chain variable region and the light chain variable region of the BMP-7-binding protein. In some aspects, the nucleic acid encodes the light chain variable region of the BMP-7-binding protein. In some aspects, the nucleic acid encodes the heavy chain variable region of the BMP-7- binding protein. [0035] In some aspects, the nucleic acid encoding the light chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. In some aspects, the nucleic acid encoding the light chain variable region comprises a nucleotide sequence having at least 85%
202424720.3 10 identity to SEQ ID NO:13. In some aspects, the nucleic acid encoding the light chain variable region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. In some aspects, the nucleic acid encoding the light chain variable region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. In some aspects, the nucleic acid encoding the light chain variable region comprises SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. [0036] In some aspects, the nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid encoding the heavy chain variable region comprises SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. [0037] In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:13; and a heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:15. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 85% identity to SEQ ID NO:13, and a heavy chain variable region comprising a nucleotide sequence having at least 85% identity to SEQ ID NO:15. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 90% identity to SEQ ID NO:13, and a heavy chain variable region comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:15. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence
202424720.3 11 having 95% identity to SEQ ID NO:13, and a heavy chain variable region comprising a nucleotide sequence having at least 95% identity to SEQ ID NO:15. In some aspects, the nucleic acid encodes a light chain variable region comprising SEQ ID NO:13, and a heavy chain variable region comprising SEQ ID NO:15. [0038] In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:33; and a heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:39. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 85% identity to SEQ ID NO:33, and a heavy chain variable region comprising a nucleotide sequence having at least 85% identity to SEQ ID NO:39. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 90% identity to SEQ ID NO:33, and a heavy chain variable region comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:39. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 95% identity to SEQ ID NO:33, and a heavy chain variable region comprising a nucleotide sequence having at least 95% identity to SEQ ID NO:39. In some aspects, the nucleic acid encodes a light chain variable region comprising SEQ ID NO:33, and a heavy chain variable region comprising SEQ ID NO:39. [0039] In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:35; and a heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 85% identity to SEQ ID NO:35, and a heavy chain variable region comprising a nucleotide sequence having at least 85% identity to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 90% identity to SEQ ID NO:35, and a heavy chain variable region
202424720.3 12 comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 95% identity to SEQ ID NO:35, and a heavy chain variable region comprising a nucleotide sequence having at least 95% identity to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising SEQ ID NO:35, and a heavy chain variable region comprising SEQ ID NO:41. [0040] In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:37; and a heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 85% identity to SEQ ID NO:37, and a heavy chain variable region comprising a nucleotide sequence having at least 85% identity to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 90% identity to SEQ ID NO:37, and a heavy chain variable region comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 95% identity to SEQ ID NO:37, and a heavy chain variable region comprising a nucleotide sequence having at least 95% identity to SEQ ID NO:41. In some aspects, the nucleic acid encodes a light chain variable region comprising SEQ ID NO:37, and a heavy chain variable region comprising SEQ ID NO:41. [0041] In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:78; and a heavy chain variable region comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:80. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 85% identity to SEQ ID NO:78, and a heavy chain variable region comprising a nucleotide sequence having at least 85% identity to SEQ
202424720.3 13 ID NO:80. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 90% identity to SEQ ID NO:78, and a heavy chain variable region comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:80. In some aspects, the nucleic acid encodes a light chain variable region comprising a nucleotide sequence having 95% identity to SEQ ID NO:78, and a heavy chain variable region comprising a nucleotide sequence having at least 95% identity to SEQ ID NO:80. In some aspects, the nucleic acid encodes a light chain variable region comprising SEQ ID NO:78, and a heavy chain variable region comprising SEQ ID NO:80. [0042] Also disclosed is a vector comprising one or more of the nucleic acids disclosed herein. In some aspects, the nucleic acid(s) comprised in the vector is operably linked to an expression control sequence. [0043] Certain aspects are directed to a cell comprising a polypeptide that specifically binds BMP-7 (the BMP-7-binding protein), a nucleic acid, and/or a vector of the present disclosure. In some aspects, the cell is an immune cell. In some aspects, the cell comprises the BMP-7-binding protein and can secrete the BMP-7-binding protein outside the cell. In some aspects, the cell is a B cell. In some aspects, the cell comprises the BMP-7-binding protein, and the BMP-7-binding protein is attached to a surface of the cell. In some aspects, the BMP-7- binding protein is a chimeric antigen receptor. In some aspects, the BMP-7-binding protein is a T cell receptor. In some aspects, the cell is a T cell. [0044] Certain aspects are directed to a composition comprising a BMP-7-binding protein, such as a BMP-7-binding protein described herein, and one or more additional therapeutics. In some aspects, the one or more additional therapeutics comprise one or more immunotherapies. In some aspects, the one or more immunotherapies comprise a cytotoxic T-lymphocyte- associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor. In some aspects, the one or more immunotherapies comprise a CAR-T cell therapy. In some aspects, the CAR- T cell therapy comprises EGFR-targeted CAR-T cells. [0045] Certain aspects are directed to a composition comprising a BMP-7-binding protein, such as a BMP-7-binding protein described herein, a nucleic acid encoding for the BMP-7- binding protein, a vector comprising the nucleic acid, or a cell comprising the BMP-7-binding protein, the nucleic acid, or the vector; and a pharmaceutically acceptable excipient. In some aspects, the composition further comprises an additional therapeutic. In some aspects, the additional therapeutic comprises a biotherapeutic, a chemotherapeutic, an immunotherapeutic,
202424720.3 14 a toxin, an antisense oligonucleotide, a small inhibitory RNA (siRNA), an enzyme, a protein (e.g., a toxin, an enzyme, etc.), a viral vector, or a nanodrug. [0046] In some aspects, the immunotherapeutic comprises a cytotoxic T-lymphocyte- associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor. In some aspects, the CTLA4 inhibitor is an anti-CTLA4 antibody. In some aspects, the PD1 inhibitor is an anti- PD1 antibody. In some aspects, the PDL-1 inhibitor is an anti-PDL-1 antibody. In some aspects, the LAG3 inhibitor is an anti-LAG3 antibody. In some aspects, the TIM-3 inhibitor is an anti-TIM-3 antibody. [0047] In some aspects, the one or more immunotherapies comprise a CAR-T cell therapy. In some aspects, the CAR-T cell therapy comprises EGFR-targeted CAR-T cells. In some aspects, the CAR-T cell therapy comprises CAR-T cells genetically modified to have disrupted or reduced expression of one or more Bone Morphogenetic Protein (BMP) receptors (e.g., Bone Morphogenetic Protein Receptor Type 1A (BMPR1A), Bone Morphogenetic Protein Receptor Type 1B (BMPR1B), Activin A receptor type I (ACVR1), Activin receptor type 2A (ACVR2A), Activin receptor type 2B (ACVR2B), Bone morphogenetic protein receptor type 2 (BMPR2), or any combination thereof). [0048] Other aspects are directed to a use of a composition comprising a polypeptide that specifically binds BMP-7 which in some aspects comprises an additional therapeutic agent, in the manufacture of a medicament for the treatment or prevention of cancer. Certain aspects are directed to use of the composition comprising the BMP-7-binding protein in the manufacture of a medicament for the treatment or prevention of cancer. [0049] A further aspect is directed to a polypeptide that specifically binds BMP-7 prepared by a method described herein. Some aspects are directed to a method for generating a BMP-7- binding protein, such as a BMP-7-binding protein described herein, by culturing a cell described herein under conditions sufficient to express a BMP-7-binding protein disclosed herein in the cell. Some aspects are directed to a method for generating a BMP-7-binding protein, such as a BMP-7-binding protein described herein, comprising (a) providing to a cell a nucleic acid encoding for the BMP-7-binding protein; and (b) subjecting the cell to conditions sufficient to express the nucleic acid in the cell. [0050] In some aspects, provided herein is a method for treating cancer in a subject comprising providing to the subject a therapeutically effective amount of a BMP-7-binding protein or a composition, such as a pharmaceutical composition, comprising a BMP-7-binding
202424720.3 15 protein, such as a BMP-7-binding protein described herein; a nucleic acid encoding for the BMP-7-binding protein; an expression vector comprising the nucleic acid; or a cell comprising the BMP-7-binding protein, the nucleic acid, or the vector. In some aspects, the method further comprises, prior to administering to the subject the therapeutically effective amount of a BMP- 7-binding protein or a composition, such as a pharmaceutical composition, comprising a BMP- 7-binding protein, such as a BMP-7-binding protein described herein; the nucleic acid encoding for the BMP-7-binding protein; the expression vector comprising the nucleic acid; or the cell comprising the BMP-7-binding protein, the nucleic acid, or the vector, diagnosing the subject with cancer. [0051] In some aspects, the subject was previously treated for the cancer with a cancer therapy. In some aspects, the cancer therapy comprises one or more immunotherapies. In some aspects, the one or more immunotherapies comprise a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor. In some aspects, the subject was determined to be resistant to the cancer therapy. [0052] In some aspects, the cancer is a solid tumor. In some aspects, the cancer is lung cancer, gastric cancer, brain cancer, colon cancer, melanoma, or breast cancer. In some aspects, the cancer is lung cancer. In some aspects, the lung cancer is non-small cell lung cancer. In some aspects, the cancer is breast cancer. In some aspects, the breast cancer is triple-negative breast cancer. [0053] In some aspects, the cancer cells overexpress BMP-7 compared to non-cancerous cells. In some aspects, the cancer is pancreatic cancer, osteosarcoma, prostate cancer, colorectal cancer, malignant melanoma, breast cancer, ovarian cancer, esophageal cancer, gastric cancer, or renal cell cancer. In some aspects, the cancer is pancreatic cancer. [0054] In some aspects, the method further comprises providing to the subject one or more additional therapies. In some aspects, the one or more additional therapies are radiotherapy, chemotherapy, or immunotherapy. [0055] Also disclosed herein, in some aspects, is a method of increasing the efficacy of a cancer therapy administered to a subject for treatment of cancer, the method comprising administering to the subject a therapeutically effective amount of the cancer therapy and a BMP-7-binding protein or a composition, such as a pharmaceutical composition, comprising a BMP-7-binding protein, such as a BMP-7-binding protein described herein; a nucleic acid encoding for the BMP-7-binding protein; an expression vector comprising the nucleic acid; or
202424720.3 16 a cell comprising the BMP-7-binding protein, the nucleic acid, or the vector. In some aspects, the cancer therapy and the a BMP-7-binding protein or a composition, such as a pharmaceutical composition, comprising a BMP-7-binding protein, such as a BMP-7-binding protein described herein; a nucleic acid encoding for the BMP-7-binding protein; an expression vector comprising the nucleic acid; or a cell comprising the BMP-7-binding protein, the nucleic acid, or the vector, are comprised in the same composition. In some aspects, the cancer therapy and a BMP-7-binding protein or a composition, such as a pharmaceutical composition, comprising a BMP-7-binding protein, such as a BMP-7-binding protein described herein; a nucleic acid encoding for the BMP-7-binding protein; an expression vector comprising the nucleic acid; or a cell comprising the BMP-7-binding protein, the nucleic acid, or the vector, are comprised in the different compositions. In some aspects, the cancer therapy comprises an immunotherapy. In some aspects, the immunotherapy comprises a CAR-T cell therapy. In some aspects, the immunotherapy comprises EGFR-targeted CAR-T cells. In some aspects, the cancer cells overexpress BMP-7 compared to non-cancerous cells. In some aspects, the cancer is pancreatic cancer, osteosarcoma, prostate cancer, colorectal cancer, malignant melanoma, breast cancer, ovarian cancer, esophageal cancer, gastric cancer, or renal cell cancer. In some aspects, the cancer is pancreatic cancer. [0056] Some aspects are directed to a monoclonal antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0057] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID
202424720.3 17 NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0058] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0059] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0060] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0061] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%,
202424720.3 18 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0062] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0063] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0064] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 95% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0065] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0066] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least, at most, exactly, or
202424720.3 19 between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0067] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0068] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0069] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0070] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
202424720.3 20 [0071] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14 and the VH comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16. In some aspects, the VL has at least 85% identity to SEQ ID NO:14 and the VH has at least 85% identity to SEQ ID NO:16. In some aspects, the VL has at least 90% identity to SEQ ID NO:14 and the VH has at least 90% identity to SEQ ID NO:16. In some aspects, the VL has at least 95% identity to SEQ ID NO:14 and the VH has at least 95% identity to SEQ ID NO:16. In some aspects, the VL comprises SEQ ID NO:14 and the VH comprises SEQ ID NO:16. In some aspects, the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7). [0072] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:34 and the VH comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:40. In some aspects, the VL has at least 85% identity to SEQ ID NO:34 and the VH has at least 85% identity to SEQ ID NO:40. In some aspects, the VL has at least 90% identity to SEQ ID NO:34 and the VH has at least 90% identity to SEQ ID NO:40. In some aspects, the VL has at least 95% identity to SEQ ID NO:34 and the VH has at least 95% identity to SEQ ID NO:40. In some aspects, the VL comprises SEQ ID NO:34 and the VH comprises SEQ ID NO:40. In some aspects, the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7). [0073] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value
202424720.3 21 derivable therein, to SEQ ID NO:36 and the VH comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:42. In some aspects, the VL has at least 85% identity to SEQ ID NO:36 and the VH has at least 85% identity to SEQ ID NO:42. In some aspects, the VL has at least 90% identity to SEQ ID NO:36 and the VH has at least 90% identity to SEQ ID NO:42. In some aspects, the VL has at least 95% identity to SEQ ID NO:36 and the VH has at least 95% identity to SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:36 and the VH comprises SEQ ID NO:42. In some aspects, the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7). [0074] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:38 and the VH comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:42. In some aspects, the VL has at least 85% identity to SEQ ID NO:38 and the VH has at least 85% identity to SEQ ID NO:42. In some aspects, the VL has at least 90% identity to SEQ ID NO:38 and the VH has at least 90% identity to SEQ ID NO:42. In some aspects, the VL has at least 95% identity to SEQ ID NO:38 and the VH has at least 95% identity to SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:38 and the VH comprises SEQ ID NO:42. In some aspects, the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7). [0075] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:79 and the VH comprises an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:81. In some aspects, the VL has at least 85%
202424720.3 22 identity to SEQ ID NO:79 and the VH has at least 85% identity to SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:79 and the VH has at least 90% identity to SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:79 and the VH has at least 95% identity to SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:79 and the VH comprises SEQ ID NO:81. In some aspects, the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7). [0076] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0077] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24; and a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30. In some aspects, the VL comprises SEQ ID NO:14. In some aspects, the VH comprises SEQ ID NO:16. [0078] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65. In some aspects, the VL comprises SEQ ID NO:34. In some aspects, the VH comprises SEQ ID NO:40. In some aspects, the VL comprises SEQ ID NO:36. In some aspects, the VH comprises SEQ ID NO:42.
202424720.3 23 In some aspects, the VL comprises SEQ ID NO:38. In some aspects, the VH comprises SEQ ID NO:42. [0079] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77. In some aspects, the VL comprises SEQ ID NO:79. In some aspects, the VH comprises SEQ ID NO:81. [0080] Throughout this application, the terms “about” and “approximately” and “substantially” are used according to their plain and ordinary meaning in the area of cell and molecular biology to indicate a deviation of ±10% of the value(s) to which it is attached. Therefore, in any disclosed aspect, the terms may be substituted with “within [a percentage] of” what is specified. In one non-limiting aspect, the percentage includes 0.1, 0.5, 1, 5, and 10 percent. [0081] The use of the word “a,” “an,” and “the” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Thus, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. [0082] The phrase “and/or” means “and” or “or”. To illustrate, A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and/or” operates as an inclusive or. [0083] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Thus, as used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the disclosure, yet open to the inclusion of unspecified elements, whether essential or not. [0084] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. As
202424720.3 24 used herein the term “consisting essentially of” refers to those elements required for a given aspect. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that aspect of the disclosure. With respect to pharmaceutical compositions, the term “consisting essentially of” includes the active ingredients recited, excludes any other active ingredients, but does not exclude any pharmaceutical excipients or other components that are not therapeutically active. The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the aspect. [0085] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “use of” any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect. [0086] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different aspects may be combined. [0087] Reference throughout this specification to “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” or “a further aspect” or combinations thereof means that a particular feature, structure, or characteristic described in connection with the aspect is included in at least one aspect of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects. [0088] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects are discussed throughout this application. Any aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. The aspects in the Example section are understood to be aspects that are applicable to all aspects of the technology described herein. [0089] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating certain aspects of the disclosure, are given by way of illustration only, since various changes and modifications
202424720.3 25 within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS [0090] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein. [0091] FIG.1 shows titration of an antibody generated to specifically target human BMP- 7 (BMP-7 mAb #5). [0092] FIG. 2 shows functional validation of BMP-7 mAb in macrophages treated with BMP-7 protein for 48 hours. Macrophages were treated with BMP-7 for 48 hours and p38 alpha (MAPK14) expression were evaluated by using quantitative PCR. BMP-7 mAb number 344- 5-1 demonstrated highest upregulation of p38 alpha, a marker for Th1 cytokines/chemokines. [0093] FIGs.3A-3B show tumor growth and survival analysis of mice 129 Sv (Females) with 344SQR tumors. FIG.3A. example FIG.3B. Mouse survival rates were analyzed by the Kaplan–Meier method and compared with log-rank tests. [0094] FIGs. 4A-4B show tumor growth and survival analysis of mice 129 Sv (Males) with 344SQR tumors. FIG.4A. Mice were treated with IgG ctrl (n=5) or anti-PD1 (10 mg/kg) (n=5) and/or Human BMP-7344-5-1 (10 mg/kg) (n=5) 3 times a week for 2 weeks. FIG.4B. Mouse survival rates were analyzed by the Kaplan–Meier method and compared with log-rank tests. [0095] FIG. 5 shows functional validation of BMP-7 mAb antibodies in macrophages treated with BMP-7 antibody for 48 hours. [0096] FIG.6 shows representative images of immunohistochemical stains of CD8 T cells (CD8 – brown dots) and M2 macrophages (CD163 – M2 macrophage marker) in formalin- fixed paraffin-embedded tissue sections from 344SQR tumors treated with BMP-7 mAb plus anti-PD1. [0097] FIGs. 7A-7B show tumor growth analysis of mice 129 Sv with 344SQR tumors (non-small cell lung cancer; FIG.7A) or mice BALB/c with 4T1 tumors (triple negative breast cancer’ FIG.7B) treated with IgG ctrl (n=5) or anti-PD1 (10 mg/kg) (n=5) or Human BMP-7 mAbs (10 mg/kg) (n=5) 3 times a week for 2 weeks.
202424720.3 26 [0098] FIGs. 8A-8B show tumor growth of mice 129 Sv with 344SQR primary tumors (FIG.8A) and secondary tumors (FIG.8B) treated with IgG ctrl (n=5) or anti-PD1 (10 mg/kg) (n=5) or Human BMP-7 mAb (10 mg/kg) (n=5) 3 times a week for 2 weeks. [0099] FIG.9 shows sensor-grams of chimeric antibody binding to human BMP-7. [0100] FIGs. 10A-10B show alignment of sequenced humanized BMP-7 monoclonal antibody VH (FIG.10A) and VL chains (FIG.10B). [0101] FIGs. 11A-11B show alignment of selected, sequenced humanized BMP-7 monoclonal antibody VH (FIG.11A) and VL chains (FIG.11B). [0102] FIG. 12 shows 99% purification of selected humanized BMP-7 monoclonal antibodies by SDS-PAGE. Loading order: Marker (M); AHF17901, reduced (1); AHF17906, reduced (2); AHF17909, reduced (3); AHF17901, non-reduced (4); AHF17906, non-reduced (5); AHF17909, non-reduced (6); human IgG. [0103] FIG.13 shows sensor-grams of chimeric BMP-7 monoclonal antibody binding to human BMP-7. [0104] FIG. 14 shows BMP-7 gene expression in Capan-2 pancreatic cancer cells, GSU gastic cancer cells, MCF-7 breast cancer cells, and MDA-MB-231 breast cancer cells. BMP- 7 gene expression was measured using quantitative PCR using primers specific for BMP-7 gene. [0105] FIG.15 shows apoptosis of Capan-2 pancreatic cancer cells by CAR-T cells after treatment with an anti-BMP-7 antibody. Apoptosis was measured using the Caspase 3/7 green, fluorescent dye (Essen Bioscience) analyzed by the INCUCYTE® Live-Cell Analysis System. A higher Y-axis value indicates increased apoptosis and killing capability of the CAR-T cells. [0106] FIG. 16 shows that addition of BMP7 protein blocks the effect of apoptosis of Capan-2 pancreatic cancer cells by CAR-T cells after treatment with an anti-BMP-7 antibody. Apoptosis was measured using the Caspase 3/7 green, fluorescent dye (Essen Bioscience) analyzed by the INCUCYTE® Live-Cell Analysis System. A higher Y-axis value indicates increased apoptosis and killing capability of the CAR-T cells. [0107] FIG. 17 shows that addition of BMP7 protein blocks the effect of apoptosis of Capan-2 pancreatic cancer cells by CAR-T cells after treatment with an anti-BMP-7 antibody. Apoptosis was measured using the Caspase 3/7 green, fluorescent dye (Essen Bioscience) analyzed by the INCUCYTE® Live-Cell Analysis System. A higher Y-axis value indicates increased apoptosis and killing capability of the CAR-T cells.
202424720.3 27 [0108] FIG. 18 shows predicted target-potential vs. off-target risk for different gRNAs targeting the BMPR1B gene locus, with unique gRNAs predicted to have high target specificity highlighted in light grey. [0109] FIG. 19 shows predicted target-potential vs. off-target risk for different gRNAs targeting the BMPR1A gene locus, with unique gRNAs predicted to have high target specificity highlighted in light grey. [0110] FIG. 20 is a plasmid map of a lentiviral vector (VB200329-1029jwu) encoding a hCas9:T2A:Puro construct and two gRNAs (#29757 and #29702) targeting the hBMPR1B gene locus. [0111] FIG. 21 is a plasmid map of a lentiviral vector (VB200329-1005ytw) encoding a hCas9 endonuclease and two gRNAs (#29757 and #29702) targeting the hBMPR1B gene locus. [0112] FIG. 22 is a plasmid map of a lentiviral vector (VB200328-7336rqg) encoding a hCas9:T2A:Puro construct and two gRNAs (#14262 and #14258) targeting the hBMPR1A gene locus. [0113] FIG. 23 is a plasmid map of a lentiviral vector (VB200328-7298grx) encoding a hCas9 endonuclease and two gRNAs (#14262 and #14258) targeting the hBMPR1A gene locus. DETAILED DESCRIPTION [0114] Immunotherapies have been produced which provide durable disease control in some cancer patients. However, most cancer patients do not respond to immunotherapy, and even patients responding to immunotherapy will often develop resistance to the immunotherapy. Thus, there remains a need for further therapeutics that target mechanisms underlying this resistance and help to enhance antitumor immune function to overcome resistance to immunotherapies. [0115] The present disclosure is based, at least in part, on the surprising discovery that cancer resistant to immunotherapy is responsive to a therapy comprising a BMP-7 binding protein. Further, administering a BMP-7 binding protein therapy was surprisingly found to re- sensitize tumors to immunotherapeutics. As disclosed herein, administration of a BMP-7 binding protein therapy, when associated with cancer, for example, a solid tumor cancer, relieves immunosuppression and enables efficacy of immunotherapy. The BMP-7 binding
202424720.3 28 proteins described can interact with functional BMP-7 protein secreted by cancer cells, for example, solid tumor cells, to neutralize BMP-7 protein and initiate downstream signaling events associated with a reduction in pro-inflammatory signaling and re-sensitization of tumors to immunotherapeutics. [0116] Accordingly, in some aspects, disclosed are methods and compositions for treating cancer comprising administering a BMP-7 binding protein therapy to a subject having or suspected or having cancer. In some aspects, the cancer is a solid tumor cancer. In some aspects, the subject has or has previously had resistance to immunotherapy. Further aspects disclose methods for stratifying cancer patients based on a history of resistance to immunotherapy. For example, aspects are directed to methods for identifying a subject as being a candidate for a BMP-7 binding protein therapy by identifying the subject as having or having previously had resistance to immunotherapy. [0117] The clinical relevance of a novel BMP-7 antibody and the use of such antibody for therapeutic applications, such as treatment of resistance to immunotherapies in solid tumors, is described. Antibodies of the present disclosure and derivatives thereof (e.g., antibody fragments, antibody-like molecules, etc.) may therefore be useful in the treatment of various conditions including, for example, cancer. In some aspects, antibodies of the present disclosure are useful in the treatment of cancers which overexpress BMP-7, for example, solid tumor cancers such as lung cancer, including non-small cell lung cancer and small-cell lung cancer, breast cancer, including triple negative breast cancer, brain cancer, colon cancer, melanoma, and gastric cancer. I. Examples of Definitions [0118] “Individual, “subject,” and “patient” are used interchangeably and can refer to a human or non-human. [0119] The terms “lower,” “lowered,” “reduce,” “reduced,” “reduction,” “decrease,” “decreased,” “inhibit,” “inhibited,” or “inhibition” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “lower,” “lowered,” “reduce,” “reduced,” “reduction,” “decrease,” “decreased,” “inhibit,” “inhibited,” or “inhibition” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%
202424720.3 29 or up to and including a 100% decrease (i.e., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level. [0120] The terms “increased,” “increase,” “enhanced,” “enhance,” “activated,” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, “increased,” “increase,” “enhanced,” “enhance,” “activated,” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. [0121] As used herein, the terms “reference,” “standard,” or “control” describe a value relative to which a comparison is performed. For example, an agent, subject, population, sample, or value of interest is compared with a reference, standard, or control agent, subject, population, sample, or value of interest. A reference, standard, or control may be tested and/or determined substantially simultaneously and/or with the testing or determination of interest for an agent, subject, population, sample, or value of interest and/or may be determined or characterized under comparable conditions or circumstances to the agent, subject, population, sample, or value of interest under assessment. [0122] A “nucleic acid,” as used herein, is a molecule comprising nucleic acid components and refers to DNA or RNA molecules. The term “nucleic acid” may be used interchangeably with the term “polynucleotide.” A nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone. Nucleic acids may also encompass modified nucleic acid molecules, such as base-modified, sugar-modified, backbone-modified, etc. DNA or RNA molecules. Nucleic acids may exist in a variety of forms such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding polypeptides, e.g., antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof; polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide; anti-sense nucleic acids for inhibiting expression of a polynucleotide; mRNA; saRNA; and complementary sequences of the foregoing described
202424720.3 30 herein. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and/or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids). [0123] Nucleic acids may be single-stranded or double-stranded and may comprise RNA and/or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids). In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. A tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide. [0124] The term “polynucleotide” refers to a nucleic acid molecule that may be recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (i.e., nucleic acids 100 residues or less in length) and recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or a combination thereof. Additional coding or non- coding sequences may, but need not, be present within a polynucleotide. [0125] A “gene,” “polynucleotide,” “coding region,” “sequence,” “segment,” “fragment,” or “transgene” which “encodes” a particular protein, is a nucleic acid molecule which is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences. As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. The coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5’ (amino) terminus (N-terminus) and a translation stop codon at the 3’ (carboxy) terminus (C-terminus). A gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will
202424720.3 31 usually be located 3’ to the gene sequence. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein. [0126] As used herein, the term “expression” of a nucleic acid sequence refers to the generation of any gene product from the nucleic acid sequence. In some aspects, a gene product may be a transcript. In some aspects, a gene product may be a polypeptide. In some aspects, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein. [0127] In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. [0128] The term “DNA,” as used herein, means a nucleic acid molecule comprising nucleotides such as deoxy-adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate and deoxy-cytidine-monophosphate monomers which are composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, and polymerize by a characteristic backbone structure. The backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, e.g., deoxyribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, e.g., the order of the bases linked to the sugar/phosphate-backbone, is called the DNA sequence. DNA may be single stranded or double stranded. In the double stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g., by A/T-base-pairing and G/C-base-pairing. DNA may contain all, or a majority of, deoxyribonucleotide residues. As used herein, the term “deoxyribonucleotide” means a nucleotide lacking a hydroxyl group at the 2′ position of a β-D-ribofuranosyl group. Without any limitation, DNA may encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, DNA that is recombinantly produced, and modified DNA.
202424720.3 32 [0129] The term “RNA,” as used herein, means a nucleic acid molecule comprising nucleotides such as adenosine-monophosphate, uridine-monophosphate, guanosine- monophosphate and cytidine-monophosphate monomers which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, e.g., ribose, of a first and a phosphate moiety of a second, adjacent monomer. RNA may be obtainable by transcription of a DNA-sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo, transcription of DNA may result in premature RNA which is processed into messenger-RNA (mRNA). Processing of the premature RNA, e.g., in eukaryotic organisms, comprises various posttranscriptional modifications such as splicing, 5′ capping, polyadenylation, and/or export from the nucleus or the mitochondria. Mature messenger RNA is processed and provides the nucleotide sequence that may be translated into an amino acid sequence of a peptide or protein. A mature mRNA may comprise a 5′ cap, a 5′ UTR, an open reading frame, a 3′ UTR and a poly-A tail sequence. RNA may contain all, or a majority of, ribonucleotide residues. As used herein, the term “ribonucleotide” means a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribofuranosyl group. In one aspect, RNA may be messenger RNA (mRNA) that relates to an RNA transcript which encodes a peptide or protein. As known to those of skill in the art, mRNA generally contains a 5′ untranslated region (5′ UTR), a polypeptide coding region, and a 3′ untranslated region (3′ UTR). Without any limitation, RNA may encompass double stranded RNA, antisense RNA, single stranded RNA, isolated RNA, synthetic RNA, RNA that is recombinantly produced, and modified RNA (modRNA). [0130] The terms “protein,” “polypeptide,” or “peptide” are used herein as synonyms and refer to a polymer of amino acid monomers, e.g., a molecule comprising at least two amino acid residues. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. Polypeptides may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. A protein comprises one or more peptides or polypeptides and may be folded into a 3-dimensional form, which may be required for the protein to exert its biological function. [0131] As used herein, the terms “wild type” or “WT” or “native” refer to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild type versions of a protein or polypeptide are employed, however, in other aspects of the disclosure, a modified protein or polypeptide is employed. The terms described above may be used interchangeably.
202424720.3 33 [0132] A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide having a chemical structure, particularly an amino acid sequence, that is altered with respect to the wild type protein or polypeptide. In some aspects, a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild type activity or function in other respects, such as immunogenicity. [0133] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild type) or recombinant (modified) protein. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, produced by solid-phase peptide synthesis (SPPS) (i.e., an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution), liquid phase peptide synthesis (i.e., sequential addition of monomer building blocks in a liquid phase), a combination of solid and liquid phase peptide synthesis, or other in vitro methods. Assembling nucleic acids by a ligase may also be used to promote intermolecular ligation of the 5ʹ and 3′ ends of polynucleotide chains through the formation of a phosphodiester bond. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antigen or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule. [0134] The term “fragment,” with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, e.g., a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus. A fragment shortened at the C- terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3′-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5′-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90%, or at least 99% of the amino acid residues from an amino acid sequence. In the present disclosure, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least, at most, exactly, or between (inclusive or
202424720.3 34 exclusive) any two of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. [0135] In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 70% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 80% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 85% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 90% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 95% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 97% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 99% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. [0136] As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some aspects, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some aspects, a variant polypeptide or nucleic acid may differ from a
202424720.3 35 reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). [0137] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antibody or antibody derivative) that it encodes. Mutations can be introduced using any technique known in the art. In one aspect, one or more particular amino acid residues are changed using, for example, a site- directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. However it is made, a mutant polypeptide can be expressed and screened for a desired property. [0138] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. See, e.g., Romain Studer et al., Biochem. J.449:581-594 (2013), incorporated herein by reference. For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody. [0139] Variant polypeptides encoded by nucleic acids of the disclosure may contain amino acid changes that confer any of a number of desirable properties. Variant polypeptides can be made using routine mutagenesis techniques and assayed as appropriate to determine whether they possess the desired property. The stability of protein(s) encoded by a variant nucleic acid may be measured by assaying thermal stability or stability upon urea denaturation or may be measured using in silico prediction. Methods for such experiments and in silico determinations are known in the art. [0140] In some aspects, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some aspects, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some aspects, a reference polypeptide or nucleic acid has one or more biological activities. In some aspects, a variant polypeptide or nucleic acid shares one or more of the biological
202424720.3 36 activities of the reference polypeptide or nucleic acid. In some aspects, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some aspects, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. [0141] In some aspects, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence inserted, or deleted, as compared to the reference, with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen- binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines its functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. [0142] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. The variant polypeptide or nucleic acid sequence has at least one modification compared to the reference polypeptide or nucleic acid sequence, e.g., from 1 to about 50 modifications. A variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type. In one aspect, the variant polypeptide or nucleic acid sequence has from 1 to about 50 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has from 1 to about 40 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has from 1 to about 30 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has from 1 to about 20 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has from 1 to about 10 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has from 1 to about 5 modifications compared to the reference polypeptide or nucleic acid sequence. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or
202424720.3 37 deleted, as compared to the reference. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. [0143] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5’ or 3’ nucleic acid sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5’ or 3’ portions of the coding region. [0144] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids. [0145] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein. [0146] Insertional mutants typically involve the addition of amino acid residues at a non- terminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein. [0147] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. [0148] “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative replacements (also “conservative substitutions” or “conservative amino acid substitutions”) are those that take place within a family of amino acids that possess similar biochemical properties, including charge, hydrophobicity, and size. Genetically encoded amino acids are generally divided into
202424720.3 38 families based on the chemical nature of the side chain, e.g., acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Thus, a conservative replacement may comprise replacement of an amino acid in one family for an amino acid in the same family (e.g., replacement of a lysine with an arginine, replacement of an aspartate for a glutamate, etc.). Alternatively, or in addition, amino acid similarity may be determined using a Blocks Substitution Matrix (BLOSUM), such as BLOSUM62 (Henikoff S and Henikoff JG, Proc. Natl. Acad. Sci. U.S.A 89(22): 10915-9 (1992)). In this case, a conservative replacement may be a substitution of amino acids having a non-negative value on a BLOSUM62 matrix. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Standard ELISA, Surface Plasmon Resonance (SPR), or other antibody binding assays can be performed by one skilled in the art to make a quantitative comparison of antigen binging affinity between the unmodified antibody and any polypeptide derivatives with conservative substitutions generated through any of several methods available to one skilled in the art. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties. [0149] Alternatively, substitutions may be “non-conservative” (also “nonconservative”). In some aspects, a non-conservative substitution affects a function or activity of the polypeptide. In some aspects, a non-conservative substitution does not affect a function or activity of the polypeptide. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class. [0150] In some aspects, a reference polypeptide or nucleic acid is a “wild type” or “WT” or “native” sequence found in nature, including allelic variations. A wild type polypeptide or nucleic acid sequence has a sequence that has not been intentionally modified. For the purposes of the present disclosure, “variants” of an amino acid sequence (peptide, protein, or polypeptide) comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants. “Variants” of a nucleotide sequence comprise nucleotide insertion variants, nucleotide addition variants, nucleotide deletion variants and/or nucleotide substitution variants. The term “variant” includes all mutants, splice
202424720.3 39 variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular, those which are naturally occurring. The term “variant” includes, in particular, fragments of an amino acid or nucleic acid sequence. [0151] A fragment or variant of an amino acid sequence (peptide or protein) may be a “functional fragment” or “functional variant.” The term “functional fragment” or “functional variant” of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, e.g., it is functionally equivalent. With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived. The term “functional fragment” or “functional variant,” as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence. In one aspect, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. [0152] An amino acid sequence (peptide, protein, or polypeptide) “derived from” a designated amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences. [0153] Changes may be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antigen or antibody or antibody derivative) that it encodes. Mutations may be introduced using any technique known in the art. In one aspect, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. In some aspects, however it is made, a mutant polypeptide may be expressed and screened for a desired property.
202424720.3 40 [0154] Mutations may be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one may make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations may be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. For example, the mutation may quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody. [0155] “Sequence similarity” indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. “Sequence identity” between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. The terms “% identical,” “% identity,” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison,” in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads. App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (FOGSAA, GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group). In some aspects, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website. [0156] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100. [0157] In some aspects, the degree of similarity or identity is given for a region that is at least, at most, exactly, or between (inclusive or exclusive) any two of about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the entire length of the reference
202424720.3 41 sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least, at most, exactly, or between any two of about 100, about 120, about 140, about 160, about 180, or about 200 nucleotides, in some aspects, continuous nucleotides. In some aspects, the degree of similarity or identity is given for the entire length of the reference sequence. [0158] Homologous amino acid sequences may exhibit at least, at most, exactly, or between (inclusive or exclusive) any two of 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity of the amino acid residues. In one aspect, homologous amino acid sequences exhibit at least 95% identity of the amino acid residues. In one aspect, homologous amino acid sequences exhibit at least 98% identity of the amino acid residues. In one aspect, homologous amino acid sequences exhibit at least 99% identity of the amino acid residues. [0159] In the present disclosure, a vector refers to a nucleic acid molecule, such as an artificial nucleic acid molecule. A vector may be used to incorporate a nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame. Vectors include, but are not limited to, storage vectors, expression vectors, cloning vectors, and transfer vectors. A vector may be an RNA vector or a DNA vector. In some aspects the vector is a DNA molecule. In some aspects, the vector is a plasmid vector. In some aspects, the vector is a viral vector. Typically, an expression vector will contain a desired coding sequence and appropriate other sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired fragment (typically a DNA fragment) and may lack functional sequences needed for expression of the desired fragment(s). [0160] “Treating” or “treatment,” as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest, and includes: (i) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; (ii) inhibiting the disease or condition, i.e., arresting its development; (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms “disease” and “condition” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore
202424720.3 42 not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians. [0161] “Prevent” or “prevention,” as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder, or condition has been delayed for a predefined period of time. [0162] As will be understood from context, “risk” of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition. In some aspects, risk is expressed as a percentage. In some aspects, risk is at least, at most, exactly, or between (inclusive or exclusive) any two of from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some aspects risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some aspects, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some aspects a reference sample or group of reference samples are from individuals comparable to a particular individual. In some aspects, risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and/or condition. In some aspects, risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes. [0163] An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some aspects, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some aspects, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some aspects, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some aspects, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some aspects, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition. [0164] As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers approved by the
202424720.3 43 United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Pharmaceutical compositions may be immunogenic compositions. In some aspects, active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some aspects, pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation. II. BMP-7 [0165] Bone morphogenic protein 7 (BMP-7), also known as osteogenic protein-1 (OP-1), is a secreted protein that belongs to the transforming growth factor beta (TGFB) superfamily and regulates proliferation, differentiation, and apoptosis in many different cell types by altering target gene transcription. The complete mRNA sequence of human BMP-7 has the GENBANK® Accession Number NM_001719, incorporated by reference herein in its entirety. [0166] BMPs can act as either tumor suppressors or oncogenes depending on the cellular context and tumor type. Secreted BMP-7 impinges on effector T-cell functions, favoring the generation of immunosuppressive cells, and precluding response to immunotherapy. As shown herein, BMP-7 is upregulated in mouse and human tumors resistant to anti-PD1 therapy, and BMP-7 levels are higher not only in blood from mice bearing resistant tumors but also in pretreatment blood from patients exhibiting disease progression while on anti-PD1 and radiotherapy. BMP-7 has also been reported in a wide range of human cancers and is associated with metastasis and poor prognosis. In lung cancer, BMP-7 overexpression is associated with lymph node involvement and an indicator of bone metastasis. BMPs regulate immune cell responses and are immunosuppressive in cancer. For example, BMPs have been shown to regulate activation, growth, and cytokine secretion in macrophages and to promote PDL1 and PDL2 upregulation in dendritic cells. Treatment with BMP-7 in vitro and in vivo significantly enhanced monocyte polarization into M2 macrophages. [0167] BMP ligands bind to and form heteromeric complexes with two types of serine/threonine kinase receptors on the cell surface, which then activate the “small mothers against decapentaplegic” (SMAD) proteins 1 and 5 in cells. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. In response to BMP ligands, SMAD proteins can be phosphorylated and activated by the BMP
202424720.3 44 receptor kinase. Phosphorylated SMAD proteins form oligomeric complexes, which are important for their function in the transcription regulation. Specifically, binding of BMP to its receptor leads to the phosphorylation of intracellular SMADs, which then bind to co-SMAD4 and translocate into the nucleus to regulate gene expression. [0168] Secreted BMP-7 promotes resistance to immunotherapy by repressing macrophage- mediated inflammatory responses and Th1-associated cytokines in the tumor microenvironment. BMP-7 downregulates MAPK14 and MAPK14-regulated cytokines and chemokines including IL1A, IL1B, TNF, and CCL5 via SMAD1 activation. At the same time, BMP-7 decreases CD4+ T-cell activation by downregulating IFNG and IL2 expression via SMAD1/MAPK14 signaling. [0169] BMP-7 can specifically downregulate MAPK14 at the mRNA and protein levels. MAPK14 can act as a tumor suppressor by regulating cell cycle progression and induction of apoptosis or as an oncogene by promoting invasion, inflammation, and angiogenesis. MAPK14 is a member of the p38 MAPK family. It is downregulated not only in tumors but also in TILs from anti-PD1-resistant tumors. P38 proteins are important participants in inflammatory signaling pathways and are activated in response to a variety of cellular stresses, including osmotic shock, lipopolysaccharides, and inflammatory cytokines. MAPK14 is the critical isoform in inflammatory responses and is involved in the expression of proinflammatory mediators in macrophages such as IL1B, TNF, and IL12 as well as CCL5, COX-2, IL8, IL6, IL3, IL2, and IL1, all of which contain AU-rich elements in their 3′-untranslated regions to which MAPK14 binds. MAPK14 participates in the regulation of IFNG expression and its mRNA stabilization in immune cells. [0170] MAPK14 is known to be regulated by BMP-7 via SMAD activation. BMP-7 can either promote or inhibit MAPK14 activation depending on the cellular context and BMP-7 dose. MAPK14-regulated inflammatory cytokines IL1A, IL1B, and TNF are downregulated in tumor-infiltrating leukocytes (TILs) collected from 344SQR tumors treated with anti-PD1 versus 344SQP. Cytokines and chemokines regulated by MAPK14 including IL1A, IL1B, TNF, CCL5, IFNG, and IL2 are also downregulated in blood from mice bearing 344SQR tumors compared with parental tumors. Thus, BMP-7 regulates MAPK14 expression not only in tumors resistant to anti-PD1 but also in TILs in the tumor microenvironment, and BMP-7 also regulates expression of proinflammatory cytokines and chemokines in TILs via MAPK14 regulation. [0171] Similar to results observed in tumors, BMP-7 regulates MAPK14 via SMAD1 activation in immune cells. Previous studies in macrophages isolated from a vivo model of
202424720.3 45 atherosclerosis treated with intravenous injections of BMP-7 or liposomal clodronate demonstrated that BMP-7 significantly reduced the number of proinflammatory macrophages and decreased MAPK14 activation while increasing SMAD1/5/8 phosphorylation in macrophages. Other studies have shown that BMP-7 promotes M2 polarization in human and mouse macrophages in vitro and in vivo models. [0172] MAPK14 signaling promotes not only M2 monocytes polarization into M1-type cells in response to lipopolysaccharides but also is central in the activation of proinflammatory gene transcription. In macrophages, MAPK14 is activated by lipopolysaccharide and Toll-like receptor-4, which subsequently activates proinflammatory cytokines, including IL1 and TNF. Murine macrophages co-cultured with 344SQR cells have lower expression of MAPK14, IL1A, IL1B, TNF, and CCL5 compared with cells co-cultured with 344SQP cells. That these findings depend on BMP-7 has been confirmed by co-culturing macrophages with BMP-7-knockdown 344SQR cells, which results in higher expression levels of MAPK14, IL1A, IL1B, TNF, and CCL5 compared with control cells. Thus, BMP-7 suppresses the proinflammatory cytokine expression regulated by MAPK14 in macrophages. [0173] P38 signaling is known to be activated in T cells stimulated via TCR signaling and reduced in anergic T cells. MAPK14 also participates in the regulation of IFNG expression in CD4+ T cells and promotes the 3′-untranslated region stabilization of IFNG mRNA in NK cells. Further, the inhibition of MAPK14 in Th1 cells differentiated in vitro blocked the IFNG expression induced by IL12/IL18 and CD3/CD28 stimulation. Previous studies showed that treating cells with SB203580, a specific inhibitor of MAPK14, suppressed the transcriptional activation of the IL2 promoter in T lymphocytes. SMAD regulatory pathways regulate different aspects of immune activation and immune suppression in T cells. For example, TGFB promotes the differentiation of CD4+ T cells into suppressive FOXP3+ T regulatory cells via SMAD activation. BMP-7 regulates MAPK14 expression via SMAD1 signaling not only in tumors and macrophages but also in CD4+ T cells. Activated CD4+ T cells incubated with BMP-7 had lower MAPK14, IFNG, and IL2 expression compared with untreated cells and cells treated with BMP-7 plus follistatin, a natural inhibitor of BMP-7. In agreement with these findings, other studies have also correlated IL2 activation with MAPK14 signaling in T cells. Thus, BMP-7 decreases IFNG and IL2 expression in CD4+ T cells via SMAD1/MAPK14 signaling. [0174] Impacts of BMP-7 expression on cancer and immunotherapy resistance are described in, for example, Cortez, M.A., et al. Nat Commun 11, 4840 (2020), incorporated by reference herein in its entirety.
202424720.3 46 III. Antibodies [0175] Aspects of the disclosure relate to BMP-7-binding proteins. The terms “polypeptide that specifically binds BMP-7” and “BMP-7-binding protein” are used interchangeably herein. In some aspects, the BMP-7-binding protein specifically binds to human BMP-7. [0176] In some aspects, the BMP-7-binding protein is an antibody, an antibody-like molecule, or an antigen-binding fragment thereof. In some aspects, the BMP-7-binding protein is an antibody, a nanobody, a minibody, an scFv fragment, or a Fab fragment. In some aspects, the BMP-7-binding protein is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, or a polyclonal antibody. In some aspects, the BMP-7-binding protein is a monoclonal antibody. In some aspects, the BMP-7-binding protein is a murine antibody. In some aspects, the BMP-7-binding protein is a chimeric antibody. In some aspects, the BMP-7- binding protein is a humanized antibody. [0177] In some aspects, the disclosed BMP-7-binding proteins include antibodies comprising a light chain variable region (VL) having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and a heavy chain variable region (VH) having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. [0178] In some aspects, the disclosure relates to a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having 85%, 86%, 87%,
202424720.3 47 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7). [0179] Some aspects are directed to an antibody comprising a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. Some aspects are directed to an antibody comprising a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6. Some aspects are directed to an antibody comprising a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24. Some aspects are directed to an antibody comprising a VL comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57. Some aspects are directed to an antibody comprising a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71. [0180] Some aspects are directed to an antibody comprising a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-
202424720.3 48 H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. Some aspects are directed to an antibody comprising a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12. Some aspects are directed to an antibody comprising a VH comprising a CDR- H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32. Some aspects are directed to an antibody comprising a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30. Some aspects are directed to an antibody comprising a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65. Some aspects are directed to an antibody comprising a VH comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77. [0181] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0182] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24; and a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30. In some aspects, the VL comprises SEQ ID NO:14. In some aspects, the VH comprises SEQ ID NO:16.
202424720.3 49 [0183] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65. In some aspects, the VL comprises SEQ ID NO:34. In some aspects, the VH comprises SEQ ID NO:40. In some aspects, the VL comprises SEQ ID NO:36. In some aspects, the VH comprises SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:38. In some aspects, the VH comprises SEQ ID NO:42. [0184] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77. In some aspects, the VL comprises SEQ ID NO:79. In some aspects, the VH comprises SEQ ID NO:81. [0185] As used herein, the term “antibody” refers to an intact immunoglobulin of any class or isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen. An isotype refers to the genetic variations or differences in the constant regions of the heavy and light chains of an antibody. In humans, there are five heavy chain isotypes: IgA, IgD, IgG, IgE, and IgM and two light chain isotypes: kappa and lambda. The IgG class is divided into four isotypes: IgG1, IgG2, IgG3 and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice. They share more than 95% homology in the amino acid sequences of the Fc regions but show major differences in the amino acid composition and structure of the hinge region. [0186] The term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody derivative, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, a multi-specific antibody, a DARPin, or a derivative or fragment of each thereof. Also contemplated are antibodies having specificity for more than one antigen or target, including bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and other multispecific antibodies. [0187] As used herein, an “antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. As used herein, the terms “antibody” or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response
202424720.3 50 of an animal, including IgM, IgD, IgG, IgA, IgE, and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen-binding activity. Examples of such include but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region or any portion thereof or at least one portion of a binding protein. In certain aspects, the antibody or antigen binding fragment specifically binds human BMP-7. [0188] In one aspect, the anti-BMP-7 antibody or antigen binding fragment thereof is a neutralizing antibody or antigen-binding fragment thereof. The term “neutralizing” in the context of a BMP-7 neutralizing antibody refers to an antibody that may do one or more of: interfere with the BMP-7 receptor/BMP-7 interaction; reduce the concentration of BMP-7 receptor/BMP-7 interacted species in a subject or a cell; prevent the BMP-7 receptor/BMP-7 interaction in a subject or a cell; and/or reduce the biological function of BMP-7, which may include, but is not limited to, one or more of: promoting tumorigenesis or tumor progression; promoting cell growth and metastases; promoting inflammation, cell differentiation, adhesion, tumorigenesis, migration, invasion, and angiogenesis; inducing phosphorylation and activation of SMAD proteins; or downregulating MAPK14. [0189] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody. An antigen may possess one or more epitopes that can interact with different antibodies. [0190] The term “epitope” refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. Epitope determinants may include a plurality of chemical atoms or groups on an antigen surface, such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In some aspects, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some aspects, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some aspects, at least some such chemical atoms or groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Generally, antibodies specific for a particular target antigen would recognize an epitope on the target antigen within a complex mixture. [0191] The epitope regions of a given polypeptide can be identified using many different epitope mapping techniques well known in the art, including: x-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, and hydrogen-deuterium exchange see, e.g., Epitope Mapping Protocols, (Johan Rockberg and
202424720.3 51 Johan Nilvebrant, Ed., 2018) Humana Press, New York, N.Y. Such techniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol.23:709-715 (1986), each of which is incorporated by reference herein in their entirety. Additionally, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydropathy plots. [0192] The term “immunogenic sequence” means a molecule that includes an amino acid sequence of at least one epitope such that the molecule can stimulate the production of antibodies in an appropriate host. The term “immunogenic composition” means a composition that comprises at least one immunogenic molecule (e.g., an antigen or carbohydrate). [0193] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains, such as antibodies naturally occurring in camelids that may comprise only heavy chains. Antibodies as disclosed herein may be derived solely from a single source or may be “chimeric,” that is, different portions of the antibody may be derived from two different antibodies. For example, for chimeric antibodies, the variable regions may be derived from a rat or murine source, while the constant region is derived from a different animal source, such as a human. The antibodies or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes derivatives, variants, fragments, and muteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol.2013; 4: 302; 2013). [0194] The term “variable region” refers to a portion of the antibody that gives the antibody its specificity for binding antigen. The variable region is typically located at the ends of the heavy and light chains. Variable loops of β-strands, three each on the light (VL) and heavy (VH) chains are responsible for binding to the antigen. These loops are referred to as the “complementarity determining regions” (CDRs). In general, the CDR residues are directly and most substantially involved in influencing antigen binding. [0195] The term “constant region” refers to a portion of the antibody that is identical in all antibodies of the same isotype. The constant region differs in antibodies of different isotypes. [0196] The term “light chain” may describe a full-length light chain or fragments thereof. A full-length light chain has a molecular weight of around 25,000 Daltons and includes a variable region domain (abbreviated herein as VL), and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, identified as kappa (κ) and lambda (λ). The term “VL fragment” means a fragment of the light chain of a monoclonal antibody that
202424720.3 52 includes all or part of the light chain variable region, including CDRs. A VL fragment can further include light chain constant region sequences. The variable region domain of the light chain is at the amino-terminus of the polypeptide. [0197] The term “heavy chain” may describe a full-length heavy chain or fragments thereof. For example, a full-length heavy chain for human IgG1 has a molecular weight of around 50,000 Daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as CH1, CH2, and CH3). The term “VH fragment” means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the heavy chain variable region, including CDRs. A VH fragment can further include heavy chain constant region sequences. The number of heavy chain constant region domains will depend on the isotype. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE and is defined by the heavy chains present of which there are five classifications: mu (µ), delta (d), gamma (γ), alpha (α), or epsilon (ε) chains, respectively. Human IgG has several subtypes, including, IgG1, IgG2, IgG3, and IgG4. A. Types of Antibodies [0198] Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies with specificity to two or more antigens. They may also be fragments (e.g., F(ab’)2, Fab’, Fab, Fv, and the like), including hybrid fragments. An immunoglobulin also includes natural, synthetic, or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins. [0199] The term “monomer” means an antibody containing only one immunoglobulin unit. Monomers are the basic functional units of antibodies. The term “dimer” means an antibody containing two immunoglobulin units attached to one another via constant domains of the antibody heavy chains (the Fc, or fragment crystallizable, region). The complex may be stabilized by a joining (J) chain protein. The term “multimer” means an antibody containing more than two immunoglobulin units attached to one another via constant domains of the antibody heavy chains (the Fc region). The complex may be stabilized by a joining (J) chain protein. [0200] The term “bivalent antibody” means an antibody that comprises two antigen- binding sites. The two binding sites may have the same antigen specificities, or they may be bi-specific, meaning the two antigen-binding sites have different antigen specificities.
202424720.3 53 [0201] Bispecific antibodies are a class of antibodies that have paratopes (i.e., antigen- binding sites) for two or more distinct epitopes. Bispecific antibodies can be biparatopic, wherein a bispecific antibody may specifically recognize a different epitope from the same antigen. Bispecific antibodies can be constructed from a pair of different single domain antibodies termed “nanobodies.” Single domain antibodies may be sourced and modified from cartilaginous fish and camelids. Nanobodies can be joined together by a linker using techniques typical to a person skilled in the art; such methods for selection and joining of nanobodies are described in PCT Publication No. WO2015044386A1, No. WO2010037838A2, and Bever et al., Anal Chem. 86:7875–7882 (2014), each of which are specifically incorporated herein by reference in their entirety. [0202] Bispecific antibodies can be constructed as: a whole IgG, Fab’2, Fab’PEG, a diabody, or alternatively as a single chain variable fragment (scFv). Diabodies and scFvs can be constructed without an Fc region, using only variable domains. Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab’ fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol.79:315- 321 (1990); Kostelny et al., J. Immunol.148:1547-1553 (1992), each of which are specifically incorporated by reference in their entirety. [0203] In certain aspects, the antigen-binding domain may be multispecific or heterospecific by multimerizing with VH and VL region pairs that bind a different antigen. For example, the antibody may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Accordingly, aspects may include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof that are directed to epitopes and to other targets, such as Fc receptors on effector cells. [0204] Multispecific antibodies can be used and directly linked via a short flexible polypeptide chain, using routine methods known in the art. One such example is diabodies that are bivalent, bispecific antibodies with two antigen-binding sites in which the VH and VL domains are expressed on a single polypeptide chain and utilize a linker that is too short to allow for pairing between domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain creating two antigen binding sites. The linker functionality is applicable for aspects of triabodies, tetrabodies, and higher order antibody multimers. (see, e.g., Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001), each of which is incorporated herein by reference in their entirety).
202424720.3 54 [0205] The part of the Fv fragment of an antibody molecule that binds with high specificity to the epitope of the antigen is referred to herein as the “paratope.” The paratope consists of the amino acid residues that contact the epitope of an antigen to facilitate antigen recognition. Each of the two Fv fragments of an antibody is composed of the two variable domains, VH and VL, in dimerized configuration. The primary structure of each of the variable domains includes three hypervariable loops separated by, and flanked by, framework regions (FRs). The hypervariable loops are the regions of highest primary sequences variability among the antibody molecules from any mammal. The term hypervariable loop is sometimes used interchangeably with the term “complementarity determining region” (CDR). The length of the hypervariable loops (or CDRs) varies between antibody molecules. The framework regions of all antibody molecules from a given mammal have high primary sequence similarity/consensus. The consensus of framework regions can be used by one skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) which are interspersed among the framework regions. The hypervariable loops are given identifying names which distinguish their position within the polypeptide, and on which domain they occur. CDRs in the VL domain are identified as L1, L2, and L3, with L1 occurring at the most distal end and L3 occurring closest to the CL domain. The CDRs may also be given the names CDR-L1, CDR-L2, and CDR-L3. The L3 (CDR-L3) is generally the region of highest variability among all antibody molecules produced by a given organism. The CDRs are regions of the polypeptide chain arranged linearly in the primary structure and separated from each other by FRs. The amino terminal (N-terminal) end of the VL chain is named FR1. The region identified as FR2 occurs between L1 and L2 hypervariable loops. FR3 occurs between L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. This structure and nomenclature is repeated for the VH chain, which includes three CDRs identified as H1, H2, and H3, or CDR-H1, CDR-H2 and CDR-H3. The majority of amino acid residues in the variable domains, or Fv fragments (VH and VL), are part of the FRs (approximately 85%). [0206] Several methods have been developed and can be used by one skilled in the art to identify the exact amino acids that constitute each of these regions. This can be done using any of a number of multiple sequence alignment methods and algorithms, which identify the conserved amino acid residues that make up the framework regions, therefore identifying the CDRs that may vary in length but are located between framework regions. Three commonly used methods have been developed for identification of the CDRs of antibodies: Kabat (as described in T. T. Wu and E. A. Kabat, J Exp Med, 132(2): 211–50 (1970)); Chothia (as described in C. Chothia et al., Nature, 342(6252): 877–83 (1989)); and IMGT (as described in
202424720.3 55 M.-P. Lefranc et al., Developmental & Comparative Immunology, 27(1): 55–77 (2003)). These methods each include unique numbering systems for the identification of the amino acid residues that constitute the variable regions. In most antibody molecules, the amino acid residues that actually contact the epitope of the antigen occur in the CDRs, although in some cases, residues within the framework regions contribute to antigen binding. Depending on the type and size of the antigen, different CDR residues may contact the antigen. See Almagro JC. J Mol Recognit.17(2):132-43 (2004), incorporated herein by reference. [0207] One skilled in the art can use any of several methods to determine the paratope of an antibody. These methods include: [0208] 1) Computational predictions of the tertiary structure of the antibody/epitope binding interactions based on the chemical nature of the amino acid sequence of the antibody variable region and composition of the epitope. [0209] 2) Hydrogen-deuterium exchange and mass spectroscopy. [0210] 3) Polypeptide fragmentation and peptide mapping approaches in which one generates multiple overlapping peptide fragments from the full length of the polypeptide and evaluates the binding affinity of these peptides for the epitope. [0211] 4) Antibody Phage Display Library analysis in which the antibody Fab fragment encoding genes of the mammal are expressed by bacteriophage in such a way as to be incorporated into the coat of the phage. This population of Fab expressing phage are then allowed to interact with the antigen which has been immobilized or may be expressed in by a different exogenous expression system. Non-binding Fab fragments are washed away, thereby leaving only the specific binding Fab fragments attached to the antigen. The binding Fab fragments can be readily isolated and the genes which encode them determined. This approach can also be used for smaller regions of the Fab fragment including Fv fragments or specific VH and VL domains as appropriate. [0212] In certain aspects, affinity matured antibodies are enhanced with one or more modifications in one or more CDRs thereof (and/or one or more FRs thereof) that result in an improvement in the affinity of the antibody for a target antigen as compared to a parent antibody that does not possess those alteration(s). Certain affinity matured antibodies will have nanomolar or picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art, e.g., Marks et al., Bio/Technology 10:779 (1992) describes affinity maturation by VH and VL domain shuffling, random mutagenesis of CDR and/or framework residues employed in phage display is described by Rajpal et al., PNAS.24: 8466-8471 (2005) and Thie et al., Methods Mol Biol.525:309-22 (2009) in conjugation with
202424720.3 56 computation methods as demonstrated in Tiller et al., Front. Immunol.8:986 (2017), each of which references are incorporated herein by reference in their entirety. [0213] Chimeric immunoglobulins are the products of fused genes derived from different species (the various domains of the antibodies’ heavy and light chains are coded for by DNA from more than one species; see, e.g., U.S. Pat. No. 4,816,567); “humanized” antibodies generally have the FRs from human immunoglobulins and one or more CDRs are from a non- human source (e.g., murine). [0214] As used herein, the term “humanized antibody” or “humanized immunoglobulin” refers to a human/non-human chimeric antibody that contains a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a variable region of the recipient are replaced by residues from a variable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity and capacity. Humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. The humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, a non-human antibody containing one or more amino acids in a framework region, a constant region or a CDR, that have been substituted with a correspondingly positioned amino acid from a human antibody. In general, humanized antibodies are expected to produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody. The humanized antibodies may have conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions. Conservative substitutions groupings include: glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine and asparagine- glutamine. Humanization or engineering of antibodies can be performed using any known method such as, but not limited to, those described in U.S. Pat. Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567; each incorporated by reference herein in its entirety. [0215] Minimizing the antibody polypeptide sequence from the non-human species can optimize chimeric antibody function and reduces immunogenicity. Specific amino acid residues of the non-human antibody are modified to be homologous to corresponding residues in a human antibody. One example is the “CDR-grafted” antibody, in which an antibody comprises one or more CDRs from a particular species or belonging to a specific antibody class
202424720.3 57 or subclass, while the remainder of the antibody chain(s) is identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. In some instances, corresponding non-human (e.g., murine) residues replace framework region residues of the human immunoglobulin. Replacement of human framework region residues with non-human framework region residues may serve to improve and/or restore antigen binding. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody to further refine performance. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol.2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239:1534-36 (1988); Almagro et al., Front Immunol 8; 1751 (2018); and Payes et al., “Genetic Engineering of Antibody Molecules,” In: Reviews in Cell Biology and Molecular Medicine. John Wiley & Sons, Inc., Hoboken, New Jersey, USA. 1(3): 1-52 (2015), each of which is incorporated by reference herein in its entirety. [0216] Intrabodies are intracellularly localized immunoglobulins that bind to intracellular antigens as opposed to secreted antibodies, which bind antigens in the extracellular space. [0217] Antibodies also include “linear antibodies.” The procedure for making linear antibodies is known in the art and described in Zapata et al., 1995. Briefly, these antibodies comprise a pair of tandem Ed segments (VH-CH1-VH-CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. [0218] The terms “polyclonal antibody” or “polyclonal antibody composition” as used herein refer to a preparation of antibodies that are derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope. Thus, polyclonal antibody preparations typically include different antibodies against different determinants (epitopes). In order to produce polyclonal antibodies, a host, such as a rabbit or goat, is immunized with the antigen or antigen fragment, generally with an adjuvant and, if necessary, coupled to a carrier. Antibodies to the antigen are subsequently collected from the sera of the host. The polyclonal antibody can be affinity purified against the antigen rendering it monospecific. [0219] A monoclonal antibody or “mAb” refers to an antibody obtained from a population of substantially homogeneous antibodies from an exclusive parental cell, e.g., the population is identical except for naturally occurring mutations that may be present in minor amounts.
202424720.3 58 Each monoclonal antibody is directed against a single antigenic determinant (epitope). Monoclonal antibodies are highly specific, as each monoclonal antibody is directed against a single determinant on the antigen. B. Functional Antibody Fragments and Antigen-Binding Fragments 1. Antigen-Binding Fragments [0220] Certain aspects relate to antibody fragments, such as antibody fragments that bind to antigen. The term functional antibody fragment includes antigen-binding fragments of an antibody that retain the ability to specifically bind to an antigen. These fragments are constituted of various arrangements of the variable region heavy chain (VH) and/or light chain (VL) and can include constant region heavy chain 1 (CH1) and light chain (CL). In some aspects, they lack the Fc region constituted of heavy chain 2 (CH2) and 3 (CH3) domains. Aspects of antigen binding fragments and the modifications thereof may include: (i) the Fab fragment type constituted with the VL, VH, CL, and CH1 domains; (ii) the Fd fragment type constituted with the VH and CH1 domains; (iii) the Fv fragment type constituted with the VH and VL domains; (iv) the single domain fragment type, dAb, (Holt et al. Trends Biotechnol. 21(11):484-90 (2003)) constituted with a single VH or VL domain; (v) isolated complementarity determining region (CDR) regions. Such terms are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, N.Y. (1990); Antibodies, 4:259-277 (2015), each of which are incorporated by reference in their entirety. [0221] Antigen-binding fragments also include fragments of an antibody that retain exactly, at least, or at most 1, 2, or 3 CDRs from a light chain variable region. Fusions of CDR- containing sequences to an Fc region (or a CH2 or CH3 region thereof) are included within the scope of this definition including, for example, scFv fused, directly or indirectly, to an Fc region are included herein. [0222] The term Fab fragment means a monovalent antigen-binding fragment of an antibody containing the variable (VL and VH) and the constant (CL and CH1) domains. The term Fab’ fragment means a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab’ fragment includes the VL, VH, CL and CH1
202424720.3 59 domains and all or part of the hinge region. The term F(ab’)2 fragment means a bivalent antigen-binding fragment of a monoclonal antibody comprising two Fab’ fragments linked by a disulfide bridge at the hinge region. An F(ab’)2 fragment includes, for example, all or part of the two VH and VL domains and can further include all or part of the two CL and CH1 domains. [0223] The term Fd fragment means a fragment of the heavy chain of a monoclonal antibody, which includes all or part of the VH, including the CDRs. An Fd fragment can further include CH1 region sequences. [0224] The term Fv fragment means a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of the VL and VH, and absent of the CL and CH1 domains. The VL and VH include, for example, the CDRs. Single-chain antibodies (sFv or scFv) are Fv molecules in which the VL and VH regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding fragment. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are herein incorporated by reference. The term (scFv)2 means bivalent or bispecific sFv polypeptide chains that include oligomerization domains at their C-termini, separated from the sFv by a hinge region. The oligomerization domain comprises self-associating α-helices, e.g., leucine zippers, which can be further stabilized by additional disulfide bonds. (scFv)2 fragments are also known as “miniantibodies” or “minibodies.” [0225] A single domain antibody is an antigen-binding fragment containing only a VH or the VL domain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens. 2. Fragment Crystallizable (Fc) Region [0226] An Fc region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing a hinge region that promotes dimerization are included. 3. Cell Surface Receptors
202424720.3 60 [0227] Antigen-binding proteins of the present disclosure may be expressed on the surface of a cell. In some aspects, antigen-binding proteins are cell surface receptors comprising antigen binding domains (e.g., BMP-7-binding domains) disclosed herein. In some aspects, described herein are cell surface receptors comprising a BMP-7-binding domain and one or more additional components or domains. Examples of cell surface receptors of the present disclosure include chimeric antigen receptor (CARs) and T-cell receptors (TCRs). A BMP-7- specific cell surface receptor may comprise one or more of an antigen binding domain, a signal peptide, an extracellular spacer, a transmembrane domain, a cytoplasmic region, and a linker. In some aspects, a cell surface receptor of the present disclosure comprises an antigen binding domain having or comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with one or more of SEQ ID NOs:1-16 and 19-81. Cells expressing a BMP-7-specific cell surface receptor may be useful in treating one or more BMP-7-associated conditions, as described elsewhere herein. C. Polypeptides with Antibody CDRs & Scaffolding Domains that Display the CDRs [0228] Antigen-binding peptide scaffolds, such as CDRs, are used to generate protein- binding molecules in accordance with the aspects. Generally, a person skilled in the art can determine the type of protein scaffold on which to graft at least one of the CDRs. It is known that scaffolds, optimally, must meet a number of criteria such as: good phylogenetic conservation; known three-dimensional structure; small size; few or no post-transcriptional modifications; and/or be easy to produce, express, and purify. Skerra, J Mol Recognit, 13:167- 87 (2000). [0229] The protein scaffolds can be sourced from but are not limited to fibronectin type III FN3 domain (known as “monobodies”), fibronectin type III domain 10, lipocalin, anticalin, Z- domain of protein A of Staphylococcus aureus, thioredoxin A or proteins with a repeated motif such as the “ankyrin repeat”, the “armadillo repeat”, the “leucine-rich repeat” and the “tetratricopeptide repeat”. Such proteins are described in US Patent Publication Nos. 2010/0285564, 2006/0058510, 2006/0088908, 2005/0106660, and PCT Publication No. WO2006/056464, each of which are specifically incorporated herein by reference in their entirety. Scaffolds derived from toxins from scorpions, insects, plants, mollusks, etc., and the protein inhibiters of neuronal nitric oxide synthase (PIN) may also be used. D. Antibody Binding
202424720.3 61 [0230] The term “selective-binding agent”, “antigen-binding agent”, or “antigen-binding protein” refers to a molecule that binds to an antigen. Non-limiting examples include antibodies, antigen-binding fragments, scFv, Fab, Fab’, F(ab’)2, single chain antibodies, peptides, peptide fragments and proteins. [0231] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. “Immunologically reactive” means that the selective binding agent or antibody of interest will bind with antigens present in a biological sample. The term “immune complex” refers the combination formed when an antibody or selective binding agent binds to an epitope on an antigen. 1. Affinity/Avidity [0232] The term “affinity” refers the strength with which an antibody or selective binding agent binds an epitope. In antibody binding reactions, this is expressed as the affinity constant (Ka or ka sometimes referred to as the association constant) for any given antibody or selective binding agent. Affinity is measured as a comparison of the binding strength of the antibody to its antigen relative to the binding strength of the antibody to an unrelated amino acid sequence. The terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and/or selective binding agent. [0233] There are several experimental methods that can be used by one skilled in the art to evaluate the binding affinity of any given antibody or selective binding agent for its antigen. This is generally done by measuring the equilibrium dissociation constant (KD or Kd), using the equation KD = koff / kon = [A][B]/[AB]. The term koff is the rate of dissociation between the antibody and antigen per unit time and is related to the concentration of antibody and antigen present in solution in the unbound form at equilibrium. The term kon is the rate of antibody and antigen association per unit time and is related to the concentration of the bound antigen- antibody complex at equilibrium. The units used for measuring the KD are mol/L (molarity, or M), or concentration. The Ka of an antibody is the inverse of the KD and is determined by the equation Ka = 1/ KD. Examples of some experimental methods that can be used to determine the KD value are enzyme-linked immunosorbent assays (ELISA), isothermal titration calorimetry (ITC), fluorescence anisotropy, surface plasmon resonance (SPR), and affinity capillary electrophoresis (ACE).
202424720.3 62 [0234] Antibodies deemed useful in certain aspects may have an equilibrium dissociation constant of at least, at most, exactly, or between (inclusive or exclusive) any two of about 10- 6, 10-7, 10-8, 10-9, 10-10 M, 10-11 M, 10-12 M, or any range derivable therein. These values are reported for antibodies discussed herein and the same assay may be used to evaluate the binding properties of such antibodies. An antibody of the disclosure is said to “specifically bind” its target antigen when the dissociation constant (KD) is about 10-8 M. The antibody specifically binds antigen with “high affinity” when the KD is about 5×10-9 M, and with “very high affinity” when the KD is about 5×10-12 M. 2. Epitope Specificity [0235] The epitope of an antigen is the specific region of the antigen for which an antibody has binding affinity. In the case of protein or polypeptide antigens, the epitope is the specific residues (or specified amino acids or protein segment) that the antibody binds with high affinity. An antibody does not necessarily contact every residue within the protein. Nor does every single amino acid substitution or deletion within a protein necessarily affect binding affinity. For purposes of this specification and the accompanying claims, the terms “epitope” and “antigenic determinant” are used interchangeably to refer to the site on an antigen to which B and/or T cell receptors respond or recognize. Polypeptide epitopes can be formed from both contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a polypeptide. In some aspects, an epitope includes at least 3, for example 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, in a unique spatial conformation. [0236] Epitope specificity of an antibody can be determined in a variety of ways. One approach, for example, involves testing a collection of overlapping peptides of about 15 amino acids spanning the full sequence of the protein and differing in increments of a small number of amino acids (e.g., 3 to 30 amino acids). The peptides are immobilized in separate wells of a microtiter dish. Immobilization can be accomplished, for example, by biotinylating one terminus of the peptides. This process may affect the antibody affinity for the epitope, therefore different samples of the same peptide can be biotinylated at the N and C terminus and immobilized in separate wells for the purposes of comparison. This is useful for identifying end-specific antibodies. Optionally, additional peptides can be included terminating at a particular amino acid of interest. This approach is useful for identifying end-specific antibodies to internal fragments. An antibody or antigen-binding fragment is screened for binding to each
202424720.3 63 of the various peptides. The epitope is defined as a segment of amino acids that is common to all peptides to which the antibody shows high affinity binding. [0237] It also is possible to determine without undue experimentation, whether an antibody has the same specificity as the antibody of this disclosure by determining whether the antibody being tested prevents an antibody of this disclosure from binding the protein or polypeptide with which the antibody is normally reactive. If the antibody being tested competes with the antibody of the disclosure as shown by a decrease in binding by the monoclonal antibody of this disclosure, then it is likely that the two antibodies bind to the same or a closely related epitope. Alternatively, one can pre-incubate the antibody of this disclosure with a protein with which it is normally reactive, and determine if the antibody being tested is inhibited in its ability to bind the antigen. If the antibody being tested is inhibited then, likely, it has the same, or a closely related, epitopic specificity as the antibody of this disclosure. 3. Modification of Antigen-Binding Domains [0238] It is understood that the antibodies of the present disclosure may be modified to yield variant antibodies. Variant antibodies are substantially identical to the antibody polypeptide sequences, or fragments thereof, and still bind the epitopes of the present disclosure. Polypeptide sequences are “substantially identical” when optimally aligned using such programs as Clustal Omega, IGBLAST, GAP, or BESTFIT using default gap weights, they share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity or any range therein. [0239] As discussed herein, minor variations in the amino acid sequences of antibodies or antigen-binding regions thereof are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and most preferably at least 99% sequence identity. In some aspects, conservative amino acid replacements are contemplated. [0240] The variable region of the antibodies of the present disclosure can be modified by mutating amino acid residues within the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in
202424720.3 64 vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions. [0241] Framework modifications can be made to the antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to the corresponding germline sequence. [0242] In addition, the antibodies of the disclosure may be engineered to include modifications within the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Such modifications include, but are not limited to, alterations of the number of cysteine residues in the hinge region to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody (U.S. Pat. No. 5,677,425) and amino acid mutations in the Fc hinge region to decrease the biological half-life of the antibody (U.S. Pat. No.6,165,745). [0243] Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. Certain preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Standard methods to identify protein sequences that fold into a known three-dimensional structure are available to those skilled in the art; Dill and McCallum., Science 338:1042-1046 (2012). Several algorithms for predicting protein structures and the gene sequences that encode these have been developed, and many of these algorithms can be found at the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov/guide/proteins/) and at the Bioinformatics Resource Portal (on the World Wide Web at expasy.org/proteomics). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the disclosure. [0244] It is also contemplated that the antigen-binding domain may be multi-specific or multivalent by multimerizing the antigen-binding domain with VH and VL region pairs that bind either the same antigen (multi-valent) or a different antigen (multi-specific). E. Enzymatic or Chemical Modification of Antibodies
202424720.3 65 [0245] Additionally, the antibodies of the disclosure may be enzymatically or chemically modified to produce further derivatives of the antibodies and antigen binding fragments that are described herein. The term “antibody derivative” can include post-translational modification to the linear polypeptide sequence of the antibody or fragment. The derivatized antibody or fragment thereof may comprise any molecule or substance that imparts a desired property to the antibody or fragment. [0246] The derivatized antibody can comprise, for example, a chemical post-translational modification, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead), a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antibody for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses). An antibody or fragment thereof can be covalently attached to a molecule or substance, such as a labeling moiety or a therapeutic moiety; covalent attachment does not prevent the antibody from generating an anti-idiotypic response. An antibody or fragment thereof can be non-covalently attached to a molecule or substance, such as a labeling moiety or a therapeutic moiety. [0247] Optionally, an antibody or an antigen-binding fragment can be chemically conjugated to, or expressed as, a fusion protein with other proteins. In some aspects, polypeptides may be chemically modified by conjugating or fusing the polypeptide to serum protein, such as human serum albumin, to increase half-life of the resulting molecule. See, e.g., EP 0322094 and EP 0486525. In some aspects, the polypeptides may be conjugated to a diagnostic agent and used diagnostically, for example, to monitor the development or progression of a disease and determine the efficacy of a given treatment regimen. In some aspects, the polypeptides may also be conjugated to a therapeutic agent to provide a therapy in combination with the therapeutic effect of the polypeptide. [0248] In some aspects, disclosed are antibodies and antibody-like molecules that are linked to at least one agent to form an antibody conjugate or payload. To increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides and the like. By contrast, a reporter molecule is defined as any moiety that may be detected
202424720.3 66 using an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands. 1. Post-Translational Modifications [0249] The antigen-binding protein can have or lack one or more post-translational modifications such as myristoylation, palmitoylation, isoprenylation or prenylation, farnesylation, geranylgeranylation, glypiation, acylation, acetylation, formylation, alkylation, methylation, amide bond formation, amidation at C-terminus, arginylation, polyglutamylation, polyglycylation, butyrylation, glycosylation, glycation, polysialylation, malonylation, hydroxylation, iodination, phosphorylation, adenylylation, propionylation, S- glutathionylation, S-nitrosylation, S-sulfenylation (aka S-sulphenylation), succinylation, sulfation, biotinylation, pegylation, SUMOylation, ubiquitination, neddylation, pupylation, disulfide bridges, or racemization. The antigen-binding protein can have reduced or increased amounts of one or more post-translational modifications as compared to the same antigen- binding protein expressed in the cell that is native to the encoded gene. The reduction or increase may be by at least or at most 25, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500% or more (or any range derivable therein). [0250] In some aspects, contemplated are glycosylation variants of antibodies, wherein the number and/or type of glycosylation site(s) has been altered compared to the amino acid sequences of the parent polypeptide. Glycosylation of the polypeptides can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861, incorporated herein by reference). Antibody protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native antibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence will prevent addition of an N-linked carbohydrate chain present in the native polypeptide. For example, the glycosylation can be reduced by the deletion of an Asn or by substituting the Asn
202424720.3 67 with a different amino acid. In other aspects, one or more new N-linked glycosylation sites are created. [0251] Additional antibody variants include cysteine variants, wherein one or more cysteine residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g., serine). Cysteine variants are useful, inter alia, when antibodies must be refolded into a biologically active conformation. Cysteine variants may have fewer cysteine residues than the native antibody and typically have an even number to minimize interactions resulting from unpaired cysteines. [0252] In some aspects, the polypeptides can be pegylated to increase biological half-life by reacting the polypeptide with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the polypeptide. Polypeptide pegylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). Methods for pegylating proteins are known in the art and can be applied to the polypeptides of the disclosure to obtain PEGylated derivatives of antibodies. See, e.g., EP 0154316 and EP 0401384, incorporated herein by reference. In some aspects, the antibody is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyrrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols, and polyvinyl alcohols. As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins. 2. Conjugates [0253] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. “Detectable labels” are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to be detected, and/or further quantified if desired. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for
202424720.3 68 high-throughput screening. The label may be simply detected, or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property. Examples of detectable labels include, but not limited to, radioactive isotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like. Examples of labels are, but not limited to, horseradish peroxidase (HRP), fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, nicotinamide adenine dinucleotide phosphate (NADPH), and α- or ß-galactosidase. [0254] In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce signals include but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases. Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl- coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.). [0255] In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent. [0256] Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker. Suitable binding pairs for use in indirectly
202424720.3 69 linking the fluorescent label to the intermediate include, but are not limited to, antigens/antibodies, e.g., rhodamine/anti-rhodamine, biotin/avidin and biotin/streptavidin. [0257] Antibodies may also be coupled to low molecular weight haptens to increase the sensitivity of the antibody in an assay. The haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See, Harlow and Lane (1988) supra. [0258] Antibody conjugates also include those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme to generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and/or avidin and streptavidin compounds. The uses of such labels are well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241; each incorporated herein by reference. Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light. [0259] Additional suitable conjugated molecules include ribonuclease (RNase), DNase I, an antisense oligonucleotide, an inhibitory RNA molecule such as a siRNA molecule, an immunostimulatory nucleic acid, aptamers, ribozymes, triplex forming molecules, and external guide sequences (e.g., guide RNAs). Aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets, and can bind small molecules, such as ATP (U.S. Pat. No. 5,631,146) and theophiline (U.S. Pat. No.5,580,737), as well as large molecules, such as reverse transcriptase (U.S. Pat. No.5,786,462) and thrombin (U.S. Pat. No.5,543,293). Ribozymes are nucleic acid molecules that can catalyze a chemical reaction, either intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. Triplex forming function nucleic acid molecules can interact with double-stranded or single-stranded nucleic acid by forming a triplex, in which three strands of DNA form a complex dependent on both Watson-Crick and Hoogsteen base- pairing. Triplex molecules can bind target regions with high affinity and specificity. The functional nucleic acid molecules may act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules may possess a de novo activity independent of any other molecules.
202424720.3 70 [0260] The antibodies of the disclosure or antigen-binding regions thereof can also be linked to another functional molecule such as another antibody or ligand for a receptor to generate a bi-specific or multi-specific molecule that binds to at least two or more different binding sites or target molecules. Linking of the antibody to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, can be done, for example, by chemical coupling, genetic fusion, or noncovalent association. Multi- specific molecules can further include a third binding specificity, in addition to the first and second target epitope. [0261] The antibodies or fragments thereof of the present disclosure may be linked to a moiety that is toxic to a cell to which the antibody is bound to form “depleting” antibodies. [0262] The antibodies of the disclosure may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. [0263] The antibodies also can be bound to many different carriers. Thus, this disclosure also provides compositions containing the antibodies and another substance, active or inert. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the disclosure. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation. [0264] In some aspects, contemplated are immunoconjugates comprising an antibody or antigen-binding fragment thereof conjugated (e.g., covalently attached) to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In this way, the agent of interest can be targeted directly to cells bearing the targeted cell surface antigen. The antibody and agent may be associated through non-covalent interactions such as through electrostatic forces, or by covalent bonds. Various linkers, known in the art, can be employed to form the immunoconjugate. Additionally, the immunoconjugate can be provided in the form of a genetic fusion protein. In one aspect, an antibody may be conjugated to various therapeutic substances to target the cell surface antigen. Examples of conjugated agents include, but are not limited to, metal chelate complexes, drugs, toxins, and other effector molecules, such as cytokines,
202424720.3 71 lymphokines, chemokines, immunomodulators, radiosensitizers, asparaginase, carboranes, and radioactive halogens. [0265] In antibody drug conjugates (ADC), an antibody is conjugated to one or more drug moieties through a linker. The ADC may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form antibody- L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody. ADC may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. Alternatively, a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate. [0266] Examples of ADC known to a person skilled in the art are pro-drugs useful for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605-614 (1999); Niculescu- Duvaz and Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278). In contrast, systematic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the target tumor cells (Baldwin et al., Lancet 1:603-5 (1986); Thorpe, “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,” In: Monoclonal Antibodies ‘84: Biological and Clinical Applications, A. Pincera et al., (eds.) pp. 475-506) (1985). Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., Cancer Immunol. Immunother. 21:183-87 (1986)). [0267] In certain aspects, ADCs include covalent or aggregative conjugates of antibodies, or antigen-binding fragments thereof, with other proteins or peptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an antibody polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, e.g., the yeast alpha-factor leader, or a peptide such as an epitope tag (e.g., V5-His). Antibody-containing fusion proteins may comprise peptides added to facilitate purification or identification of the antibody (e.g., poly-His). An antibody
202424720.3 72 polypeptide also can be linked to the FLAG® (Sigma-Aldrich, St. Louis, Mo.) peptide as described in Hopp et al., Bio/Technology 6:1204 (1988), and U.S. Pat. No.5,011,912. [0268] The conjugated agents can be linked to the antibody directly or indirectly, using any of a large number of available methods. Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety (Amon et al., 1985; Hellstrom et al., 1987; Thorpe, 1985; Baldwin et al., 1985; Thorpe et al., 1982). [0269] Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro- 3-6-diphenylglycouril-3 attached to the antibody (U.S. Patent Nos.4,472,509 and 4,938,948, each incorporated herein by reference). [0270] Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. [0271] Conjugates may also be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). [0272] In some aspects, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site, are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No.5,196,066, incorporated herein by reference). [0273] Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region has also been disclosed in the literature (O’Shannessy et al., J. Immunol. Methods 99(2):153-61 (1987)). [0274] Bi-specific and multi-specific molecules can be prepared using methods known in the art. For example, each binding unit of the hi-specific molecule can be generated separately and then conjugated to one another. When the binding molecules are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5’-dithiobis(2-nitroberizoic acid) (DTNB), o-phenylenedimaleimide (oRDM), N-
202424720.3 73 succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohaxane-I-carboxylate (sulfo-SMCC) (Karpovsky et al., 1984; Liu et al., 1985). When the binding molecules are antibodies, they can be conjugated by sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. F. Proteins [0275] In certain aspects the size of a protein or polypeptide (wild-type or modified) may comprise, but is not limited to, at least, at most, exactly, or between (inclusive or exclusive) any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art. [0276] The polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids (e.g., amino acid substitutions) or be at least, at most, exactly, or between (inclusive or exclusive) any two of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with at least, at most, exactly, or between (inclusive or exclusive) any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
202424720.3 74 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, or more contiguous amino acids or nucleic acids, or any range derivable therein, of any of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 53, 57, 59, 62, 65, 67, 69, 71, 73, 75, 77, 79, or 81. [0277] In some aspects, the protein or polypeptide may comprise amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 (or any derivable range therein) of any of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 53, 57, 59, 62, 65, 67, 69, 71, 73, 75, 77, 79, or 81. [0278] In some aspects, the protein or polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 (or any derivable range therein) contiguous amino acids of any of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 53, 57, 59, 62, 65, 67, 69, 71, 73, 75, 77, 79, or 81. [0279] In some aspects, the polypeptide or protein may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 (or any derivable range therein) contiguous amino acids that are at least, at most, exactly, or between (inclusive or exclusive) any two of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with one of any of SEQ ID
202424720.3 75 NOs:2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 53, 57, 59, 62, 65, 67, 69, 71, 73, 75, 77, 79, or 81. [0280] In some aspects there is a polypeptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 of any of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 53, 57, 59, 62, 65, 67, 69, 71, 73, 75, 77, 79, or 81 and comprising at least, at most, exactly, or between (inclusive or exclusive) any two of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 (or any derivable range therein) contiguous amino acids or nucleotides of any of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 50, 53, 57, 59, 62, 65, 67, 69, 71, 73, 75, 77, 79, or 81. [0281] Nucleotide as well as protein, polypeptide, and peptide sequences for various genes have been previously disclosed and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s GENBANK® and GENPEPT® databases (on the World Wide Web at ncbi.nlm.nih.gov) and The Universal Protein Resource (UNIPROT®; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art. [0282] It is contemplated that in compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide per ml. The concentration of polypeptide in a composition can be at least, at most, exactly, or between (inclusive or exclusive) any two of about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein). 1. Sequences [0283] Amino acid sequences from 12 light chain variable region CDRs from the BMP-7- binding proteins (e.g., antibodies) of the present disclosure are provided in SEQ ID NOs:2, 4,
202424720.3 76 6, 20, 22, 24, 50, 53, 57, 67, 69, and 71 as follows and in Table 1: CDR-L1 (SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67), CDR-L2 (SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69), CDR-L3 (SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71). Table 1
Figure imgf000078_0001
[0284] Amino acid sequences from 13 heavy chain variable region CDRs from the BMP- 7-binding proteins (e.g., antibodies) of the present disclosure are provided in SEQ ID NOs:8, 10, 12, 26, 28, 30, 32, 59, 62, 65, 73, 75, and 77 as follows and in Table 2: CDR-H1 (SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73), CDR-H2 (SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75), and CDR-H3 (SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77). Table 2
Figure imgf000078_0002
202424720.3 77
Figure imgf000079_0001
[0285] The amino acid sequence for a first full light chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:14 as follows: [0286] DIVMTQSPSSMYASLGERVTITCKASQDITGYLGWFQQKPGKSPKTLIYRA DRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO:14). [0287] The amino acid sequence for a second full light chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:34 as follows: [0288] DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYY TSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDYSSPPTFGGGTKLEIK (SEQ ID NO:34). [0289] The amino acid sequence for a third full light chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:36 as follows: [0290] DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGQAPKLLIYY TSNRFTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQDYSSPPTFGGGTKLEIK (SEQ ID NO:36). [0291] The amino acid sequence for a fourth full light chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:38 as follows: [0292] DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGQAPKLLIYY TSNRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQDYSSPPTFGGGTKLEIK (SEQ ID NO:38). [0293] The amino acid sequence for a fifth full light chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:79 as follows: [0294] SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYY TSNRFTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPPTFGGGTKLEIK (SEQ ID NO:79). [0295] The amino acid sequence for a first full heavy chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:16 as follows: [0296] SGPELKKPGETVKISCKASGYTFTDSSMHWVKQAPGKGLKWMGWINTET GEPTYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCSRDYDWFAYWGQGTL VTVSA (SEQ ID NO:16).
202424720.3 78 [0297] The amino acid sequence for a second full heavy chain variable region of a BMP- 7-binding protein disclosed herein is provided in SEQ ID NO:40 as follows: [0298] QVQLVQSGSELKKPGASVKVSCKASGYTFTDSSMHWVRQAPGQGLKWM GWINTETGEPTYVDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYDWFAY WGQGTLVTVSS (SEQ ID NO:40). [0299] The amino acid sequence for a third full heavy chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:42 as follows: [0300] QVQLVQSGSELKKPGASVKVSCKASGYTFTDSSMHWVRQAPGQGLEWM GWINTETGEPTYVDDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYDWFAY WGQGTLVTVSS (SEQ ID NO:42). [0301] The amino acid sequence for a fourth full heavy chain variable region of a BMP-7- binding protein disclosed herein is provided in SEQ ID NO:81 as follows: [0302] IQLVQSGPELKKPGETVKISCKASGYTFTDSSMHWVKQAPGKGLKWMG WINTETGEPTYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCSRDYDWFAY WGQGTLVTVSA (SEQ ID NO:81). [0303] In some aspects, the VH and the VL are on the same polypeptide. In some aspects, the VH and the VL are on different polypeptides. [0304] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79. Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody- like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP-7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79. In some aspects, the VH has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 90% identity
202424720.3 79 to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79. In some aspects, the VH has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79. In some aspects, the VH has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79. In some aspects, the VH comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. [0305] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79; and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79; and the VH has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79; and the VH has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79; and the VH has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79; and the VH comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. [0306] Some aspects are directed to antigen-binding proteins (e.g., antibodies, antibody- like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP-7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%,
202424720.3 80 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14; and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16. In some aspects, the VL has at least 85% identity to SEQ ID NO:14, and the VH has at least 85% identity to SEQ ID NO:16. In some aspects, the VL has at least 90% identity to SEQ ID NO:14, and the VH has at least 90% identity to SEQ ID NO:16. In some aspects, the VL has at least 95% identity to SEQ ID NO:14, and the VH has at least 95% identity to SEQ ID NO:16. In some aspects, the VL comprises SEQ ID NO:14, and the VH comprises SEQ ID NO:16. [0307] Some aspects are directed to antigen-binding proteins (e.g., antibodies, antibody- like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP-7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:34; and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:40. In some aspects, the VL has at least 85% identity to SEQ ID NO:34, and the VH has at least 85% identity to SEQ ID NO:40. In some aspects, the VL has at least 90% identity to SEQ ID NO:34, and the VH has at least 90% identity to SEQ ID NO:40. In some aspects, the VL has at least 95% identity to SEQ ID NO:34, and the VH has at least 95% identity to SEQ ID NO:40. In some aspects, the VL comprises SEQ ID NO:34, and the VH comprises SEQ ID NO:40. [0308] Some aspects are directed to antigen-binding proteins (e.g., antibodies, antibody- like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP-7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:36; and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable
202424720.3 81 therein, to SEQ ID NO:42. In some aspects, the VL has at least 85% identity to SEQ ID NO:36, and the VH has at least 85% identity to SEQ ID NO:42. In some aspects, the VL has at least 90% identity to SEQ ID NO:36, and the VH has at least 90% identity to SEQ ID NO:42. In some aspects, the VL has at least 95% identity to SEQ ID NO:36, and the VH has at least 95% identity to SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:36, and the VH comprises SEQ ID NO:42. [0309] Some aspects are directed to antigen-binding proteins (e.g., antibodies, antibody- like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP-7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:38; and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:42. In some aspects, the VL has at least 85% identity to SEQ ID NO:38, and the VH has at least 85% identity to SEQ ID NO:42. In some aspects, the VL has at least 90% identity to SEQ ID NO:38, and the VH has at least 90% identity to SEQ ID NO:42. In some aspects, the VL has at least 95% identity to SEQ ID NO:38, and the VH has at least 95% identity to SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:38, and the VH comprises SEQ ID NO:42. [0310] The amino acid sequence for a full light chain constant region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:44 as follows: [0311] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:44). [0312] The amino acid sequence for a full heavy chain constant region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:46 as follows: [0313] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
202424720.3 82 PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:46). [0314] In some aspects, the CH and the CL are on the same polypeptide. In some aspects, the CH and the CL are on different polypeptides. [0315] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain constant region (CL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:44. Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP-7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen- binding fragment thereof) comprising a heavy chain constant region (CH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:46. In some aspects, the CL has at least 85% identity to SEQ ID NO:44. In some aspects, the CH has at least 85% identity to SEQ ID NO:46. In some aspects, the CL has at least 90% identity to SEQ ID NO:44. In some aspects, the CH has at least 90% identity to SEQ ID NO:46. In some aspects, the CL has at least 95% identity to SEQ ID NO:44. In some aspects, the CH has at least 95% identity to SEQ ID NO:46. In some aspects, the CL comprises SEQ ID NO:44. In some aspects, the CH comprises SEQ ID NO:46. [0316] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain constant region (CL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:44; and a heavy chain constant region (CH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:46. In some aspects, the CL has at least 85% identity to SEQ ID NO:44; and the CH has at least 85% identity to SEQ ID NO:46. In some aspects, the CL has at least 90% identity to SEQ ID NO:44; and the CH has at least 90% identity to SEQ ID NO:46.
202424720.3 83 In some aspects, the CL has at least 95% identity to SEQ ID NO:44; and the CH has at least 95% identity to SEQ ID NO:46. In some aspects, the CL comprises SEQ ID NO:44; and the CH comprises SEQ ID NO:46. [0317] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; and a light chain constant region (CL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:44. Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP-7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:40 or SEQ ID NO:42; and a heavy chain constant region (CH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:46. In some aspects, the VL has at least 85% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; and the CL has at least 85% identity to SEQ ID NO:44. In some aspects, the VH has at least 85% identity to SEQ ID NO:40 or SEQ ID NO:42; and the CH has at least 85% identity to SEQ ID NO:46. In some aspects, the VL has at least 90% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; and the CL has at least 90% identity to SEQ ID NO:44. In some aspects, the VH has at least 90% identity to SEQ ID NO:40 or SEQ ID NO:42; and the CH has at least 90% identity to SEQ ID NO:46. In some aspects, the VL has at least 95% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; and the CL has at least 95% identity to SEQ ID NO:44. In some aspects, the VH has at least 95% identity to SEQ ID NO:40 or SEQ ID NO:42; and the CH has at least 95% identity to SEQ ID NO:46. In some aspects, the VL comprises SEQ ID NO:34, SEQ ID NO:36, or SEQ ID
202424720.3 84 NO:38; and the CL comprises SEQ ID NO:44. In some aspects, the VH comprises SEQ ID NO:40 or SEQ ID NO:42; and the CH comprises SEQ ID NO:46. [0318] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; a light chain constant region (CL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:44; a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:40 or SEQ ID NO:42; and a heavy chain constant region (CH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:46. In some aspects, the VL has at least 85% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; the CL has at least 85% identity to SEQ ID NO:44; the VH has at least 85% identity to SEQ ID NO:40 or SEQ ID NO:42; and the CH has at least 85% identity to SEQ ID NO:46. In some aspects, the VL has at least 90% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; the CL has at least 90% identity to SEQ ID NO:44; the VH has at least 90% identity to SEQ ID NO:40 or SEQ ID NO:42; and the CH has at least 90% identity to SEQ ID NO:46. In some aspects, the VL has at least 95% identity to SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; the CL has at least 95% identity to SEQ ID NO:44; the VH has at least 95% identity to SEQ ID NO:40 or SEQ ID NO:42; and the CH has at least 95% identity to SEQ ID NO:46. In some aspects, the VL comprises SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; the CL comprises SEQ ID NO:44; the VH comprises SEQ ID NO:40 or SEQ ID NO:42; and the CH comprises SEQ ID NO:46. [0319] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or
202424720.3 85 between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID SEQ ID NO:79. Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP-7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:81. In some aspects, the VL has at least 85% identity to SEQ ID NO:79. In some aspects, the VH has at least 85% identity to SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:79. In some aspects, the VH has at least 90% identity to SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:79. In some aspects, the VH has at least 95% identity to SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:79. In some aspects, the VH comprises SEQ ID NO:81. [0320] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a light chain variable region (VL) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:79; and a heavy chain variable region (VH) having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:81. In some aspects, the VL has at least 85% identity to SEQ ID NO:79; and the VH has at least 85% identity to SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:79; and the VH has at least 90% identity to SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:79; and the VH has at least 95% identity to SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:79; and the VH comprises SEQ ID NO:81. [0321] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a CDR-L1 having at least, at most, exactly, or between (inclusive or
202424720.3 86 exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0322] Some aspects are directed to antigen-binding proteins (e.g., antibodies, antibody- like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP-7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 95% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0323] Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:2; a CDR-L2 having 85% sequence identity to SEQ ID NO:4; and a CDR-L3 having 85% sequence identity to SEQ ID NO:6. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to
202424720.3 87 SEQ ID NO:2; a CDR-L2 having 90% sequence identity to SEQ ID NO:4; and a CDR-L3 having 90% sequence identity to SEQ ID NO:6. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:2; a CDR-L2 having 95% sequence identity to SEQ ID NO:4; and a CDR-L3 having 95% sequence identity to SEQ ID NO:6. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:2; a CDR-L2 comprising SEQ ID NO:4; and a CDR-L3 comprising SEQ ID NO:6. [0324] Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:20; a CDR-L2 having 85% sequence identity to SEQ ID NO:22; and a CDR-L3 having 85% sequence identity to SEQ ID NO:24. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:20; a CDR-L2 having 90% sequence identity to SEQ ID NO:22; and a CDR-L3 having 90% sequence identity to SEQ ID NO:24. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:20; a CDR-L2 having 95% sequence identity to SEQ ID NO:22; and a CDR-L3 having 95% sequence identity to SEQ ID NO:24. Some aspects are directed to antigen-binding proteins comprising a CDR- L1 comprising SEQ ID NO:20; a CDR-L2 comprising SEQ ID NO:22; and a CDR-L3 comprising SEQ ID NO:24. [0325] Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:50; a CDR-L2 having 85% sequence identity to SEQ ID NO:53; and a CDR-L3 having 85% sequence identity to SEQ ID NO:57. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:50; a CDR-L2 having 90% sequence identity to SEQ ID NO:53; and a CDR-L3 having 90% sequence identity to SEQ ID NO:57. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:50; a CDR-L2 having 95% sequence identity to SEQ ID NO:53; and a CDR-L3 having 95% sequence identity to SEQ ID NO:57. Some aspects are directed to antigen-binding proteins comprising a CDR- L1 comprising SEQ ID NO:50; a CDR-L2 comprising SEQ ID NO:53; and a CDR-L3 comprising SEQ ID NO:57. [0326] Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:67; a CDR-L2 having 85% sequence identity to SEQ ID NO:69; and a CDR-L3 having 85% sequence identity to SEQ ID NO:71. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:67; a CDR-L2 having 90% sequence identity to SEQ ID NO:69; and a CDR-L3
202424720.3 88 having 90% sequence identity to SEQ ID NO:71. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:67; a CDR-L2 having 95% sequence identity to SEQ ID NO:69; and a CDR-L3 having 95% sequence identity to SEQ ID NO:71. Some aspects are directed to antigen-binding proteins comprising a CDR- L1 comprising SEQ ID NO:67; a CDR-L2 comprising SEQ ID NO:69; and a CDR-L3 comprising SEQ ID NO:71. [0327] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a CDR-H1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0328] Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR- H3 having 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. Some
202424720.3 89 aspects are directed to antigen-binding proteins comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0329] Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:8; a CDR-H2 having 85% sequence identity to SEQ ID NO:10; and a CDR-H3 having 85% sequence identity to SEQ ID NO:12. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:8; a CDR-H2 having 90% sequence identity to SEQ ID NO:10; and a CDR-H3 having 90% sequence identity to SEQ ID NO:12. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:8; a CDR-H2 having 95% sequence identity to SEQ ID NO:10; and a CDR-H3 having 95% sequence identity to SEQ ID NO:12. Some aspects are directed to antigen-binding proteins comprising a CDR- H1 comprising SEQ ID NO:8; a CDR-H2 having 85% comprising SEQ ID NO:10; and a CDR- H3 comprising SEQ ID NO:12. [0330] Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:26; a CDR-H2 having 85% sequence identity to SEQ ID NO:28; and a CDR-H3 having 85% sequence identity to SEQ ID NO:30. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:26; a CDR-H2 having 90% sequence identity to SEQ ID NO:28; and a CDR-H3 having 90% sequence identity to SEQ ID NO:30. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:26; a CDR-H2 having 95% sequence identity to SEQ ID NO:28; and a CDR-H3 having 95% sequence identity to SEQ ID NO:30. Some aspects are directed to antigen-binding proteins comprising a CDR- H1 comprising SEQ ID NO:26; a CDR-H2 comprising SEQ ID NO:28; and a CDR-H3 comprising SEQ ID NO:30. [0331] Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:26; a CDR-H2 having 85% sequence identity to SEQ ID NO:28; and a CDR-H3 having 85% sequence identity to SEQ ID NO:32. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:26; a CDR-H2 having 90% sequence identity to SEQ ID NO:28; and a CDR-H3 having 90% sequence identity to SEQ ID NO:32. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:26; a CDR-H2 having 95% sequence identity to SEQ ID NO:28; and a CDR-H3 having 95% sequence identity
202424720.3 90 to SEQ ID NO:32. Some aspects are directed to antigen-binding proteins comprising a CDR- H1 comprising SEQ ID NO:26; a CDR-H2 comprising SEQ ID NO:28; and a CDR-H3 comprising SEQ ID NO:32. [0332] Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:59; a CDR-H2 having 85% sequence identity to SEQ ID NO:62; and a CDR-H3 having 85% sequence identity to SEQ ID NO:65. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:59; a CDR-H2 having 90% sequence identity to SEQ ID NO:62; and a CDR-H3 having 90% sequence identity to SEQ ID NO:65. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:59; a CDR-H2 having 95% sequence identity to SEQ ID NO:62; and a CDR-H3 having 95% sequence identity to SEQ ID NO:65. Some aspects are directed to antigen-binding proteins comprising a CDR- H1 comprising SEQ ID NO:59; a CDR-H2 comprising SEQ ID NO:62; and a CDR-H3 comprising SEQ ID NO:65. [0333] Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 85% sequence identity to SEQ ID NO:73; a CDR-H2 having 85% sequence identity to SEQ ID NO:75; and a CDR-H3 having 85% sequence identity to SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 90% sequence identity to SEQ ID NO:73; a CDR-H2 having 90% sequence identity to SEQ ID NO:75; and a CDR-H3 having 90% sequence identity to SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR-H1 having 95% sequence identity to SEQ ID NO:73; a CDR-H2 having 95% sequence identity to SEQ ID NO:75; and a CDR-H3 having 95% sequence identity to SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR- H1 comprising SEQ ID NO:73; a CDR-H2 comprising SEQ ID NO:75; and a CDR-H3 comprising SEQ ID NO:77. [0334] Aspects of the present disclosure include antigen-binding proteins (e.g., antibodies, antibody-like molecules, or fragments thereof) (e.g., a bone morphogenetic protein-7 (BMP- 7)-binding protein, or a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof) comprising a CDR-L1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value
202424720.3 91 derivable therein, to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a CDR-H1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0335] Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a CDR-H1 having 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a CDR-H1 having 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having 95% sequence
202424720.3 92 identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having 95% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a CDR-H1 having 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0336] Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:2; a CDR-L2 having 85% sequence identity to SEQ ID NO:4; and a CDR-L3 having 85% sequence identity to SEQ ID NO:6; and a CDR-H1 having 85% sequence identity to SEQ ID NO:8; a CDR-H2 having 85% sequence identity to SEQ ID NO:10; and a CDR-H3 having 85% sequence identity to SEQ ID NO:12. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:2; a CDR-L2 having 90% sequence identity to SEQ ID NO:4; and a CDR-L3 having 90% sequence identity to SEQ ID NO:6; and a CDR-H1 having 90% sequence identity to SEQ ID NO:8; a CDR-H2 having 90% sequence identity to SEQ ID NO:10; and a CDR-H3 having 90% sequence identity to SEQ ID NO:12. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:2; a CDR-L2 having 95% sequence identity to SEQ ID NO:4; and a CDR-L3 having 95% sequence identity to SEQ ID NO:6; and a CDR-H1 having 95% sequence identity to SEQ ID NO:8; a CDR-H2 having 95% sequence identity to SEQ ID NO:10; and a CDR-H3 having 95% sequence identity to SEQ ID NO:12. Some aspects are directed to antigen-binding proteins comprising a CDR- L1 comprising SEQ ID NO:2; a CDR-L2 comprising SEQ ID NO:4; and a CDR-L3 comprising SEQ ID NO:6; and a CDR-H1 comprising SEQ ID NO:8; a CDR-H2 comprising SEQ ID NO:10; and a CDR-H3 comprising SEQ ID NO:12. [0337] Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:20; a CDR-L2 having 85% sequence identity to SEQ ID NO:22; and a CDR-L3 having 85% sequence identity to SEQ ID NO:24; and a CDR-H1 having
202424720.3 93 85% sequence identity to SEQ ID NO:26; a CDR-H2 having 85% sequence identity to SEQ ID NO:28; and a CDR-H3 having 85% sequence identity to SEQ ID NO:30. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:20; a CDR-L2 having 90% sequence identity to SEQ ID NO:22; and a CDR-L3 having 90% sequence identity to SEQ ID NO:24; and a CDR-H1 having 90% sequence identity to SEQ ID NO:26; a CDR-H2 having 90% sequence identity to SEQ ID NO:28; and a CDR- H3 having 90% sequence identity to SEQ ID NO:30. Some aspects are directed to antigen- binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:20; a CDR-L2 having 95% sequence identity to SEQ ID NO:22; and a CDR-L3 having 95% sequence identity to SEQ ID NO:24; and a CDR-H1 having 95% sequence identity to SEQ ID NO:26; a CDR-H2 having 95% sequence identity to SEQ ID NO:28; and a CDR-H3 having 95% sequence identity to SEQ ID NO:30. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:20; a CDR-L2 comprising SEQ ID NO:22; and a CDR-L3 comprising SEQ ID NO:24; and a CDR-H1 comprising SEQ ID NO:26; a CDR-H2 comprising SEQ ID NO:28; and a CDR-H3 comprising SEQ ID NO:30. [0338] Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:20; a CDR-L2 having 85% sequence identity to SEQ ID NO:22; and a CDR-L3 having 85% sequence identity to SEQ ID NO:24; and a CDR-H1 having 85% sequence identity to SEQ ID NO:26; a CDR-H2 having 85% sequence identity to SEQ ID NO:28; and a CDR-H3 having 85% sequence identity to SEQ ID NO:32. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:20; a CDR-L2 having 90% sequence identity to SEQ ID NO:22; and a CDR-L3 having 90% sequence identity to SEQ ID NO:24; and a CDR-H1 having 90% sequence identity to SEQ ID NO:26; a CDR-H2 having 90% sequence identity to SEQ ID NO:28; and a CDR- H3 having 90% sequence identity to SEQ ID NO:32. Some aspects are directed to antigen- binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:20; a CDR-L2 having 95% sequence identity to SEQ ID NO:22; and a CDR-L3 having 95% sequence identity to SEQ ID NO:24; and a CDR-H1 having 95% sequence identity to SEQ ID NO:26; a CDR-H2 having 95% sequence identity to SEQ ID NO:28; and a CDR-H3 having 95% sequence identity to SEQ ID NO:32. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:20; a CDR-L2 comprising SEQ ID NO:22; and a CDR-L3 comprising SEQ ID NO:24; and a CDR-H1 comprising SEQ ID NO:26; a CDR-H2 comprising SEQ ID NO:28; and a CDR-H3 comprising SEQ ID NO:32.
202424720.3 94 [0339] Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:50; a CDR-L2 having 85% sequence identity to SEQ ID NO:53; and a CDR-L3 having 85% sequence identity to SEQ ID NO:57; and a CDR-H1 having 85% sequence identity to SEQ ID NO:59; a CDR-H2 having 85% sequence identity to SEQ ID NO:62; and a CDR-H3 having 85% sequence identity to SEQ ID NO:65. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:50; a CDR-L2 having 90% sequence identity to SEQ ID NO:53; and a CDR-L3 having 90% sequence identity to SEQ ID NO:57; and a CDR-H1 having 90% sequence identity to SEQ ID NO:59; a CDR-H2 having 90% sequence identity to SEQ ID NO:62; and a CDR- H3 having 90% sequence identity to SEQ ID NO:65. Some aspects are directed to antigen- binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:50; a CDR-L2 having 95% sequence identity to SEQ ID NO:53; and a CDR-L3 having 95% sequence identity to SEQ ID NO:57; and a CDR-H1 having 95% sequence identity to SEQ ID NO:59; a CDR-H2 having 95% sequence identity to SEQ ID NO:62; and a CDR-H3 having 95% sequence identity to SEQ ID NO:65. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:50; a CDR-L2 comprising SEQ ID NO:53; and a CDR-L3 comprising SEQ ID NO:57; and a CDR-H1 comprising SEQ ID NO:59; a CDR-H2 comprising SEQ ID NO:62; and a CDR-H3 comprising SEQ ID NO:65. [0340] Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 85% sequence identity to SEQ ID NO:67; a CDR-L2 having 85% sequence identity to SEQ ID NO:69; and a CDR-L3 having 85% sequence identity to SEQ ID NO:71; and a CDR-H1 having 85% sequence identity to SEQ ID NO:73; a CDR-H2 having 85% sequence identity to SEQ ID NO:75; and a CDR-H3 having 85% sequence identity to SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 having 90% sequence identity to SEQ ID NO:67; a CDR-L2 having 90% sequence identity to SEQ ID NO:69; and a CDR-L3 having 90% sequence identity to SEQ ID NO:71; and a CDR-H1 having 90% sequence identity to SEQ ID NO:73; a CDR-H2 having 90% sequence identity to SEQ ID NO:75; and a CDR- H3 having 90% sequence identity to SEQ ID NO:77. Some aspects are directed to antigen- binding proteins comprising a CDR-L1 having 95% sequence identity to SEQ ID NO:67; a CDR-L2 having 95% sequence identity to SEQ ID NO:69; and a CDR-L3 having 95% sequence identity to SEQ ID NO:71; and a CDR-H1 having 95% sequence identity to SEQ ID NO:73; a CDR-H2 having 95% sequence identity to SEQ ID NO:75; and a CDR-H3 having 95% sequence identity to SEQ ID NO:77. Some aspects are directed to antigen-binding proteins comprising a CDR-L1 comprising SEQ ID NO:67; a CDR-L2 comprising SEQ ID
202424720.3 95 NO:69; and a CDR-L3 comprising SEQ ID NO:71; and a CDR-H1 comprising SEQ ID NO:73; a CDR-H2 comprising SEQ ID NO:75; and a CDR-H3 comprising SEQ ID NO:77. [0341] In some aspects, the disclosure relates to a bone morphogenetic protein-7 (BMP-7)- binding protein comprising: a light chain variable region (VL) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. In some aspects, the disclosure relates to a bone morphogenetic protein-7 (BMP-7)-binding protein comprising: a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0342] In some aspects, the disclosure relates to a bone morphogenetic protein-7 (BMP-7)- binding protein comprising: a light chain variable region (VL) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
202424720.3 96 [0343] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0344] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0345] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0346] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. In some
202424720.3 97 aspects, the disclosure relates to a BMP-7-binding protein comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0347] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0348] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0349] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0350] In some aspects, the disclosure relates to a BMP-7-binding protein comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50,
202424720.3 98 SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0351] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0352] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0353] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0354] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 having at least 95% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 95% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0355] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VL comprises a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID
202424720.3 99 NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0356] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0357] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0358] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0359] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 having at least 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0360] In some aspects, the disclosure relates to a BMP-7-binding protein, wherein the VH comprises a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID
202424720.3 100 NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0361] Some aspects are directed to a monoclonal (e.g., humanized) antibody or antigen- binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. Some aspects are directed to a monoclonal (e.g., humanized) antibody or antigen- binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0362] Some aspects are directed to a monoclonal (e.g., humanized) antibody or antigen- binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0363] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID
202424720.3 101 NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0364] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0365] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0366] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID
202424720.3 102 NO:67, SEQ ID NO:69, or SEQ ID NO:71. In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0367] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0368] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0369] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
202424720.3 103 [0370] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof comprising: a VL comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and a VH comprising three CDRs, wherein one or more of the CDRs comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. [0371] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0372] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 85% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 85% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0373] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least 90% sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 90% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0374] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 having at least 95%
202424720.3 104 sequence identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 having at least 95% sequence identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0375] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VL comprises a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. [0376] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0377] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 85% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 85% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 85% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0378] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 90% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 90% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 90% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
202424720.3 105 [0379] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 having at least 95% sequence identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 having at least 95% sequence identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 having at least 95% sequence identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0380] In some aspects, the disclosure relates to a monoclonal (e.g., humanized) antibody or antigen-binding fragment thereof, wherein the VH comprises a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0381] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or any range derivable therein, with SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or any range derivable therein, with SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or any range derivable therein, with SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR- H1 comprising an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or any range derivable therein, with SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or any range derivable therein, with SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising an amino acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or any range derivable therein, with SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
202424720.3 106 [0382] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0383] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0384] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising an amino acid sequence having at least 95%
202424720.3 107 identity with SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0385] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0386] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:2, a CDR- L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:6; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:12. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:2, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:6; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:12. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:2, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:6; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:12. Some aspects are directed to an antibody comprising: a VL comprising a CDR- L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising
202424720.3 108 SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12. [0387] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:2, a CDR- L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:6; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:32. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:2, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:6; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:32. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:2, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:4, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:6; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:8, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:10, and a CDR-H3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:32. Some aspects are directed to an antibody comprising: a VL comprising a CDR- L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32. [0388] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:20, a CDR- L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:22, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:24; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:26, a CDR-H2 comprising an amino acid sequence having at
202424720.3 109 least 85% identity with SEQ ID NO:28, and a CDR-H3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:30. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:20, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:22, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:24; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:26, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:28, and a CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:30. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:20, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:22, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:24; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:26, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:28, and a CDR-H3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:30. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24; and a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30. [0389] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:50, a CDR- L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:53, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:57; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:59, a CDR-H2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:62, and a CDR-H3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:65. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:50, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:53, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:57; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:59, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:62, and a
202424720.3 110 CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:65. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:50, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:53, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:57; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:59, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:62, and a CDR-H3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:65. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65. [0390] Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:67, a CDR- L2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:71; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:75, and a CDR-H3 comprising an amino acid sequence having at least 85% identity with SEQ ID NO:77. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:71; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:75, and a CDR-H3 comprising an amino acid sequence having at least 90% identity with SEQ ID NO:77. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:67, a CDR-L2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:69, and a CDR-L3 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:71; and a VH comprising a CDR-H1 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:73, a CDR-H2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO:75, and a CDR-H3 comprising an amino acid sequence having
202424720.3 111 at least 95% identity with SEQ ID NO:77. Some aspects are directed to an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77. [0391] In some aspects, the antibody VL comprises SEQ ID NO:14. In some aspects, the antibody VH comprises SEQ ID NO:16. G. Nucleic Acids [0392] Some aspects are directed to a nucleic acid encoding for a BMP-7-binding protein. Some aspects are directed to a nucleic acid encoding for a polypeptide or fragment thereof that specifically binds BMP-7. [0393] In some aspects, the nucleic acid encodes the heavy chain variable region and the light chain variable region of the BMP-7-binding protein. In some aspects, the nucleic acid encodes the light chain variable region of the BMP-7-binding protein. In some aspects, the nucleic acid encodes the heavy chain variable region of the BMP-7-binding protein. In some aspects, the nucleic acid encoding the light chain variable region comprises SEQ ID NO:13. In some aspects, the nucleic acid encoding the heavy chain variable region comprises SEQ ID NO:15. In some aspects, a nucleic acid encoding for a BMP-7-binding protein comprises SEQ ID NO:13 and SEQ ID NO:15. [0394] In certain aspects, there are polynucleotide variants having substantial identity to the sequences disclosed herein, e.g., those comprising at least, at most, exactly, or between (inclusive or exclusive) any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 80%, at least 85%, at least 90%, or at least 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide. [0395] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any
202424720.3 112 length. They can be, for example, at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and/or can comprise one or more additional sequences, for example, regulatory sequences, and/or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post- translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide. 1. Sequences [0396] Nucleotide sequences from 12 light chain variable region CDRs from the BMP-7- binding proteins (e.g., antibodies) of the present disclosure are provided in SEQ ID NOs:1, 3, 5, 19, 21, 23, 47-49, 51, 52, 54-56, 66, 68, and 70 as follows and in Table 3: CDR-L1 (SEQ ID NO:1, SEQ ID NO:19, SEQ ID NOs:47-49, or SEQ ID NO:66), CDR-L2 (SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:68), CDR-L3 (SEQ ID NO:5, SEQ ID NO:23, SEQ ID NOs:54-56, or SEQ ID NO: 70). Table 3
Figure imgf000114_0001
202424720.3 113
Figure imgf000115_0001
[0397] Nucleotide sequences from 13 heavy chain variable region CDRs from the BMP-7- binding proteins (e.g., antibodies) of the present disclosure are provided in SEQ ID NOs:7, 9, 11, 25, 27, 29, 31, 58, 60, 61, 63, 64, 72, 74, and 76 as follows and in Table 4: CDR-H1 (SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:58, or SEQ ID NO:72), CDR-H2 (SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:74), and CDR-H3 (SEQ ID NO:11, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO:76). Table 4
Figure imgf000115_0002
[0398] The nucleotide sequence encoding for a first full light chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:13 as follows: [0399] GATATTGTGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAG AGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTACTGGCTATTTAGGCTG
202424720.3 114 GTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAGACAG ATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTAT TCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTAC AGTATGATGAGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC (SEQ ID NO:13). [0400] The nucleotide sequence encoding for a second full light chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:33 as follows: [0401] GATATCCAGATGACCCAGTCTCCATCTAGCCTGAGCGCCAGCGTCGGA GATAGAGTGACCATCACCTGTAAAGCTTCTCAGAGCGTGTCCAACGACGTGGCCT GGTATCAGCAAAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTACACCAGCA ACCGGTTTACAGGCGTGCCTAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACT TCACCCTGACCATCAGCTCCCTGCAGCCTGAGGACTTCGCCACATACTACTGCCA GCAGGACTACAGCAGCCCCCCCACATTCGGCGGCGGAACAAAGCTGGAAATCAA G (SEQ ID NO:33). [0402] The nucleotide sequence encoding for a third full light chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:35 as follows: [0403] GACATCCAGATGACCCAGAGCCCCAGCTCCCTGAGCGCCTCTGTCGGC GACAGAGTGACCATCACCTGTAAAGCCAGCCAGTCCGTGTCCAACGACGTGGCC TGGTATCAGCAAAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTACACCAGC AACCGGTTCACCGGCGTGCCTGATAGATTCAGCGGCTCTGGCAGCGGCACAGAT TTTACACTGACCATCAGCAGCCTGCAGCCTGAGGACTTCGCTACATACTACTGCC AGCAGGACTACAGCTCTCCACCTACCTTCGGCGGAGGAACAAAGCTGGAAATCA AG (SEQ ID NO:35). [0404] The nucleotide sequence encoding for a fourth full light chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:37 as follows: [0405] GATATCCAGATGACCCAGTCCCCATCTAGCCTGAGCGCCTCTGTCGGC GACAGAGTGACCATCACCTGTAAAGCCAGCCAGAGCGTGTCCAACGACGTGGCC TGGTATCAGCAAAAGCCTGGCCAGGCCCCTAAGCTGCTGATCTACTACACCAGC AACCGGTTCACCGGCGTGCCTAGCAGATTCAGCGGCAGCGGATCTGGCACAGAT TTTACACTGACCATCAGCTCCCTGCAGCCTGAGGACTTCGCTACATACTTCTGCC AGCAGGACTACAGCAGCCCCCCCACCTTCGGCGGCGGAACAAAGCTGGAAATCA AG (SEQ ID NO:37). [0406] The nucleotide sequence encoding for a fifth full light chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:78 as follows:
202424720.3 115 [0407] AGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGA GACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCT TGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATACATCCA ATCGCTTCACTGGAGTCCCTGATCGCTTCACTGGCAGTGGGTATGGGACGGATTT CACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAG CAGGATTATAGCTCTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:78). [0408] The nucleotide sequence encoding for a first full heavy chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:15 as follows: [0409] AGTCAGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCC TGCAAGGCTTCTGGTTATACCTTCACAGACTCTTCAATGCACTGGGTGAAGCAGG CTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGC CAACATATGTAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGC CAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATA TTTCTGTTCTAGAGATTACGACTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTC ACTGTCTCCGCAG (SEQ ID NO:15). [0410] The nucleotide sequence encoding for a second full heavy chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:39 as follows: [0411] CAAGTGCAGCTGGTTCAGAGCGGCAGCGAGCTGAAAAAGCCCGGCGC TTCTGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCGACAGCAGCATG CACTGGGTCAGACAGGCCCCTGGACAGGGCCTGAAATGGATGGGCTGGATCAAC ACCGAGACAGGCGAACCTACATACGTGGACGACTTCAAGGGCAGATTCGTGTTC AGCCTGGATACCAGCGTGTCCACCGCCTACCTGCAGATCAGCAGCCTGAAGGCT GAAGATACAGCCGTGTACTTTTGTGCCCGGGACTACGACTGGTTCGCCTATTGGG GCCAGGGAACCCTGGTGACCGTGTCTTCT (SEQ ID NO:39). [0412] The nucleotide sequence encoding for a third full heavy chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:41 as follows: [0413] CAAGTGCAGCTGGTCCAGAGCGGCAGCGAGCTGAAAAAGCCCGGAGC TTCTGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCGACAGCAGCATG CACTGGGTGCGGCAGGCCCCTGGCCAGGGCCTGGAATGGATGGGCTGGATCAAC ACCGAGACAGGCGAGCCTACCTACGTGGACGACTTCAAGGGCAGATTCGTGTTC AGCCTGGACACCAGCGTGTCCACCGCCTACCTGCAGATCAGCAGCCTGAAGGCT GAAGATACAGCCGTGTACTTTTGTGCCAGAGATTACGACTGGTTCGCCTATTGGG GCCAGGGAACACTGGTTACCGTGTCTTCT (SEQ ID NO:41).
202424720.3 116 [0414] The nucleotide sequence encoding for a fourth full heavy chain variable region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:80 as follows: [0415] CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGA GACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTCTTCAATG CACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAAC ACTGAGACTGGTGAGCCAACATATGTAGATGACTTCAAGGGACGGTTTGCCTTCT CTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGA GGACACGGCTACATATTTCTGTTCTAGAGATTACGACTGGTTTGCTTACTGGGGC CAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:80). [0416] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. In some aspects, the nucleic acid comprises SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78. [0417] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. In some aspects, the nucleic acid comprises SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80. [0418] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%,
202424720.3 117 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:13 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:15. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:13 and a nucleotide sequence having at least 85% identity to SEQ ID NO:15. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:13 and a nucleotide sequence having at least 90% identity to SEQ ID NO:15. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:13 and a nucleotide sequence having at least 95% identity to SEQ ID NO:15. In some aspects, the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:13 and a nucleotide sequence comprising SEQ ID NO:15. In some aspects, the nucleic acid comprises SEQ ID NO:13 and SEQ ID NO:15. [0419] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:33 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:39. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:33 and a nucleotide sequence having at least 85% identity to SEQ ID NO:39. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:33 and a nucleotide sequence having at least 90% identity to SEQ ID NO:39. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:33 and a nucleotide sequence having at least 95% identity to SEQ ID NO:39. In some aspects, the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:33 and a nucleotide sequence comprising SEQ ID NO:39. In some aspects, the nucleic acid comprises SEQ ID NO:33 and SEQ ID NO:39. [0420] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:35 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein,
202424720.3 118 to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:35 and a nucleotide sequence having at least 85% identity to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:35 and a nucleotide sequence having at least 90% identity to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:35 and a nucleotide sequence having at least 95% identity to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:35 and a nucleotide sequence comprising SEQ ID NO:41. In some aspects, the nucleic acid comprises SEQ ID NO:35 and SEQ ID NO:41. [0421] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:37 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:37 and a nucleotide sequence having at least 85% identity to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:37 and a nucleotide sequence having at least 90% identity to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:37 and a nucleotide sequence having at least 95% identity to SEQ ID NO:41. In some aspects, the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:37 and a nucleotide sequence comprising SEQ ID NO:41. In some aspects, the nucleic acid comprises SEQ ID NO:37 and SEQ ID NO:41. [0422] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:78 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:78 and a nucleotide sequence having at least 85% identity to SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:78 and a nucleotide sequence having at least 90% identity
202424720.3 119 to SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:78 and a nucleotide sequence having at least 95% identity to SEQ ID NO:80. In some aspects, the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:78 and a nucleotide sequence comprising SEQ ID NO:80. In some aspects, the nucleic acid comprises SEQ ID NO:78 and SEQ ID NO:80. [0423] The nucleotide sequence encoding for a first full light chain constant region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:43 as follows: [0424] AGGACAGTGGCCGCCCCAAGCGTGTTCATCTTTCCCCCTTCCGACGAG CAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTC GGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCC AGGAGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCA CCCTGACACTGAGCAAGGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAG TCACCCATCAGGGGCTGTCATCACCCGTCACTAAGTCATTCAATCGCGGAGAATG C (SEQ ID NO:43). [0425] The nucleotide sequence encoding for a first full heavy chain constant region of a BMP-7-binding protein disclosed herein is provided in SEQ ID NO:45 as follows: [0426] GCCAGCACCAAAGGCCCAAGCGTATTCCCACTGGCCCCTAGCTCTAAA AGCACCAGCGGCGGAACCGCAGCCCTGGGATGTCTGGTCAAGGACTACTTCCCC GAGCCTGTGACCGTGTCTTGGAACAGCGGTGCTCTCACCAGCGGAGTGCACACCT TCCCTGCTGTGCTTCAGTCCTCCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGT GCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC AGCAATACCAAGGTGGATAAAAAGGTCGAGCCTAAGTCCTGCGACAAGACCCAC ACATGCCCTCCTTGCCCCGCCCCTGAGCTGCTGGGCGGCCCCTCCGTGTTTCTGTT CCCCCCTAAACCAAAGGATACACTGATGATCAGCAGAACCCCTGAAGTGACCTG CGTGGTCGTGGACGTGTCTCACGAGGATCCTGAGGTGAAGTTCAACTGGTACGTG GACGGCGTGGAAGTTCATAATGCCAAGACCAAGCCTAGGGAGGAACAGTACAAC AGCACATATAGAGTGGTGTCAGTGCTGACAGTCCTGCACCAGGACTGGCTGAAC GGCAAGGAATACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCTCCTATCGAG AAGACCATTTCTAAGGCCAAAGGACAACCAAGAGAACCCCAGGTGTACACACTG CCTCCTAGCCGCGAAGAGATGACCAAGAACCAGGTGAGTCTGACATGTCTGGTG AAAGGCTTCTACCCTTCTGACATCGCCGTTGAATGGGAGAGCAATGGCCAGCCA GAGAACAACTACAAGACAACACCTCCTGTGCTGGATAGCGACGGCTCTTTTTTCC TGTACAGCAAGCTGACCGTGGATAAGAGCAGATGGCAACAGGGCAACGTGTTCA
202424720.3 120 GCTGTTCTGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAAAGCCTGA GCCTGAGCCCTGGAAAG (SEQ ID NO:45). [0427] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:43 and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:45. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:43 and a nucleotide sequence having at least 85% identity to SEQ ID NO:45. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:43 and a nucleotide sequence having at least 90% identity to SEQ ID NO:45. In some aspects, the nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO:43 and a nucleotide sequence having at least 95% identity to SEQ ID NO:45. In some aspects, the nucleic acid comprises a nucleotide sequence comprising SEQ ID NO:43 and a nucleotide sequence comprising SEQ ID NO:45. In some aspects, the nucleic acid comprises SEQ ID NO:43 and SEQ ID NO:45. [0428] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:43; and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:45. [0429] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range
202424720.3 121 derivable therein, to SEQ ID NO:13; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:15; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:43; and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:45. [0430] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:33; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:39,; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:43; and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:45. [0431] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:35; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:41; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:43; and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:45.
202424720.3 122 [0432] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:37; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:41; a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:43; and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:45. [0433] In some aspects, the nucleic acid comprises a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:78; and a nucleotide sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range derivable therein, to SEQ ID NO:80. IV. Antibody Production A. Antibody Production [0434] Methods for preparing and characterizing antibodies for use in diagnostic and detection assays, for purification, and for use as therapeutics are well known in the art as disclosed in, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745, each incorporated herein by reference (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). These antibodies may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab’)2 fragments, Fab fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof which bind to the antigen in question. In certain aspects, polypeptides, peptides, and proteins
202424720.3 123 and immunogenic fragments thereof can also be synthesized in solution or on a solid support in accordance with conventional techniques. [0435] Unless specified otherwise, the antibodies can be isolated from any suitable biological source, e.g., murine, rat, rabbit, goat, camelid, sheep, or canine. [0436] In an example, a polyclonal antibody is prepared by immunizing an animal with an antigen or a portion thereof and collecting antisera from that immunized animal. The antigen may be altered compared to an antigen sequence found in nature. A variant or altered antigenic peptide or polypeptide can be employed to generate antibodies. Inocula are typically prepared by dispersing the antigenic composition in a physiologically tolerable diluent to form an aqueous composition. Antisera is subsequently collected by methods known in the arts, and the serum may be used as-is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). [0437] Methods of making monoclonal antibodies are also well known in the art (e.g., U.S. Patent 4,196,265, herein incorporated by reference in its entirety for all purposes). Typically, this technique involves immunizing a suitable animal with a selected immunogenic composition, e.g., a purified or partially purified protein, polypeptide, peptide, or domain. Resulting antibody-producing B-cells from the immunized animal, or all dissociated splenocytes, are then induced to fuse with cells from an immortalized cell line to form hybridomas. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing and have high fusion efficiency and enzyme deficiencies that render then incapable of growing in certain selective media that support the growth of only the desired fused cells (hybridomas). Typically, the fusion partner includes a property that allows selection of the resulting hybridomas using specific media. For example, fusion partners can be hypoxanthine/aminopterin/thymidine (HAT)-sensitive. [0438] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Next, selection of hybridomas can be performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. Fusion procedures for making hybridomas, immunization protocols, and techniques for isolation of immunized splenocytes for fusion are known in the art.
202424720.3 124 [0439] For example, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8,653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MIA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 313, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O) or the like, or heteromyelomas, fusion products thereof, or any cell or fusion cell derived there from, or any other suitable cell line as known in the art, with antibody producing cells, such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or any other cells expressing heavy or light chain constant or variable or framework or CDR sequences, either as endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the like or any combination thereof. Antibody producing cells can also be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest. Any other suitable host cell can also be used for expressing-heterologous or endogenous nucleic acid encoding an antibody, specified fragment or variant thereof, of the present disclosure. The fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods. [0440] Other techniques for producing monoclonal antibodies include the viral or oncogenic transformation of B-lymphocytes, a molecular cloning approach may be used to generate a nucleic acid or polypeptide, the selected lymphocyte antibody method (SLAM) (see, e.g., Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996), the preparation of combinatorial immunoglobulin phagemid libraries from RNA isolated from the spleen of the immunized animal and selection of phagemids expressing appropriate antibodies, or producing a cell expressing an antibody from a genomic sequence of the cell comprising a modified immunoglobulin locus using Cre-mediated site-specific recombination (see, e.g., U.S. 6,091,001). [0441] Monoclonal antibodies may be further purified using filtration, centrifugation, and various chromatographic methods such as high-performance liquid chromatography (HPLC). Monoclonal antibodies may be further screened or optimized for properties relating to specificity, avidity, half-life, immunogenicity, binding association, binding disassociation, or
202424720.3 125 overall functional properties relative to being a treatment for infection. Thus, monoclonal antibodies may have alterations in the amino acid sequence of CDRs, including insertions, deletions, or substitutions with a conserved or non-conserved amino acid. [0442] Chimeric, humanized, or primatized antibodies of the present disclosure can be prepared based on the sequence of a reference monoclonal antibody prepared using standard molecular biology techniques. DNA encoding the heavy and light chain immunoglobulins can be obtained from the hybridoma of interest and engineered to contain non-reference (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (U.S. Pat. No. 4,816,567). To create a humanized antibody, the murine CDR regions can be inserted into a human framework using methods known in the art (U.S. Pat. No.5,225,539 and U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,762 and 6,180,370). Similarly, to create a primatized antibody, the murine CDR regions can be inserted into a primate framework using methods known in the art (WO 93/02108 and WO 99/55369). [0443] Techniques for making partially to fully human antibodies are known in the art and any such techniques can be used. According to one aspect, fully human antibody sequences are made in a transgenic mouse which has been engineered to express human heavy and light chain antibody genes. Multiple strains of such transgenic mice have been made which can produce different classes of antibodies. B cells from transgenic mice which are producing a desirable antibody can be fused to make hybridoma cell lines for continuous production of the desired antibody. (See for example, Russel et al., 2000; Gallo et al., 2000; Green, 1999; Yang et al., 1999A; Yang, 1999B; Jakobovits, 1998; Green and Jakobovits, 1998; Jakobovits, 1998; Tsuda et al., 1997; Sherman-Gold, 1997; Mendez et al., 1997; Jakobovits, 1996; Jakobovits, 1995; Mendez et al., 1995; Jakobovits, 1994; Arbones et al., 1994; Jakobovits, 1993; Jakobovits et al., 1993; U.S. Pat. No.6,075,181). [0444] Alternatively, the antibodies of this disclosure can also be modified to create veneered antibodies. Veneered antibodies are those in which the exterior amino acid residues of the antibody of one species are judiciously replaced or “veneered” with those of a second species so that the antibodies of the first species will not be immunogenic in the second species thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein is primarily dependent on the nature of its surface, the immunogenicity of an antibody could be reduced by replacing the exposed residues which differ from those usually found in another mammalian species antibodies. This judicious replacement of exterior residues should have
202424720.3 126 little, or no, effect on the interior domains, or on the interdomain contacts. Thus, ligand binding properties should be unaffected because of alterations which are limited to the variable region framework residues. The process is referred to as “veneering” since only the outer surface or skin of the antibody is altered, the supporting residues remain undisturbed. [0445] The procedure for “veneering” makes use of the available sequence data for human antibody variable domains compiled by Kabat et al. (1987) Sequences of Proteins of Immunological Interest, 4th ed., Bethesda, Md., National Institutes of Health, updates to this database, and other accessible U.S. and foreign databases (both nucleic acid and protein). Non- limiting examples of the methods used to generate veneered antibodies include EP 519596; U.S. Pat. No.6,797,492; and described in Padlan et al., 1991. [0446] Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, cDNA, or the like, display library; e.g., as available from various commercial vendors such as MorphoSys (Martinsreid/Planegg, Del.), BioInvent (Lund, Sweden), Affitech (Oslo, Norway) using methods known in the art. Art known methods are described in the patent literature some of which include U.S. Pat. Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternative methods rely upon immunization of transgenic animals (e.g., SCID mice) (Nguyen et al., 1977; Sandhu et al., 1996); Eren et al., 1998), that can produce a repertoire of human antibodies, as known in the art and/or as described herein. Such techniques, include, but are not limited to, ribosome display (Wanes et al., 1997; Hanes et al., 1998); single cell antibody producing technologies (e,g., selected lymphocyte antibody method (“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al., 1987; Babcook et al., 1996); gel microdroplet and flow cytometry (Powell et al., 1990; Gray et al., 1995; Kenny et al., 1995); B-cell selection (Steenbakkers et al., 1994). [0447] The antibodies of this disclosure can be recovered and purified from recombinant cell cultures by known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be used for purification. [0448] The immunogenicity of a particular immunogen composition can be enhanced using non-specific stimulators of the immune response, known as adjuvants. Adjuvants that may be
202424720.3 127 used include, but are not limited to, interleukin-1 (IL-1), IL-2, IL-4, IL-7, IL-12, γ-interferon (INF-γ), granulocyte-macrophage colony-stimulating factor (GMCSF), Bacillus Calmette- Guérin (BCG), aluminum hydroxide, muramyl dipeptide (MDP) compounds, muramyl tripeptide phosphatidyl ethanolamine (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). Exemplary adjuvants may include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants, and/or aluminum hydroxide adjuvant. In addition to adjuvants, it may be desirable to co-administer biologic response modifiers (BRM), such as but not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/ Mead, NJ), cytokines such as INF-ß, IL-2, or IL-12, or genes encoding proteins involved in immune helper functions, such as B7-1 (CD80) or B7-2 (CD86). A phage-display system can be used to expand antibody molecule populations in vitro. Saiki, et al., Nature 324:163 (1986); Scharf et al., Science 233:1076 (1986); U.S. Pat. Nos. 4,683,195 and 4,683,202; Yang et al., J Mol Biol.254:392 (1995); Barbas, III et al., Methods: Comp. Meth Enzymol. (1995) 8:94; Barbas, III et al., Proc Natl Acad Sci USA 88:7978 (1991). B. Antibody Fragments Production [0449] Antibody fragments that retain the ability to recognize the antigen of interest will also find use herein. Several antibody fragments are known in the art that comprise antigen- binding sites capable of exhibiting immunological binding properties of an intact antibody molecule and can be subsequently modified by methods known in the arts. Functional fragments, including only the variable regions of the heavy and light chains, can also be produced using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as Fv. See, e.g., Inbar et al., Proc. Nat. Acad. Sci. USA 69:2659-2662 (1972); Hochman et al., Biochem.15:2706-2710 (1976); and Ehrlich et al., Biochem.19:4091-4096 (1980). [0450] Single-chain variable fragments (scFvs) may be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VL and VH). scFvs can form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et al., Prot. Eng. 10:423 (1997); Kort et al., Biomol. Eng. 18:95-108 (2001)). By combining different VL- and VH-comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al., Biomol. Eng.18:31-40 (2001)). Antigen-binding
202424720.3 128 fragments are typically produced by recombinant DNA methods known to those skilled in the art. Although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be made as a single chain polypeptide (known as single chain Fv (sFv or scFv); see e.g., Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988). Design criteria include determining the appropriate length to span the distance between the C-terminus of one chain and the N-terminus of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not tend to coil or form secondary structures. Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues and may include glutamic acid and lysine residues inserted to enhance solubility. Antigen-binding fragments are screened for utility in the same manner as intact antibodies. Such fragments include those obtained by N-terminal and/or C-terminal deletions, where the remaining amino acid sequence is substantially identical to the corresponding positions in the naturally occurring sequence deduced, for example, from a full-length cDNA sequence. [0451] Also contemplated herein are non-peptide compounds having properties analogous to those of a template peptide. These types of non-peptide compounds are termed “peptide mimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al., J. Med. Chem.30:1229 (1987). [0452] Also contemplated are “antibody like binding peptidomimetics” (ABiPs), which are peptide-like molecules that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods. These analogs can be peptides, non-peptides or combinations of peptide and non-peptide regions. Fauchere, Adv. Drug Res. 15:29 (1986); Veber and Freidiner, TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference in their entirety for any purpose. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect. Such compounds are often developed with the aid of computerized molecular modeling. Generally, peptidomimetics of the disclosure are proteins that are structurally similar to an antibody displaying a desired biological activity, such as the ability to bind a protein, but have one or more peptide linkages optionally replaced by a linkage selected from: —CH2NH—, —CH2S—, —CH2—CH2—, —CH-CH— (cis and trans), —COCH2—, —CH(OH)CH2—, and —CH2SO— by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used to generate more stable
202424720.3 129 proteins. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide. [0453] Once generated, a phage display library can be used to improve the immunological binding affinity of Fab molecules using known techniques. See, e.g., Figini et al., J. Mol. Biol. 239:68 (1994). The coding sequences for the heavy and light chain portions of the Fab molecules selected from the phage display library can be isolated or synthesized and cloned into any suitable vector or replicon for expression. Any suitable expression system can be used. V. Obtaining Antibodies [0454] In some aspects, there are nucleic acid molecules encoding antibody or antibody- like polypeptides (e.g., heavy or light chain, variable domain only, or full-length). These may be generated by methods known in the art, e.g., isolated from B cells of mice that have been immunized and isolated, phage display, expressed in any suitable recombinant expression system and allowed to assemble to form antibody molecules. A. Expression [0455] The nucleic acid molecules may be used to express large quantities of recombinant antibodies or to produce chimeric antibodies, single chain antibodies, antigen-binding fragments, immunoadhesins, diabodies, bi-specific antibodies, mutated antibodies, and other antibody derivatives. If the nucleic acid molecules are derived from a non-human, non- transgenic animal, the nucleic acid molecules may be used for antibody humanization. 1. Vectors [0456] In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Expression vectors comprising the nucleic acid molecules may encode the heavy chain, light chain, or the antigen- binding portion thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, and/or probes thereof.
202424720.3 130 In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well. [0457] To express the antibodies, or antigen-binding fragments thereof, DNA encoding partial or full-length light and heavy chains are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences. In some aspects, a vector that encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Such sequences and methods of using the same are well known in the art. 2. Expression Systems [0458] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and/or eukaryote-based systems can be employed to produce nucleic acid sequences, or their cognate polypeptides, proteins, and peptides. Commercially and widely available systems include, but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art can express a vector to produce a nucleic acid sequence or its cognate polypeptide using an appropriate expression system. 3. Methods of Gene Transfer [0459] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue, or an organism, as described herein
202424720.3 131 or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No.5,384,253, incorporated herein by reference); by calcium phosphate precipitation; by using DEAE dextran followed by polyethylene glycol; by direct sonic loading; by liposome mediated transfection; by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG-mediated transformation of protoplasts (U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition mediated DNA uptake. Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction. 4. Host Cells [0460] In another aspect, contemplated are the use of host cells into which a polypeptide, nucleic acid, or recombinant expression vector has been introduced. Antibodies and antibody- like molecules can be expressed in a variety of cell types. An expression construct encoding an antibody can be transfected into cells according to a variety of methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. In certain aspects, the antibody expression construct can be placed under control of a promoter that is linked to immune cell (e.g., T cell) activation. Control of antibody expression allows immune cells, such as tumor-targeting immune cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cells themselves and in surrounding endogenous immune cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors and their cognate polypeptides. Host cells which may be used to express antibodies and other antigen-binding proteins of the present disclosure include, for example, murine myeloma cells (e.g., NS0 cells,
202424720.3 132 SP2/0-Ag14 cells), Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK21) cells, human embryonic kidney 293 (HEK293) cells, fibrosarcoma HT-1080 cells, and PER.C6 cells. In some aspects, the cell is an immune cell. In some aspects, the immune cell is a T cell. In some aspects, the immune cell is a B cell. [0461] For stable transfection of mammalian cells, it is known, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. To identify and select these integrants, a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts. [0462] In some aspects, the cells disclosed herein can be used in methods to generate the BMP-7-binding proteins of the disclosure. The methods can comprise culturing the cell under conditions sufficient to express a nucleic acid encoding for a BMP-7-binding protein in the cell. Also contemplated are methods for generating the BMP-7-binding proteins of the disclosure, the method comprising (a) providing a nucleic acid encoding for the polypeptide to a cell, and (b) subjecting the cell to conditions sufficient to express the polypeptide from the nucleic acid. B. Isolation [0463] The nucleic acid molecule encoding either or both of the entire heavy and light chains of an antibody or the variable regions thereof may be obtained from any source that produces antibodies. Methods of isolating mRNA encoding an antibody are well known in the art. The sequences of human heavy and light chain constant region genes are also known in the art. Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed in a cell into which they have been introduced and the antibody isolated. VI. Genetically Modified Cells [0464] Aspects of the disclosure are directed to genetically modified cells having reduced or disrupted expression of one or more bone morphogenetic protein (BMP) receptors. In an aspect, the one or more BMP receptors may be selected from Bone Morphogenetic Protein Receptor Type 1A (BMPR1A), Bone Morphogenetic Protein Receptor Type 1B (BMPR1B), Activin A receptor type I (ACVR1), Activin receptor type 2A (ACVR2A), Activin receptor
202424720.3 133 type 2B (ACVR2B), Bone morphogenetic protein receptor type 2 (BMPR2), or any combination thereof. [0465] The genetically modified cells may be prepared using any standard gene editing method, including, but not limited to CRISPR/Cas gene editing. In an aspect, the genetically modified cells may be prepared by delivering a CRISPR associated (Cas) endonuclease and one or more gRNAs targeting a gene locus encoding a BMP receptor. In an aspect, the genetically modified cells are prepared by delivering a Cas9 endonuclease and two gRNAs targeting a gene locus encoding a BMP receptor (e.g., a BMPR1A gene locus, a BMPR1B gene locus, a ACVR1 gene locus, an ACVR2A gene locus, a ACVR2B gene locus, or a BMPR2 gene locus). Exemplary gRNA spacer sequences targeting these gene loci are provided in Table 5 below. Table 5
Figure imgf000135_0001
[0466] The genetically modified cells may be an immune cell (e.g., a T cell). The genetically modified cells may be further modified to express one or more of the BMP-7
202424720.3 134 binding proteins disclosed herein. The genetically engineered cells may be a CAR-T cell (e.g., a CAR-T cell targeting PD1/PDL1, TIM3, LAG3, and/or CTLA-4). [0467] Further aspects of the disclosure are directed to compositions comprising the genetically modified cells. These compositions may, in certain aspects, further comprise antibodies targeting PD1/PDL1 pathway, anti-TIM3 antibodies, anti-LAG3 antibodies, and/or anti-CTLA-4 antibodies. VII. Therapeutic Methods [0468] Aspects of the disclosure are directed to compositions of the disclosure and methods for therapeutic use. The compositions of the disclosure may be used for in vivo, in vitro, or ex vivo administration. The route of administration of the composition may be, for example, intratumoral, intravenous, intramuscular, intraperitoneal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, through inhalation, or through a combination of two or more routes of administration. A. Cancer Therapy [0469] In some aspects, the disclosed methods comprise administering a cancer therapy to a subject or patient. The cancer therapy may be chosen based on the expression level measurements, alone or in combination with the clinical risk score calculated for the subject. In some aspects, the cancer therapy comprises a local cancer therapy. In some aspects, the cancer therapy excludes a systemic cancer therapy. In some aspects, the cancer therapy excludes a local therapy. In some aspects, the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy. In some aspects, the cancer therapy comprises an immunotherapy, which may be a checkpoint inhibitor therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered. [0470] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain aspects, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In some aspects, the cancer is a Stage I cancer. In some aspects, the cancer is a Stage II cancer. In some aspects, the cancer is a Stage III cancer. In some aspects, the cancer is a Stage IV cancer.
202424720.3 135 [0471] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor,
202424720.3 136 malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin’s disease; hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. [0472] In some aspects, disclosed are methods for treating cancer originating from the lung. In some aspects, the cancer is lung cancer. In some aspects, the cancer is non-small cell lung cancer (NSCLC). In some aspects, disclosed are methods for treating cancer originating from the breast. In some aspects, the cancer is breast cancer. In some aspects, the cancer is triple negative breast cancer. In some aspects, the cancer is a recurrent cancer. In some aspects, the cancer is an immunotherapy-resistant cancer. [0473] Methods may involve the determination, administration, or selection of an appropriate cancer “management regimen” and predicting the outcome of the same. As used herein the phrase “management regimen” refers to a management plan that specifies the type of examination, screening, diagnosis, surveillance, care, and treatment (such as dosage, schedule and/or duration of a treatment) provided to a subject in need thereof (e.g., a subject diagnosed with cancer). [0474] The selected treatment regimen can be an aggressive one which is expected to result in the best clinical outcome (e.g., complete cure of the disease) or a more moderate one which may relieve symptoms of the disease yet may result in incomplete cure of the disease. The type of treatment can include a surgical intervention, administration of a therapeutic drug such as a BMP-7-binding protein, immunotherapy, an exposure to radiation therapy, and/or any combination thereof. The dosage, schedule and duration of treatment can vary, depending on the severity of disease and the selected type of treatment, and those of skill in the art can adjust the type of treatment with the dosage, schedule, and duration of treatment.
202424720.3 137 [0475] Biomarkers like BMP-7 that can predict the efficacy of certain therapeutic regimen and can be used to identify patients who will receive benefit of a conventional single or combined modality therapy before treatment begins or to modify or design a future treatment plan after treatment. In the same way, those patients who do not receive much benefit from such conventional single or combined modality therapy and can offer them alternative treatment(s) may be identified. [0476] It is contemplated that a cancer treatment may include or exclude any of the cancer treatments described herein. Furthermore, aspects of the disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein. In some aspects, the patient is one that has been determined to be resistant to a therapy described herein. In some aspects, the patient is one that has been determined to be sensitive to a therapy described herein. B. Radiotherapy [0477] In some aspects, a radiotherapy, such as ionizing radiation, is administered to a subject. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). A preferred non-limiting example of ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art. [0478] In some aspects, the radiotherapy can comprise external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT). In some aspects, the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy. In some aspects, the internal radiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy. In some aspects, the radiotherapy is administered to a primary tumor. [0479] In some aspects, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some aspects, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some aspects, the amount of ionizing radiation is at least, at most, exactly, or between (inclusive or exclusive) of about 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range
202424720.3 138 therein). In some aspects, the ionizing radiation is administered in at least, at most, exactly, or between (inclusive or exclusive) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any derivable range therein). When more than one dose is administered, the doses may be about 1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein. [0480] In some aspects, the amount of radiotherapy administered to a subject may be presented as a total dose of radiotherapy, which is then administered in fractionated doses. For example, in some aspects, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some aspects, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some aspects, the total dose of radiation is at least, at most, exactly, or between (inclusive or exclusive) of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 Gy (or any derivable range therein). In some aspects, the total dose is administered in fractionated doses of at least, at most, exactly, or between (inclusive or exclusive) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein). In some aspects, at least, at most, exactly, or between (inclusive or exclusive) of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractionated doses are administered (or any derivable range therein). In some aspects, at least, at most, exactly, or between (inclusive or exclusive) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day. In some aspects, at least, at most, exactly, or between (inclusive or exclusive) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week. C. Cancer Immunotherapy
202424720.3 139 [0481] In some aspects, the methods comprise administration of a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor- associated antigens (TAAs); they are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes, and cytokines. Various immunotherapies are known in the art, and examples are described below. 1. Checkpoint Inhibitors and Combination Treatment [0482] Aspects of the disclosure may include administration of immune checkpoint inhibitors, examples of which are further described below. As disclosed herein, “checkpoint inhibitor therapy” (also “immune checkpoint blockade therapy”, “immune checkpoint therapy”, “ICT,” “checkpoint blockade immunotherapy,” or “CBI”), refers to cancer therapy comprising providing one or more immune checkpoint inhibitors to a subject suffering from or suspected of having cancer. a. PD-1, PDL1, and PDL2 inhibitors [0483] PD-1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and/or PDL1 activity. [0484] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7- DC, Btdc, and CD273. In some aspects, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2. [0485] In some aspects, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1
202424720.3 140 and/or PDL2. In another aspect, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7- 1. In another aspect, a PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014/0294898, US2014/022021, and US2011/0008369, all incorporated herein by reference. [0486] In some aspects, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some aspects, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some aspects, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810. [0487] In some aspects, the immune checkpoint inhibitor is a PDL1 inhibitor such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7. [0488] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another aspect, the antibody competes for binding with and/or binds to the same epitope on PD-1, PDL1, or PDL2 as the above-
202424720.3 141 mentioned antibodies. In another aspect, the antibody has at least, at most, exactly, or between (inclusive or exclusive) of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. b. CTLA-4, B7-1, and B7-2 [0489] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the GENBANK® accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off” switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and/or B7-2 activity. In some aspects, the inhibitor blocks the CTLA-4 and B7- 1 interaction. In some aspects, the inhibitor blocks the CTLA-4 and B7-2 interaction. [0490] In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. [0491] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No.6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. WO2001/014424, WO2000/037504, and U.S. Patent No.8,017,114; all incorporated herein by reference.
202424720.3 142 [0492] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and YERVOY®) or antigen binding fragments and variants thereof (see, e.g., WO 01/14424). [0493] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another aspect, the antibody competes for binding with and/or binds to the same epitope on PD-1, B7-1, or B7- 2 as the above- mentioned antibodies. In another aspect, the antibody has at least, at most, exactly, or between (inclusive or exclusive) of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above- mentioned antibodies. c. LAG3 [0494] Another immune checkpoint that can be targeted in the methods provided herein is the lymphocyte-activation gene 3 (LAG3), also known as CD223 and lymphocyte activating 3. The complete mRNA sequence of human LAG3 has the GENBANK® accession number NM_002286. LAG3 is a member of the immunoglobulin superfamily that is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG3’s main ligand is MHC class II, and it negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD-1 and has been reported to play a role in Treg suppressive function. LAG3 also helps maintain CD8+ T cells in a tolerogenic state and, working with PD-1, helps maintain CD8 exhaustion during chronic viral infection. LAG3 is also known to be involved in the maturation and activation of dendritic cells. Inhibitors of the disclosure may block one or more functions of LAG3 activity. [0495] In some aspects, the immune checkpoint inhibitor is an anti-LAG3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. [0496] Anti-human-LAG3 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-LAG3 antibodies can be used. For example, the anti-LAG3 antibodies can include: GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017, or GSK2831781. The anti-LAG3 antibodies disclosed in: US 9,505,839
202424720.3 143 (BMS-986016, also known as relatlimab); US 10,711,060 (IMP-701, also known as LAG525); US 9,244,059 (IMP731, also known as H5L7BW); US 10,344,089 (25F7, also known as LAG3.1); WO 2016/028672 (MK-4280, also known as 28G-10); WO 2017/019894 (BAP050); Burova E., et al., J. ImmunoTherapy Cancer, 2016; 4(Supp.1):P195 (REGN3767); Yu, X., et al., mAbs, 2019; 11:6 (LBL-007) can be used in the methods disclosed herein. These and other anti-LAG-3 antibodies useful in the claimed invention can be found in, for example: WO 2016/028672, WO 2017/106129, WO 2017062888, WO 2009/044273, WO 2018/069500, WO 2016/126858, WO 2014/179664, WO 2016/200782, WO 2015/200119, WO 2017/019846, WO 2017/198741, WO 2017/220555, WO 2017/220569, WO 2018/071500, WO 2017/015560; WO 2017/025498, WO 2017/087589 , WO 2017/087901, WO 2018/083087, WO 2017/149143, WO 2017/219995, US 2017/0260271, WO 2017/086367, WO 2017/086419, WO 2018/034227, and WO 2014/140180. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 also can be used. [0497] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-LAG3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-LAG3 antibody. In another aspect, the antibody has at least, at most, exactly, or between (inclusive or exclusive) of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. d. TIM-3 [0498] Another immune checkpoint that can be targeted in the methods provided herein is the T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2) and CD366. The complete mRNA sequence of human TIM-3 has the GENBANK® accession number NM_032782. TIM-3 is found on the surface IFNγ-producing CD4+ Th1 and CD8+ Tc1 cells. The extracellular region of TIM-3 consists of a membrane distal single variable immunoglobulin domain (IgV) and a glycosylated mucin domain of variable length located closer to the membrane. TIM-3 is an immune checkpoint and, together with other inhibitory receptors including PD-1 and LAG3, it mediates the T-cell exhaustion. TIM-3 has also been shown as a CD4+ Th1-specific cell surface protein that
202424720.3 144 regulates macrophage activation. Inhibitors of the disclosure may block one or more functions of TIM-3 activity. [0499] In some aspects, the immune checkpoint inhibitor is an anti-TIM-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. [0500] Anti-human-TIM-3 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-TIM-3 antibodies can be used. For example, anti-TIM-3 antibodies including: MBG453, TSR-022 (also known as cobolimab), and LY3321367 can be used in the methods disclosed herein. These and other anti-TIM-3 antibodies useful in the claimed invention can be found in, for example: US 9,605,070, US 8,841,418, US2015/0218274, and US 2016/0200815. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to TIM-3 also can be used. [0501] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-TIM-3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-TIM-3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-TIM-3 antibody. In another aspect, the antibody has at least, at most, exactly, or between (inclusive or exclusive) of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range or value therein) variable region amino acid sequence identity with the above-mentioned antibodies. 2. CAR-T cell therapy [0502] Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy. [0503] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for
202424720.3 145 them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted. [0504] Example CAR-T therapies include Tisagenlecleucel (KYMRIAH) and Axicabtagene ciloleucel (YESCARTA®). [0505] In some aspects, the CAR-T cell therapy comprises CAR-T cells genetically modified to have disrupted or reduced expression of one or more bone morphogenic protein (BMP) receptors. In various aspects, the one or more bone morphogenic protein (BMP) receptors may comprise Bone Morphogenetic Protein Receptor Type 1A (BMPR1A), Bone Morphogenetic Protein Receptor Type 1B (BMPR1B), Activin A receptor type I (ACVR1), Activin receptor type 2A (ACVR2A), Activin receptor type 2B (ACVR2B), Bone morphogenetic protein receptor type 2 (BMPR2), or any combination thereof. In an aspect, the CAR-T cell therapy comprises CAR-T cells having disrupted or reduced expression of a BMPR1A receptor, a BMPR1B receptor or any combination thereof. 3. Inhibition of co-stimulatory molecules [0506] In some aspects, the immunotherapy comprises an inhibitor of a co-stimulatory molecule. In some aspects, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids. 4. Dendritic cell therapy [0507] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.
202424720.3 146 [0508] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and/or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF). [0509] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF. [0510] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor-specific peptide/protein or a tumor cell lysate (a solution of broken-down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response. Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets. 5. Cytokine therapy [0511] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins. [0512] Interferons are produced by the immune system. They are usually involved in anti- viral response, but also have use for cancer. They fall in three groups: type I (IFNα and IFNβ), type II (IFNγ) and type III (IFNλ). [0513] Interleukins have an array of immune system effects. IL-2 is an example interleukin cytokine therapy. 6. Adoptive T-cell therapy
202424720.3 147 [0514] Adoptive T cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically, they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death. [0515] Multiple ways of producing and obtaining tumor targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens. D. Oncolytic virus [0516] In some aspects, the additional therapy comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapy E. Polysaccharides [0517] In some aspects, the additional therapy comprises polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants. F. Neoantigens [0518] In some aspects, the additional therapy comprises neoantigen administration. Many tumors express mutations. These mutations potentially create new targetable antigens
202424720.3 148 (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, as identified using RNA sequencing data, is higher in tumors with a high mutational burden. The level of transcripts associated with cytolytic activity of natural killer cells and T cells positively correlates with mutational load in many human tumors. G. Chemotherapies [0519] In some aspects, the additional therapy comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon-α), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adrenocortical suppressants (e.g., TAXOL® and mitotane). In some aspects, cisplatin is a particularly suitable chemotherapeutic agent. [0520] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg/m2 to about 20 mg/m2 for 5 days every three weeks for a total of three courses being contemplated in certain aspects. In some aspects, the amount of cisplatin delivered to the cell and/or subject in conjunction with the construct comprising an Egr-1 promoter operatively linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.
202424720.3 149 [0521] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., paclitaxel (“TAXOL®”) and doxorubicin hydrochloride (“doxorubicin”). The combination of an Egr-1 promoter/TNFα construct delivered via an adenoviral vector and doxorubicin was determined to be effective in overcoming resistance to chemotherapy and/or TNF-α, which suggests that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-α. [0522] Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain aspects, appropriate intravenous doses for an adult include about 60 mg/m2 to about 75 mg/m2 at about 21-day intervals or about 25 mg/m2 to about 30 mg/m2 on each of 2 or 3 successive days repeated at about 3-week to about 4-week intervals or about 20 mg/m2 once a week. The lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs. [0523] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and/or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria), is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg/kg/day to about 5 mg/kg/day, intravenous doses include, for example, initially about 40 mg/kg to about 50 mg/kg in divided doses over a period of about 2 days to about 5 days or about 10 mg/kg to about 15 mg/kg about every 7 days to about 10 days or about 3 mg/kg to about 5 mg/kg twice a week or about 1.5 mg/kg/day to about 3 mg/kg/day. Because of adverse gastrointestinal effects, the intravenous route is preferred. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities. [0524] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode- oxyuridine; FudR). 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg/m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains. [0525] The amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable aspect, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered
202424720.3 150 with the construct. In other aspects, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000-fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20-fold less, about 500-fold less or even about 5000- fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples. H. Surgery [0526] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present aspects, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery). [0527] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well. I. Other Agents [0528] It is contemplated that other agents may be used in combination with certain aspects of the present aspects to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase
202424720.3 151 the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other aspects, cytostatic or differentiation agents can be used in combination with certain aspects of the present aspects to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present aspects. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present aspects to improve the treatment efficacy. VI. Cancer Treatment [0529] Compositions (e.g., antigen-binding proteins) or methods described herein may be administered to any patient having a condition in which targeting BMP-7 may have therapeutic benefit. Conditions in which targeting BMP-7 may have a therapeutic benefit include, for example, a condition associated with activation of BMP receptors and a condition in which targeting BMP-7 may be used to specifically deliver a therapeutic. Such conditions include, for example, cancer. Thus, aspects of the present disclosure are directed to methods comprising treatment of a subject suffering from, or suspected of having, cancer. In some aspects, the cancer is lung cancer, breast cancer, brain cancer, colon cancer, melanoma, or gastric cancer. In some aspects, the cancer is lung cancer. In some aspects, the lung cancer is non-small cell lung cancer. In certain aspects, the disclosed methods comprise treating a subject who currently has or has previously had resistance to immunotherapy. A subject may be identified as having resistance to immunotherapy using tests and diagnostic methods known in the art. [0530] The therapy provided herein may comprise administration of a therapeutic agent (e.g., a BMP-7-binding protein). In some aspects, therapy provided herein comprises administration of a BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, a vector comprising the nucleic acid encoding for the BMP-7-binding protein, or a cell comprising the BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, or a vector comprising the nucleic acid encoding for the BMP-7-binding protein, and a pharmaceutically acceptable excipient. [0531] In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with a BMP-7-binding protein. As disclosed herein, cancers associated with resistance to immunotherapy are surprisingly and unexpectedly sensitive to treatment with a BMP-7- binding protein. Further, administering a BMP-7-binding protein can decrease
202424720.3 152 immunosuppression of a systemic response to an immunotherapy treatment. Accordingly, in some aspects, disclosed is a method for treating a subject suffering from cancer with a BMP- 7-binding protein. In some aspects, the cancer is a cancer characterized as being resistant to immunotherapy (e.g., a solid tumor cancer). In some aspects, the cancer is lung cancer. In some aspects, the cancer is breast cancer. [0532] In some aspects, the disclosed BMP-7-binding proteins include antibodies comprising a light chain variable region (VL) having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and a heavy chain variable region (VH) having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 90% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. [0533] In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with a monoclonal antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 85% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 85% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at
202424720.3 153 least 90% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 90% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL has at least 95% identity to SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH has at least 95% identity to SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the VL comprises SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:79, and the VH comprises SEQ ID NO:16, SEQ ID NO:40, SEQ ID NO:42, or SEQ ID NO:81. In some aspects, the disclosed monoclonal antibody or antigen-binding fragment thereof is a BMP-7-binding protein (i.e., are capable of binding to BMP-7). [0534] In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71. [0535] In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a
202424720.3 154 VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VH comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77. [0536] In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77. [0537] In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR- H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32. In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24; and a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30. In some aspects, the VL comprises SEQ ID NO:14. In some aspects, the VH comprises SEQ ID NO:16.
202424720.3 155 [0538] In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65. In some aspects, the VL comprises SEQ ID NO:34. In some aspects, the VH comprises SEQ ID NO:40. In some aspects, the VL comprises SEQ ID NO:36. In some aspects, the VH comprises SEQ ID NO:42. In some aspects, the VL comprises SEQ ID NO:38. In some aspects, the VH comprises SEQ ID NO:42. [0539] In some aspects, the disclosed methods comprise treating a subject suffering from a cancer with an antibody comprising: a VL comprising a CDR-L1 comprising SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71; and a VH comprising a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77. In some aspects, the VL comprises SEQ ID NO:79. In some aspects, the VH comprises SEQ ID NO:81. [0540] In some aspects, therapy provided herein comprises administration of a combination of therapeutic agents, such as a BMP-7-binding protein and an additional therapeutic agent, optionally comprised in a composition. In some aspects, the additional therapeutic comprises a biotherapeutic, a chemotherapeutic, an immunotherapeutic, a toxin, an antisense oligonucleotide, a small inhibitory RNA (siRNA), an enzyme, a protein (e.g., a toxin, an enzyme, etc.), a viral vector, or a nanodrug. [0541] In some aspects, the immunotherapeutic comprises a cytotoxic T-lymphocyte- associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor. Accordingly, in some aspects, therapy provided herein comprises administration of a combination of a BMP- 7-binding protein and a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor, optionally comprised in a composition. [0542] In some aspects, the immunotherapeutic comprises a CAR-T cell therapy. The CAR-T cell therapy may be CAR-T cells targeted to one or more tumor markers or antigens that are present in or produced by cancer cells or other cells of the body in response to cancer or certain benign (noncancerous) conditions, where the cancer cells or other cells of the body also express BMP-7.
202424720.3 156 [0543] Non-limiting examples of tumor markers or antigens to which CAR-T cells may be targeted include: EBNA, CD123, HER1, HER2, CA-125, CA 19-9, CA 72-4, CA 15-3\CA 27.29\BCAA, CA-195, CA-242, CA-50, CA LX, MN-CA IX, TRAIL/DR4, CD2, CD5, CD7, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v6, CD47, CD56, CD68/P1, CD70, CD97, CD99, CD123, CD171, CD179, CD200, CD319 (CS1), HLA-G, carcinoembryonic antigen, alphafetoprotein, b-human chorionic gonadotropin, AKT, Her3, epithelial tumor antigen, ROR1, folate binding protein, folate receptor, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, HERV-K, Il-6, IL-11Rα, IL-13Rα, kappa chain, lambda chain, CSPG4, CLL-1, U5snRNP200, BAFF-R, BCMA, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE- A6, MAGE-A10, MAGE-A12, MART-1, glioma-associated antigen, melanoma-associated antigen, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE- 5, GAGE-6, GAGE-7B, pi 5, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, Tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, MUC16, MUC18, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, P16, RU1, RU2, SART-1, SART-3, Wilms’ tumor antigen (WT1), AFP, β- catenin, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, Tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), VEGFR2, cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notch1-4), NY ESO 1, pl85erbB2, pl80erbB-3, c-Met, nm-23H1, beta-HCG, BCA225, BTAA, CAM 17.1, CAM43, L1 CAM, NCAM, NuMa, 43-9F, 791Tgp72, CO-029, FGF-5, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1 , RCASI, SDCCAG16, TA- 90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, EBMA-1, BARF-1, CS-1, ADRB3, thyroglobulin, EVT6-AML, TGS5, plysialic acid, neutrophil elastase, intestinal carboxyl esterase, prostase, prostein, lewisY, LY6K, PAP, OR51
202424720.3 157 E2, PANX3, SSEA-4, TARP, CXORF61, Flt3, TEM1, TEM7R, TSHR, UPK2, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs), and their regulatory subunits, k-ras, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, EphnnB2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, cyclin B1, polysialic acid, M- CSF, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, GPRC5D, GPR20, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, HAVCR1, AKAP-4, SSX2, XAGE 1, B7H3, B7H6, Kit, legumain, TN Ag, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, TSP-180, and LRRN1. Examples of sequences for antigens are known in the art, for example, in the GENBANK® database: CD19 (Accession No. NG_007275.1), EBNA (Accession No. NG_002392.2), WT1 (Accession No. NG_009272.1), CD123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No. NG_007503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No. NG_009272.1), Mage-A3 (Accession No. NG_013244.1), Mage-A4 (Accession No. NG_013245.1), Mage-A10 (Accession No. NC_000023.11), TRAIL/DR4 (Accession No. NC_000003.12), and/or CEA (Accession No. NC_000019.10). [0544] Accordingly, in some aspects, therapy provided herein comprises administration of a BMP-7-binding protein and CAR-T cells targeted to one or more tumor markers or antigens, optionally comprised in a composition. In some aspects, administration of a BMP-7-binding protein with CAR-T cells targeted to one or more tumor markers or antigens improves or increases killing by the CAR-T cells (i.e., apoptosis of cancer cells expressing the markers or antigens to which the CAR-T cells are targeted). As a non-limiting example, in certain aspects, the cancer is pancreatic cancer, the CAR-T cells are targeted to EGFR, and administration of a BMP-7-binding protein with EGFR-targeted CAR-T cells improves or increases killing by the CAR-T cells of the pancreatic cancer cells. As another non-limiting example, in certain aspects, the CAR-T cells are genetically modified to have disrupted or reduced expression of one or more Bone Morphogenetic Protein (BMP) receptors (e.g., a Bone Morphogenetic Protein Receptor Type 1A (BMPR1A), Bone Morphogenetic Protein Receptor Type 1B (BMPR1B), Activin A receptor type I (ACVR1), Activin receptor type 2A (ACVR2A), Activin
202424720.3 158 receptor type 2B (ACVR2B), Bone morphogenetic protein receptor type 2 (BMPR2), or any combination thereof). [0545] In some aspects, the subject was previously treated for the cancer with a cancer therapy. In some aspects, the subject was determined to be resistant to the cancer therapy. In some aspects, the cancer therapy comprises one or more immunotherapies. In some aspects, the one or more immunotherapies comprise a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death- ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor. [0546] In some aspects, the CTLA4 inhibitor is an anti-CTLA4 antibody. In some aspects, the PD1 inhibitor is an anti-PD1 antibody. In some aspects, the PDL-1 inhibitor is an anti-PDL- 1 antibody. In some aspects, the LAG3 inhibitor is an anti-LAG3 antibody. In some aspects, the TIM-3 inhibitor is an anti-TIM-3 antibody. [0547] In some aspects, the cancer is a solid tumor. In some aspects, the cancer is lung cancer, gastric cancer, brain cancer, colon cancer, melanoma, or breast cancer. In some aspects, the cancer is lung cancer. In some aspects, the lung cancer is non-small cell lung cancer. In some aspects, the cancer is breast cancer. In some aspects, the breast cancer is triple-negative breast cancer. [0548] In some aspects, the disclosed methods comprise identifying one or more subjects as being candidates for treatment with a BMP-7-binding protein, based on current or former resistance to treatment with an immunotherapy. For example, in some aspects, disclosed is a method comprising identifying a subject having cancer as being a candidate for treatment with a BMP-7-binding protein by determining that the subject currently has or previously had resistance to treatment with an immunotherapy. In some aspects, the disclosed methods comprise determining an optimal cancer treatment for a subject with resistance to treatment with an immunotherapy. In some aspects, a subject is given multiple types of cancer therapy, for example a cancer immunotherapy and a chemotherapy. [0549] Also disclosed in some aspects are pharmaceutical compositions comprising a BMP-7-binding protein. In some aspects, the pharmaceutical compositions can further comprise one or more additional therapeutics, for example, an immunotherapeutic, including but not limited to the immunotherapeutics disclosed herein. VIII. Administration of Therapeutic Compositions
202424720.3 159 [0550] The therapy provided herein may comprise administration of a combination of therapeutic agents, such as a first cancer therapy (e.g., a BMP-7-binding protein therapy comprising a BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, a vector comprising the nucleic acid encoding for the BMP-7-binding protein, or a cell comprising the BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, or a vector comprising the nucleic acid encoding for the BMP-7-binding protein), and a second cancer therapy (e.g., an immunotherapy, for example, a checkpoint inhibitor therapy). The therapies may be administered in any suitable manner known in the art. For example, the first and second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time or approximately the same time; also “simultaneously” or “substantially simultaneously”). In some aspects, the first and second cancer treatments are administered in a separate composition. In some aspects, the first and second cancer treatments are in the same composition. [0551] In some aspects, the BMP-7-binding protein therapy and the immunotherapy are administered substantially simultaneously. In some aspects, the BMP-7-binding protein therapy and the immunotherapy are administered substantially simultaneously. In some aspects, the BMP-7-binding protein therapy and the immunotherapy are administered sequentially. In some aspects, the BMP-7-binding protein therapy, the immunotherapy, and the chemotherapy are administered sequentially. In some aspects, the BMP-7-binding protein therapy is administered before administering the immunotherapy. In some aspects, the BMP- 7-binding protein therapy is administered after administering the immunotherapy. [0552] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed. [0553] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the cancer therapy is administered intratumorally, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
202424720.3 160 [0554] An effective amount of therapeutic or prophylactic composition is determined based on the intended goal. The treatments may include various “unit doses.” The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, and the particular route and formulation is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose. [0555] In some aspects, the BMP-7-binding protein therapy comprises a BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, a vector comprising the nucleic acid encoding for the BMP-7-binding protein, or a cell comprising the BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, or a vector comprising the nucleic acid encoding for the BMP-7-binding protein. In some aspects, a single dose of the BMP-7-binding protein therapy is administered. In some aspects, multiple doses of the BMP-7-binding protein are administered. In some aspects, the BMP-7-binding protein is administered at a dose of between 1 mg/kg and 5000 mg/kg. In some aspects, the BMP-7-binding protein is administered at a dose that is at least, at most, exactly, or between (inclusive or exclusive) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,
202424720.3 161 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg/kg. [0556] In some aspects, a single dose of the immunotherapy is administered. In some aspects, multiple doses of the immunotherapy are administered. In some aspects, the immunotherapy is administered at a dose of between 1 mg/kg and 100 mg/kg. In some aspects, the immunotherapy is administered at a dose of at least, at most, exactly, or between (inclusive or exclusive) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg/kg. [0557] An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain aspects, it is contemplated that doses include doses of at least, at most, exactly, or between (inclusive or exclusive) of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 µg/kg, mg/kg, µg/day, or mg/day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
202424720.3 162 [0558] In certain aspects, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 µM to 150 µM. In another aspect, the effective dose provides a blood level of about 4 µM to 100 µM.; or about 1 µM to 100 µM; or about 1 µM to 50 µM; or about 1 µM to 40 µM; or about 1 µM to 30 µM; or about 1 µM to 20 µM; or about 1 µM to 10 µM; or about 10 µM to 150 µM; or about 10 µM to 100 µM; or about 10 µM to 50 µM; or about 25 µM to 150 µM; or about 25 µM to 100 µM; or about 25 µM to 50 µM; or about 50 µM to 150 µM; or about 50 µM to 100 µM (or any range derivable therein). In other aspects, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: at least, at most, exactly, or between (inclusive or exclusive) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 µM or any range derivable therein. In certain aspects, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent. [0559] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing. [0560] It will be understood by those skilled in the art and made aware that dosage units of µg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of µg/ml or µM (blood levels), such as 4 µM to 100 µM. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein. [0561] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.
202424720.3 163 [0562] In some aspects, pharmaceutical compositions are administered to a subject. Different aspects may involve administering an effective amount of a composition to a subject. In some aspects, an antibody or antigen binding fragment capable of binding to BMP-7 may be administered to the subject to protect against or treat a condition (e.g., cancer). Alternatively, a nucleic acid or an expression vector encoding one or more such antibodies or polypeptides or peptides may be given to a subject as a preventative treatment. In some aspects, a cell comprising the BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, or a vector comprising the nucleic acid encoding for the BMP-7-binding protein may be administered to the subject to protect against or treat a condition (e.g., cancer). Additionally, such compositions can be administered in combination with an additional therapeutic agent (e.g., a chemotherapeutic, an immunotherapeutic, a biotherapeutic, etc.). Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. [0563] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions. [0564] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified. [0565] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and
202424720.3 164 must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. [0566] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. [0567] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [0568] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof. [0569] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically
202424720.3 165 acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. [0570] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. IX. Kits [0571] Certain aspects of the present disclosure also concern kits containing compositions of the disclosure or compositions to implement methods of the disclosure. In some aspects, kits can be used to evaluate one or more biomarkers. In certain aspects, a kit contains, contains at least, at most, exactly, or between (inclusive or exclusive) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more therapeutic agents, e.g., a BMP-7-binding protein therapy comprising a BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, a vector comprising the nucleic acid encoding for the BMP-7-binding protein, or a cell comprising the BMP-7-binding protein, a nucleic acid encoding for the BMP-7-binding protein, or a vector comprising the nucleic acid encoding for the BMP-7-binding protein, or an immunotherapy, or any value or range and combination derivable therein. In some aspects, there are kits for evaluating biomarker activity in a cell. [0572] Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means. [0573] Individual components may also be provided in a kit in concentrated amounts; in some aspects, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as 1X, 2X, 5X, 10X, or 20X or more. [0574] Kits for using therapeutic agents of the disclosure for prognostic or diagnostic applications are included as part of the disclosure. Specifically contemplated are any such molecules corresponding to any biomarker identified herein, which includes nucleic acid primers/primer sets and probes that are identical to or complementary to all or part of a biomarker, which may include noncoding sequences of the biomarker, as well as coding sequences of the biomarker. In certain aspects, negative and/or positive control nucleic acids, probes, and inhibitors are included in some kit aspects. Aspects of the disclosure include kits
202424720.3 166 for analysis of a pathological sample by assessing biomarker profile for a sample comprising, in suitable container means, two or more biomarker probes, wherein the biomarker probes detect one or more of the biomarkers identified herein. The kit can further comprise reagents for labeling nucleic acids in the sample. The kit may also include labeling reagents, including at least one of amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer. [0575] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different aspects may be combined. The claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims. [0576] Any aspect of the disclosure involving specific therapeutic agents by name is contemplated also to cover aspects involving therapeutic agents whose sequences are at least, at most, exactly, or between (inclusive or exclusive) of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the specified nucleic acid. VII. Examples [0577] The following examples are included to demonstrate aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 – Monoclonal Antibody to BMP-7 to treat resistance to immunotherapies [0578] Antibody generation. Hybridoma cells were provided. Total RNA was extracted from the hybridoma clone cell pellet using RNA extraction protocol (Zymo Research). RT- PCR cDNA was created from the RNA by reverse-transcription with random primers. PCR reactions using variable domain primers to amplify both the VH and VL regions of the monoclonal antibody DNA. The VH and VL products were cloned into the Invitrogen sequencing vector pCR2.1-TOPO (SEQ ID NO:17 corresponding to VL in pCR.2.1-TOPO, SEQ ID NO:18 corresponding to VH in pCR.2.1-TOPO) and transformed into DH5a cells and screened by PCR for positive transformants. Selected colonies were picked and analyzed by
202424720.3 167 DNA sequencing. FIG.1 shows titration of an antibody generated to specifically target human BMP-7 (BMP-7 mAb 344-5-1, having a VL comprising SEQ ID NO:14 and a CDR-L1 comprising SEQ ID NO:2 or SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:4 or SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:6 or SEQ ID NO:24; and having a VH corresponding to SEQ ID NO:16 and a CDR-H1 comprising SEQ ID NO:8 or SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:10 or SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, or SEQ ID NO:32). [0579] Functional validation of BMP-7 mAb antibody. Viable cells were counted with a hemocytometer (0.4% Trypan blue solution) and diluted to 40,000 cells per well in 24-wells plates. RAW 264.7 cells (macrophages) were then cultured in complete medium (RPMI-1640 supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% heat- inactivated fetal bovine serum) and incubated at 37 °C in 5% CO2 for 24 or 48 h, after which cells were treated with 250 ng of BMP-7 (R&D Systems, Catalog #5666-BP-010). RNA was then isolated from RAW 264.7 cells and analyzed for p38 alpha (MAPK14) expression with quantitative PCR. As shown in FIG.2, BMP-7 mAb 344-5-1 treatment produced the highest upregulation of p38 alpha, a marker for Th1 cytokines/chemokines (pro-inflammatory phenotype). Therefore, the specific inhibition of BMP7 by BMP-7 mAb 344-5-1 promotes release of cytokines and chemokines by M1-like macrophages to induce inflammation, which can lead to anti-tumor immune response. [0580] Tumor growth and survival analysis of mice treated twice a week. All mice were bred and maintained in a specific pathogen-free mouse colony. Tumors were established by subcutaneous injection of 344SQR cells (0.5 × 106 in 100 μL of sterile PBS) into the leg of syngeneic 129 Sv/Ev mice (female, 12–16 weeks old). Mice were then given intraperitoneal injections of anti-PD1 or control IgG antibodies (10 mg/kg) (Bio X cell). Mice (129 Sv) with 344SQR tumors were treated with IgG ctrl (n=5) or anti-PD1 (10 mg/kg) (n=5) and/or human BMP-7344-5-1 (10 mg/kg) (n=5) twice a week for 2 weeks, and tumor growth was measured (FIG.3A). Tumors were measured with calipers three times per week and recorded as tumor volume (in mm3) = (width2  × length)/2. Mouse survival rates were analyzed by the Kaplan– Meier method and compared with log-rank tests (FIG.3B). Collectively, these findings suggest that BMP7 inhibition may represent a potential therapeutic approach to overcome resistance to immune checkpoint blockage. [0581] Tumor growth and survival analysis of mice treated three times a week. All mice were bred and maintained in a specific pathogen-free mouse colony. Tumors were established by subcutaneous injection of 344SQR or 4T1 cells (0.5 × 106 in 100 μL of sterile
202424720.3 168 PBS) into the leg of syngeneic 129 Sv/Ev mice (male, 12–16 weeks old). Mice (129 Sv) with 344SQR tumors were treated with IgG ctrl (10 mg/kg) (n=5) or anti-PD1 (10 mg/kg) (n=5) and/or human BMP-7344-5-1 (10 mg/kg) (n=5) three times a week for two weeks, and tumor growth was measured (FIG. 4A). Tumors were measured with calipers three times per week and recorded as tumor volume (in mm3) = (width2  × length)/2. Mouse survival rates were analyzed by the Kaplan–Meier method and compared with log-rank tests (FIG. 4B). BMP-7 inhibition by BMP-7 mAb 344-5-1 was found to re-sensitize tumors to anti-PD1 and extended mouse survival relative to the control group. [0582] Tumor growth analysis of mice treated three times a week. Tumors were established by subcutaneous injection of 344SQR or 4T1 cells (0.5 × 106 in 100 μL of sterile PBS) into the leg of syngeneic 129 Sv/Ev mice or BALB/c mice (female, 12–16 weeks old). Mice (129 Sv) with 344SQR tumors were treated with IgG ctrl (10 mg/kg) (n=5) or anti-PD1 (10 mg/kg) (n=5) and/or human BMP-7344-5-1 (10 mg/kg) (n=5) three times a week until the endpoint, and tumor growth was measured (FIG.5A). Mice (BALB/c) with 4T1 tumors were treated with IgG ctrl (10 mg/kg) (n=5) or anti-PD1 (10 mg/kg) (n=5) and/or human BMP-7 344-5-1 (10 mg/kg) (n=5) three times a week until the endpoint, and tumor growth was measured (FIG.5B). Tumors were measured with calipers three times per week and recorded as tumor volume (in mm3) = (width2  × length)/2. BMP-7 inhibition by BMP-7 mAb 344-5-1 re-sensitizes tumors to anti-PD1 and extends mouse survival relative to the control group. In addition, these results demonstrate that BMP7-knockdown sensitizes 4T1 mouse mammary carcinoma tumors to anti-PD1 therapy and extends mouse survival relative to the control group. Collectively, these findings suggest that BMP7 inhibition may represent a potential therapeutic approach to overcome resistance to immunotherapies. Example 2 –BMP-7 Antibody Humanization [0583] Overview. A mouse monoclonal antibody (mAb) antibody was humanized using a back mutation library method without sacrificing the binding affinity of the BMP-7344-5-1 parent mouse antibody. The following experiments were carried out: (1) confirmation of antigen-antibody interaction; (2) back mutation library design and construction; and (3) selection of humanized antibodies. [0584] Binding confirmation of chimeric antibody by SPR. A chimeric antibody was created from the BMP-7344-5-1 parent antibody and used for BMP-7 antibody humanization. The chimeric antibody has a VL corresponding to SEQ ID NO:79 and a CDR-L1 comprising
202424720.3 169 SEQ ID NO:67, a CDR-L2 comprising SEQ ID NO:69, and a CDR-L3 comprising SEQ ID NO:71; and a VH corresponding to SEQ ID NO:81 and a CDR-H1 comprising SEQ ID NO:73, a CDR-H2 comprising SEQ ID NO:75, and a CDR-H3 comprising SEQ ID NO:77. The affinity of chimeric antibody to antigen was determined using a Surface Plasmon Resonance (SPR) biosensor, BIACORE™ 8K (GE Healthcare). Antigen was immobilized on the CM5 sensor chip. Antibody was used as the analyte. The data of dissociation (kd) and association (ka) rate constants were obtained using BIACORE™ 8K evaluation software. The equilibrium dissociation constants (KD) were calculated from the ratio of kd over ka. [0585] The parameters of affinity validation are shown in Table 6. Table 6
Figure imgf000171_0001
[0586] The binding kinetics of chimeric antibody to antigen are summarized in Table 7. Table 7 Ligand Analyte Chi² (RU²) ka (1/Ms) kd (1/s) KD (M) Rmax (RU) human BMP-7 protein chimeric antibody 1.92E-01 2.25E+04 3.69E-03 1.64E-07 22.7 [0587] Sensor-grams were shown in FIG.6. Real-time responses are shown as gray curves. Fitting of BIACORE™ experimental data to 1:1 interaction model was shown in black. [0588] Fast Screening for Expression, Biophysical Properties, and Affinity (FASEBA) of chimeric Fab and grafted Fab. The affinity of chimeric Fab and grafted Fab to antigen were determined using FASEBA with ELISA. Microtiter ELISA plates were coated with 10 µg/ml BSA (expression detection) and 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.39063, 0.195313, 0.0976563, 0.0488281 µg/ml antigen protein (binding evaluation) in 100 μl PBS at 4 ℃ overnight, and subsequently incubated with blocking buffer at 37 ℃ for 1 hour. Then, the
202424720.3 170 plates were washed with washing buffer and incubated with diluted 50 μl FASEBA supernatant in 50 μl 0.1 % PBST at RT for 2 hours. Next, the plates were washed with washing buffer and incubated with 100 μl secondary antibody for 45 minutes. After washing, the reaction was developed with 100 μl TMB substrate for 10 minutes at room temperature and stopped by adding 50 μl of 1 M HCl. The absorbance values were measured at 450 nm using a spectrometer. [0589] The sample list and results of chimeric Fab FASEBA and grafted Fab FASEBA ELISA assays are shown in Table 8. NC refers to a non-related FASEBA supernatant control and blank refers to 2xYT medium only. The concentration of 13 µg/ml antigen was selected for further FASEBA screening. Table 8
Figure imgf000172_0001
[0590] Generation of deluxe humanized library. The humanized FASEBA screening library containing all back mutants in the inner core of antibody structure were designed as described in design report. The construction of the library was carried out following GenScript ProBio’s standard operating procedures (SOP), described below. [0591] The real size of the constructed FAB library was 7.0x107 cfu. Library in-frame rate and diversity were evaluated by DNA sequencing. A total of 47 clones were sequenced. The qualities are summarized in Tables 9-11. The sequence alignments are summarized in FIGs. 7A (VH) and 7B (VL). Table 9 Fab Fragment Sequencing Results Clone for sequencing Bad sequence In-frame rate Unique clone VH 47 6 41/47=87.23% 13 VL 47 2 45/47=95.74% 15
202424720.3 171 Table 10 VH Fragment Sequencing Analysis Analysis R38K E46K Y95F A97S VH 41 R(31),K(10) E(25),K(16) Y(21),F(20) A(24),S(17) Table 11 VL Fragment Sequencing Analysis Analysis K42Q S60D T85V Y87F VL 45 K(23),Q(22) S(23),D(22) T(22),V(23) Y(22),F(23) [0592] Selection of humanized antibody by FASEBA screening. The humanized Fab library obtained was performed with high throughput FASEBA screening against antigen protein. Two plates of 180 clones were picked for FASEBA screening against antigen protein and BSA. Individual Fab clones were expressed in 96-deep-well plates. The crude protein secreted by E. coli to the medium was assayed by ELISA against BSA and antigen protein for assessment of expression and binding activity, respectively. Microtiter ELISA plates were coated with 10 µg/ml BSA (expression detection) and 13 µg/ml antigen protein (binding evaluation) in 100 μl PBS at 4 ℃ overnight and subsequently incubated with blocking buffer at 37 ℃ for 1 hour. Then, the plates were washed with washing buffer and incubated with diluted 50 μl FASEBA supernatant in 50 μl 0.1 % PBST at RT for 2 hours. Next, the plates were washed with washing buffer and incubated with 100 μl secondary antibody for 45 minutes. After washing, the reaction was developed with 100 μl TMB substrate for 10 minutes at room temperature and stopped by adding 50 μl of 1 M HCl. The absorbance values were measured at 450 nm (OD450nm) using a spectrometer. Binding and expression data are shown in Tables 12-15. Table 12 Sample-Antigen Binding Validation by ELISA Plate 1
Figure imgf000173_0001
202424720.3 172 Table 13 Sample Expression Validation by ELISA Plate 1
Figure imgf000174_0001
Table 14 Sample-Antigen Binding Validation by ELISA Plate 2
Figure imgf000174_0002
Table 15 Sample Expression Validation by ELISA Plate 2
Figure imgf000174_0003
202424720.3 173 [0593] Based on binding and expression data, 10 humanized clones (the top 5 clones from each plate) were selected for DNA sequencing. Bolded and italicized values in Tables 12-15 correspond to binding data for humanized clones selected for DNA sequencing. Italicized values correspond to binding data for chimeric Fab FASEBA control clones. Bolded values correspond to binding data for grafted Fab FASEBA control clones. Underlined values correspond to negative (NC) and blank control. Sequence alignment results are shown in FIGs. 8A (VH) and 8B (VL). [0594] Details of the humanized clones selected for sequencing are shown in Table 16, including amino acid residue differences between the humanized anti-BMP-7 antibody clones and the wildtype antibody sequence. Table 16
Figure imgf000175_0001
[0595] Construction and production of humanized IgGs. Based on the sequencing results, three clones having the highest affinity and fewest back mutation sites were selected for construction and production of humanized IgGs. These clones were AHF17901, AHF17906, and AHF17909. Binding and expression values of these clones are bolded, italicized, and underlined in Tables 12-15. The AHF17901 antibody clone (mAb #1) had a binding OD450nm of 1.377 and an expression OD450nm of 2.183, and has a VL corresponding to SEQ ID NO:34 and a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and a VH corresponding to SEQ ID NO:40 and a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR- H3 comprising SEQ ID NO:65. The AHF17906 antibody clone (mAb #2) had a binding OD450nm of 1.1 and an expression OD450nm of 2.099, and has a VL corresponding to SEQ ID NO:36 and a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53,
202424720.3 174 and a CDR-L3 comprising SEQ ID NO:57; and a VH corresponding to SEQ ID NO:42 and a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65. The AHF17909 antibody clone (mAb #3) had a binding OD450nm of 1.243 and an expression OD450nm of 2.168, and has a VL corresponding to SEQ ID NO:38 and a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR- L3 comprising SEQ ID NO:57; and a VH corresponding to SEQ ID NO:42 and a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65. [0596] The DNA sequences encoding these 3 humanized clones were inserted into a pcDNA3.4 vector to produce expression plasmids for full-length IgGs.24 h before transfection, actively growing Expi293F cells were inoculated into grown in serum-free Expression Medium at the density of 0.9×106 cells/ml, and incubated at 37ºC with 5% CO2 in shaker. The cells were carefully examined for contamination and counted for cell density. The cell density was adjusted to about 1.8~2.0×106 cells/ml. Then cells were diluted to the density required for transfection. Pre-heated buffer was added into one centrifuge tube. The heavy chain expression plasmid and the light chain expression plasmid was added into one tube and mixed them. Then transfection reagent was added into another tube and mixed fully, incubation for 5 min , then mixed the plasmids and transfection reagent, mixed fully, followed by incubation for 10 min at room temperature. The mixture was added into the Expi293F cells and incubated for 48-72 h. Supplement was added into the cells, and then the cells were put back into the shaker. The supernatant was collected for purification after 6 days of cell culture. The recombinant IgGs secreted to the medium were purified using protein A affinity chromatography. Protein A Magnetic Beads were removed from the refrigerator. The supernatant was added to Protein A Magnetic Beads and incubated at room temperature for 2 h. After incubation, protein A Magnetic Beads was balanced with 10 CV (Column volume) binding buffer. Protein A Magnetic Beads was washed with elution buffer. Collect the elution buffer. The eluted antibody was neutralized by adding 1/10 volume of neutralizing buffer. After elution, Protein A Magnetic Beads was balanced with 10 CV (Column volume) binding buffer. The eluted and neutralized antibody was dialyzed in dialysis buffer at room temperature for 2 hours, and then dialyzed at 2-8 ºC 16 hours after buffer changing. Antibody was transferred from the dialysis bag into the tubes and filtered with 0.22 µm membrane filter. Antibody concentration was measured the by Nanodrop 2000 with extinction coefficient of 1.43. Binding confirmation was tested by Surface Plasmon Resonance (SPR) using Biacore 8K/T200.
202424720.3 175 [0597] The purified IgGs migrated as a ~150 kDa band in SDS-PAGE under non-reducing conditions, ~ 50 kDa and ~ 25 kDa bands under reducing conditions. Based on the SDS-PAGE results, the purity of humanized IgGs was determined to be around 99% (FIG.9). Details of the humanized IgGs produced are shown in Table 17, including amino acid residue differences between the three humanized anti-BMP-7 antibody clones (AHF17901, AHF17906, and AHF17909) and the wildtype antibody sequence. Table 17
Figure imgf000177_0001
[0598] Affinity measurement of purified humanized IgGs. The affinity of purified antibody binding to antigen was individually determined using a Surface Plasmon Resonance (SPR) biosensor, BIACORE™ 8K (GE Healthcare). Antigen was immobilized on the sensor chip. Antibodies were used as the analytes. The data of dissociation (kd) and association (ka) rate constants were obtained using BIACORE™ 8K evaluation software. The equilibrium dissociation constants (KD) were calculated from the ratio of kd over ka. [0599] The parameters for affinity measurement are shown in Table 18. Table 18
Figure imgf000177_0002
[0600] The antibody-antigen binding kinetics are summarized in Table 19, and sensor- grams are shown in FIG. 10. Real-time responses are shown as gray curves. Fitting of BIACORE™ experimental data to 1:1 interaction model was shown as black curves.
202424720.3 176 Table 19 Ligand Analyte Chi² (RU²) ka (1/Ms) kd (1/s) KD (M) Rmax (RU) human BMP-7 protein chimeric antibody 1.26E+00 1.12E+05 3.02E-03 2.70E-08 44.6 human BMP-7 protein AHF17901 3.75E-01 5.18E+04 3.21E-03 6.20E-08 48.6 human BMP-7 protein AHF17906 3.93E-01 5.60E+04 3.57E-03 6.38E-08 45.7 human BMP-7 protein AHF17909 7.12E-01 6.49E+04 3.55E-03 5.47E-08 61.6 [0601] All antibodies in Table 19 showed heterogeneous association and dissociation property. According to the sensorgrams showed in FIG. 10, all antibodies had fast but short binding of the antigen initially and the binding slowed down during the following injection of antibodies. In the dissociation stage, antibodies had fast but short dissociation from the antigen and then the dissociation slowed down in the following injection of running buffer. Three antibodies were successfully humanized according to the results of SPR measurement. [0602] Summary. A mouse monoclonal antibody (mAb) was successfully humanized. 3 clones were obtained from FASEBA high throughput screening. 3 selected humanized antibodies were converted to full-length IgGs that were expressed and purified for affinity determination.3 humanized antibodies with similar binding affinity to chimeric antibody were obtained. Example 3 – Humanized BMP-7 Antibody to Treat Resistance to Immunotherapies [0603] Functional validation of BMP-7 mAb antibodies in macrophages treated with BMP-7 antibody for 48 hours. Macrophages were treated with humanized BMP-7 mAb clones for 48 hours. IL1B expression (an M1 macrophage marker) was evaluated by using quantitative PCR. BMP-7 mAb #3 (AHF17906) demonstrated highest upregulation of IL1B, a marker for Th1 response (FIG. 11). Therefore, BMP-7 mAb #3 was used for in vivo experiments. This experiment demonstrates that, as found in mouse experiments, humanized BMP-7 mAb can promote an M1-phenotype in human macrophages and, thus, potentially promotes anti-tumor immune response. [0604] Immunohistochemical validation of BMP-7 mAb antibodies in macrophages treated with BMP-7 antibody. Shown in FIG. 12 are representative images of immunohistochemical stains of CD8 T cells (CD8 – black dots) and M2 macrophages (CD163 – M2 macrophage marker) in formalin-fixed paraffin-embedded tissue sections from 344SQR tumors treated twice a week for two weeks with IgG control (n = 5), anti-PD1 antibody (n =
202424720.3 177 5), chimeric human BMP-7 mAb clone 344-5-1 (10 mg/kg) (n = 5), or chimeric human BMP- 7 mAb clone 344-5-1 plus anti-PD1 antibody. A representative staining image from each cohort (n = 3 biologically independent samples) is displayed. Scale bar, 100 μm (×40 magnification). This experiment shows that treatment with BMP-7 antibody can promote or increase CD8 T cell infiltration and decrease M2 immunosuppressive macrophages, which may explain why BMP-7 inhibition can work in synergism with immune checkpoint inhibitors. [0605] Primary and secondary tumor growth in mice. Tumor growth of mice (129 Sv) with 344SQR primary tumors (FIG.13A) and secondary tumors (FIG.13B) were treated with IgG ctrl (n=5) or anti-PD1 (10 mg/kg) (n=5) or chimeric human BMP-7 mAb clone 344-5-1 (10 mg/kg) (n=5) 3 times a week for 2 weeks. Radiation therapy was provided to the primary tumors in two fractions of 12 Gys. This experiment shows that BMP7 inhibition can promote abscopal effect in lung adenocarcinoma models with or without radiotherapy, which suggests that BMP-7 mAb could potentially be used to target metastasis. Example 4 – Humanized BMP-7 Antibody May Improve CAR-T Cell Killing [0606] BMP-7 is overexpressed in many cancers, including pancreatic cancer, osteosarcoma, prostate cancer, colorectal cancer, malignant melanoma, breast cancer, ovarian cancer, esophageal cancer, gastric cancer, or renal cell cancer. In some aspects, anti-BMP7 antibodies (e.g., one or more BMP-7 antibodies disclosed herein) can increase the killing capability of the CAR-T cells (e.g., EGFR-CAR-T cells) directed to tumor markers or antigens (e.g., EGFR) expressed by these cancers (e.g., pancreatic cancer). In some aspects, the addition of BMP-7 protein can block, or inhibit, the effect of anti-BMP7 antibodies on CAR-T cell (e.g., EGFR-CAR-T cells) killing. Example 5 – Humanized BMP-7 Antibody Improves CAR-T Cell Killing [0607] BMP-7 is overexpressed in Capan-2 human pancreatic cancer cells (FIG. 14), which also overexpress epidermal growth factor receptor (EGFR). As shown in FIG.15, the addition of humanized anti-BMP-7 antibody clone AHF17901 (Ab1) or humanized anti-BMP- 7 antibody clone AHF17906 (Ab2) increased the ability of EGFR-expressing CAR-T cells to effect apoptosis of Capan-2 cells. As shown in FIG.16 and FIG.17, the addition of BMP-7 protein blocks, or inhibits, the effect of humanized anti-BMP-7 antibody clone AHF17901 (Ab1) and humanized anti-BMP-7 antibody clone AHF17906 (Ab2) on the ability of EGFR- expressing CAR-T cells to effect apoptosis of Capan-2 cells.
202424720.3 178 Example 6 – CRISPR-edited CAR T cell to treat resistance to immunotherapies [0608] CRISPR-edited CAR T cells having reduced or disrupted expression of bone morphogenic receptors (BMP receptors) were generated using standard techniques. Specifically, lentiviral vectors encoding two gRNAs targeting a gene locus for a BMP receptor and a Cas nuclease were transfected into isolated human T-cells. The Cas nuclease effected a double stranded break at two target locations, allowing for removal of the intervening nucleic acid and successful disruption of the target gene. [0609] To determine the gRNAs for the gene editing vectors, various gRNA spacer sequences targeting the hBMPR1B or hBMBPR1A gene loci were screened for on-target specificity. In FIG. 18 and FIG. 19, white squares depict sequences with high on-target potential and low off-target risk for BMPR1B and BMPR1A loci, respectively. These sequences were further screened for uniqueness to identify 8 unique sequences for BMPR1B and 1 unique sequence for BMPR1A, shown in Tables 20 and 21 below. Table 20 - Unique gRNA spacer sequences for BMPR1B
Figure imgf000180_0001
Table 21 - Unique gRNA spacer sequences for BMPR1A
Figure imgf000180_0002
202424720.3 179 [0610] A series of lentiviral vectors were constructed targeting hBMPR1B, hBMPR1A, mAcvr1, mAcvr2a, mAcvr2b, mAcvrl1, and mBmpr2 gene loci. Each vector encoded two gRNAs targeting the desired loci and a Cas9 endonuclease (either hCas9 or a hCas9:TCA:Puro construct). FIGs.20-23 depict plasmid maps for illustrative vectors targeting hBMPR1B using a hCas9:TCA:Puro construct (FIG. 20) or hCas9 (FIG. 21), targeting hBMPR1A using hCas9:TCA:Puro construct (FIG.22), or hCas9 (FIG.23). Tables 22-30 below, provide details for each vector provided including the target gene and gRNAs/endonucleases encoded. Table 22 – Illustrative CRISPR gene editing vector targeting human BMPR1B (VB200329-1029jwu)
Figure imgf000181_0001
Table 23 – Illustrative CRISPR gene editing vector targeting human BMPR1B (VB200329-1005ytw)
Figure imgf000181_0002
202424720.3 180
Figure imgf000182_0001
Table 24 – Illustrative CRISPR gene editing vector targeting human BMPR1A (VB200328·7336rqg)
Figure imgf000182_0002
Table 25 – Illustrative CRISPR gene editing vector targeting human BMPR1A (VB200328•7298grx)
Figure imgf000182_0003
Table 26 – Illustrative CRISPR gene editing vector targeting murine Acvr1 (VB200508-1093dpc)
Figure imgf000182_0004
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Figure imgf000183_0001
Table 27 - Illustrative CRISPR gene editing vector targeting murine mAcvr2a (VB200S08-1102vgy)
Figure imgf000183_0002
Table 28 – Illustrative CRISPR gene editing vector targeting murine Acvr2b (V8200508-1087prs)
Figure imgf000183_0003
202424720.3 182 Table 29 – Illustrative CRISPR gene editing vector targeting murine Acvrl1 (VB200508-1082wsj)
Figure imgf000184_0001
Table 30 – Illustrative CRISPR gene editing vector targeting murine Bmpr2 (VB200508-10911gkz)
Figure imgf000184_0002
* * * [0611] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of certain aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit
202424720.3 183 and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. [0612] The references recited in the application, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
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Claims

CLAIMS What is claimed is: 1. A bone morphogenetic protein-7 (BMP-7)-binding protein comprising: a) a light chain variable region (VL) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and b) a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs), wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identical identity with SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
2. The BMP-7 binding protein of claim 1, wherein one or more of the CDRs of the VL comprise an amino acid sequence having at least 90% identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71, and one or more of the CDRs of the VH comprise an amino acid sequence having at least 90% identity with SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77. 3. The BMP-7 binding protein of claim 1 or claim 2, wherein one or more of the CDRs of the VL comprise an amino acid sequence having at least 95% identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71, and one or more of the CDRs of the VH comprise an amino acid sequence having at least 95% identity with SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
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4. The BMP-7 binding protein of any one of claims 1-3, wherein one or more of the CDRs of the VL comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71, and one or more of the CDRs of the VH comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
5. The BMP-7 binding protein of any one of claims 1-4, wherein the VL comprises a CDR- L1 having at least 85% identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having at least 85% identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having at least 85% identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
6. The BMP-7 binding protein of claim 5, wherein the CDR-L1 comprises SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; the CDR-L2 comprises SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and the CDR-L3 comprises SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, or SEQ ID NO:71.
7. The BMP-7 binding protein of any one of claims 1-6, wherein the VH comprises a CDR- H1 having at least 85% identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having at least 85% identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having at least 85% identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
8. The BMP-7 binding protein of claim 7, wherein the CDR-H1 comprises SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; the CDR-H2 comprises SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and the CDR-H3 comprises SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
9. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 85% identity with SEQ ID NO:14 and the VH comprises an amino acid sequence with at least 85% identity with SEQ ID NO:16.
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10. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 90% identity with SEQ ID NO:14 and the VH comprises an amino acid sequence with at least 90% identity with SEQ ID NO:16.
11. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 95% identity with SEQ ID NO:14 and the VH comprises an amino acid sequence with at least 95% identity with SEQ ID NO:16.
12. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises SEQ ID NO:14 and the VH comprises SEQ ID NO:16.
13. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 85% identity with SEQ ID NO:34 and the VH comprises an amino acid sequence with at least 85% identity with SEQ ID NO:40.
14. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 90% identity with SEQ ID NO:34 and the VH comprises an amino acid sequence with at least 90% identity with SEQ ID NO:40.
15. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 95% identity with SEQ ID NO:34 and the VH comprises an amino acid sequence with at least 95% identity with SEQ ID NO:40.
16. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises SEQ ID NO:34 and the VH comprises SEQ ID NO:40.
17. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 85% identity with SEQ ID NO:36 and the VH comprises an amino acid sequence with at least 85% identity with SEQ ID NO:42.
18. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 90% identity with SEQ ID NO:36 and the VH comprises an amino acid sequence with at least 90% identity with SEQ ID NO:42.
19. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 95% identity with SEQ ID NO:36 and the VH comprises an amino acid sequence with at least 95% identity with SEQ ID NO:42.
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20. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises SEQ ID NO:36 and the VH comprises SEQ ID NO:42.
21. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 85% identity with SEQ ID NO:38 and the VH comprises an amino acid sequence with at least 85% identity with SEQ ID NO:42.
22. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 90% identity with SEQ ID NO:38 and the VH comprises an amino acid sequence with at least 90% identity with SEQ ID NO:42.
23. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 95% identity with SEQ ID NO:38 and the VH comprises an amino acid sequence with at least 95% identity with SEQ ID NO:42.
24. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises SEQ ID NO:38 and the VH comprises SEQ ID NO:42.
25. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 85% identity with SEQ ID NO:79 and the VH comprises an amino acid sequence with at least 85% identity with SEQ ID NO:81.
26. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 90% identity with SEQ ID NO:79 and the VH comprises an amino acid sequence with at least 90% identity with SEQ ID NO:81.
27. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises an amino acid sequence with at least 95% identity with SEQ ID NO:79 and the VH comprises an amino acid sequence with at least 95% identity with SEQ ID NO:81.
28. The BMP-7 binding protein of any one of claims 1-8, wherein the VL comprises SEQ ID NO:79 and the VH comprises SEQ ID NO:81.
29. The BMP-7 binding protein of any of claims 1-28, wherein the VH and the VL are on the same polypeptide.
30. The BMP-7 binding protein of any of claims 1-28, wherein the VH and the VL are on different polypeptides.
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31. The BMP-7 binding protein of any one of claims 1-30, wherein the BMP-7 binding protein binds to human BMP-7.
32. The BMP-7 binding protein of any one of claims 1-31, wherein the BMP-7 binding protein is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, or a polyclonal antibody.
33. The BMP-7 binding protein of claim 32, wherein the antibody is a monoclonal antibody.
34. The BMP-7 binding protein of claim 32, wherein the antibody is a chimeric antibody.
35. The BMP-7 binding protein of claim 32, wherein the antibody is a humanized antibody.
36. A composition comprising the BMP-7 binding protein of any of claims 1-35 and one or more additional therapeutics.
37. The composition of claim 36, wherein the one or more additional therapeutics comprise one or more immunotherapies.
38. The composition of claim 37, wherein the one or more immunotherapies comprise a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.
39. The composition of claim 38, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody.
40. The composition of claim 38, wherein the PD1 inhibitor is an anti-PD1 antibody.
41. The composition of claim 38, wherein the PDL-1 inhibitor is an anti-PDL-1 antibody.
42. The composition of claim 38, wherein the LAG3 inhibitor is an anti-LAG3 antibody.
43. The composition of claim 38, wherein the TIM-3 inhibitor is an anti-TIM-3 antibody.
44. The composition of claim 37, wherein the one or more immunotherapies comprise a CAR-T cell therapy.
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45. The composition of claim 44, wherein the CAR-T cell therapy comprises EGFR- targeted CAR-T cells.
46. The composition of claim 44 or 45, wherein the CAR-T cell therapy comprises CAR-T cells that are genetically modified to have disrupted or reduced expression of one or more Bone Morphogenetic Protein (BMP) receptors.
47. The composition of claim 46, wherein the one or more BMP receptors comprise Bone Morphogenetic Protein Receptor Type 1A (BMPR1A), Bone Morphogenetic Protein Receptor Type 1B (BMPR1B), Activin A receptor type I (ACVR1), Activin receptor type 2A (ACVR2A), Activin receptor type 2B (ACVR2B), Bone morphogenetic protein receptor type 2 (BMPR2), or any combination thereof.
48. A pharmaceutical composition comprising (i) the BMP-7-binding protein of any of claims 1-35 or the composition of any of claims 36-47 and (ii) a pharmaceutically acceptable excipient.
49. The pharmaceutical composition of claim 48, further comprising an additional therapeutic.
50. The pharmaceutical composition of claim 49, wherein the additional therapeutic is a chemotherapeutic.
51. A nucleic acid comprising a nucleotide sequence encoding for the BMP-7-binding protein of any of claims 1-35 or a fragment thereof.
52. The nucleic acid of claim 51, wherein the nucleic acid encodes the heavy chain variable region and the light chain variable region of the BMP-7-binding protein.
53. The nucleic acid of claim 51, wherein the nucleic acid encodes the heavy chain variable region of the BMP-7-binding protein.
54. The nucleic acid of claim 51, wherein the nucleic acid encodes the light chain variable region of the BMP-7-binding protein.
55. An expression vector comprising the nucleic acid of any one of claims 51-54.
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56. The expression vector of claim 55, wherein the nucleic acid is operably linked to an expression control sequence.
57. The expression vector of claim 55 or claim 56, wherein the expression vector comprises a nucleotide sequence with at least 85% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78.
58. The expression vector of claim 55 or claim 56, wherein the expression vector comprises a nucleotide sequence with at least 90% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78.
59. The expression vector of claim 55 or claim 56, wherein the expression vector comprises a nucleotide sequence with at least 95% identity to SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78.
60. The expression vector of claim 55 or claim 56, wherein the expression vector comprises SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:78.
61. The expression vector of any of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 85% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80.
62. The expression vector of any of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 90% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80.
63. The expression vector of any of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 95% identity to SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80.
64. The expression vector of any of claims 55-60, wherein the expression vector comprises SEQ ID NO:15, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:80.
65. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 85% identity to SEQ ID NO:13 and a nucleotide sequence with at least 85% identity to SEQ ID NO:15.
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66. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 85% identity to SEQ ID NO:33 and a nucleotide sequence with at least 85% identity to SEQ ID NO:39.
67. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 85% identity to SEQ ID NO:35 and a nucleotide sequence with at least 85% identity to SEQ ID NO:41.
68. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 85% identity to SEQ ID NO:37 and a nucleotide sequence with at least 85% identity to SEQ ID NO:41.
69. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 85% identity to SEQ ID NO:78 and a nucleotide sequence with at least 85% identity to SEQ ID NO:80.
70. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 90% identity to SEQ ID NO:13 and a nucleotide sequence with at least 90% identity to SEQ ID NO:15.
71. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 90% identity to SEQ ID NO:33 and a nucleotide sequence with at least 90% identity to SEQ ID NO:39.
72. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 90% identity to SEQ ID NO:35 and a nucleotide sequence with at least 90% identity to SEQ ID NO:41.
73. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 90% identity to SEQ ID NO:37 and a nucleotide sequence with at least 90% identity to SEQ ID NO:41.
74. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 90% identity to SEQ ID NO:78 and a nucleotide sequence with at least 90% identity to SEQ ID NO:80.
202424720.3 192
75. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 95% identity to SEQ ID NO:13 and a nucleotide sequence with at least 95% identity to SEQ ID NO:15.
76. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 95% identity to SEQ ID NO:33 and a nucleotide sequence with at least 95% identity to SEQ ID NO:39.
77. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 95% identity to SEQ ID NO:35 and a nucleotide sequence with at least 95% identity to SEQ ID NO:41.
78. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 95% identity to SEQ ID NO:37 and a nucleotide sequence with at least 95% identity to SEQ ID NO:41.
79. The expression vector of any one of claims 55-60, wherein the expression vector comprises a nucleotide sequence with at least 95% identity to SEQ ID NO:78 and a nucleotide sequence with at least 95% identity to SEQ ID NO:80.
80. The expression vector of any one of claims 55-60, wherein the expression vector comprises SEQ ID NO:13 and SEQ ID NO:15.
81. The expression vector of any one of claims 55-60, wherein the expression vector comprises SEQ ID NO:33 and SEQ ID NO:39.
82. The expression vector of any one of claims 55-60, wherein the expression vector comprises SEQ ID NO:35 and SEQ ID NO:41.
83. The expression vector of any one of claims 55-60, wherein the expression vector comprises SEQ ID NO:37 and SEQ ID NO:41.
84. The expression vector of any one of claims 55-60, wherein the expression vector comprises SEQ ID NO:78 and SEQ ID NO:80.
85. A cell comprising (i) the BMP-7-binding protein of any of claims 1-35, (ii) the nucleic acid of any one of claim 51-54, or (iii) the expression vector of any one of claims 55-84.
202424720.3 193
86. The cell of claim 85, wherein the cell is an immune cell.
87. The cell of claim 85 or claim 86, wherein the cell comprises the BMP-7-binding protein and wherein the cell is capable of secreting the BMP-7-binding protein outside the cell.
88. The cell of claim 87, wherein the cell is a B cell.
89. The cell of claim 85 or claim 86, wherein the cell comprises the BMP-7-binding protein and wherein the BMP-7-binding protein is attached to a surface of the cell.
90. The cell of claim 89, wherein the BMP-7-binding protein is a chimeric antigen receptor.
91. The cell of claim 89, wherein the BMP-7-binding protein is a T cell receptor.
92. The cell of any of claims 89-91, wherein the cell is a T cell.
93. The cell of any one of claims 85-92, wherein the cell is genetically modified to have disrupted or reduced expression of one or more bone morphogenetic protein (BMP) receptors.
94. The cell of claim 93, wherein the one or more BMP receptors comprise Bone Morphogenetic Protein Receptor Type 1A (BMPR1A), Bone Morphogenetic Protein Receptor Type 1B (BMPR1B), Activin A receptor type I (ACVR1), Activin receptor type 2A (ACVR2A), Activin receptor type 2B (ACVR2B), Bone morphogenetic protein receptor type 2 (BMPR2), or any combination thereof.
95. A method for generating a BMP-7-binding protein, the method comprising culturing the cell of any one of claims 85-94 under conditions sufficient to express the BMP-7-binding protein in the cell.
96. A method for generating the BMP-7-binding protein of any of claims 1-35 comprising: (a) providing to a cell a nucleic acid encoding for the BMP-7-binding protein; and (b) subjecting the cell to conditions sufficient to express the nucleic acid in the cell.
97. A method of treating a subject for cancer comprising administering to the subject a therapeutically effective amount of the BMP-7-binding protein of any of claims 1-35, the composition of any of claims 36-47, the pharmaceutical composition of any of claims 48-50, the nucleic acid of any of claims 51-54, the expression vector of any of claims 55-84, or the cell of any of claims 85-94.
202424720.3 194
98. The method of claim 97, wherein the subject was previously treated with a cancer therapy.
99. The method of claim 97 or claim 98, wherein the cancer therapy comprises one or more immunotherapies.
100. The method of claim 99, wherein the one or more immunotherapies comprise a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.
101. The method of any one of claims 98-100, wherein the subject was determined to be resistant to the cancer therapy.
102. The method of any one of claims 97-101, wherein the cancer is a solid tumor.
103. The method of claim any one of claims 97-101, wherein the cancer is lung cancer.
104. The method of claim 97, wherein cancer cells in the subject overexpress BMP-7 compared to non-cancerous cells.
105. The method of claim 104, wherein the cancer is pancreatic cancer, osteosarcoma, prostate cancer, colorectal cancer, malignant melanoma, breast cancer, ovarian cancer, esophageal cancer, gastric cancer, or renal cell cancer.
106. The method of claim 104 or 105, wherein the cancer is pancreatic cancer.
107. The method of any one of claims 97-106, further comprising administering to the subject an additional therapy.
108. The method of claim 107, wherein the additional therapy is radiotherapy, chemotherapy, or immunotherapy.
109. A monoclonal antibody or antigen-binding fragment thereof comprising: a) a VL comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ
202424720.3 195 ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71; and b) a VH comprising three CDRs, wherein one or more of the CDRs comprise an amino acid sequence having at least 85% identical identity with SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
110. The monoclonal antibody or antigen-binding fragment of claim 109, wherein one or more of the CDRs of the VL comprise an amino acid sequence having at least 90% identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71, and one or more of the CDRs of the VH comprise an amino acid sequence having at least 90% identity with SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
111. The monoclonal antibody or antigen-binding fragment of claim 109 or claim 110, wherein one or more of the CDRs of the VL comprise an amino acid sequence having at least 95% identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71, and one or more of the CDRs of the VH comprise an amino acid sequence having at least 95% identity with SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
112. The monoclonal antibody or antigen-binding fragment of any one of claims 109-111, wherein one or more of the CDRs of the VL comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71, and one or more of the CDRs of the VH comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:77.
202424720.3 196
113. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-112, wherein the VL comprises a CDR-L1 having at least 85% identity to SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 having at least 85% identity to SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 having at least 85% identity to SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, SEQ ID NO:71.
114. The monoclonal antibody or antigen-binding fragment thereof of claim 113, wherein the CDR-L1 comprises SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; the CDR-L2 comprises SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and the CDR-L3 comprises SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, SEQ ID NO:71.
115. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-114, wherein the VH comprises a CDR-H1 having at least 85% identity to SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 having at least 85% identity to SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 having at least 85% identity to SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
116. The monoclonal antibody or antigen-binding fragment thereof of claim 115, wherein the CDR-H1 comprises SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; the CDR-H2 comprises SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and the CDR-H3 comprises SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
117. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 85% identity with SEQ ID NO:14 and the VH comprises an amino acid sequence with at least 85% identity with SEQ ID NO:16.
118. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 90% identity with SEQ ID NO:14 and the VH comprises an amino acid sequence with at least 90% identity with SEQ ID NO:16.
119. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 95% identity with
202424720.3 197 SEQ ID NO:14 and the VH comprises an amino acid sequence with at least 95% identity with SEQ ID NO:16.
120. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises SEQ ID NO:14 and the VH comprises SEQ ID NO:16.
121. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 85% identity with SEQ ID NO:34 and the VH comprises an amino acid sequence with at least 85% identity with SEQ ID NO:40.
122. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 90% identity with SEQ ID NO:34 and the VH comprises an amino acid sequence with at least 90% identity with SEQ ID NO:40.
123. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 95% identity with SEQ ID NO:34 and the VH comprises an amino acid sequence with at least 95% identity with SEQ ID NO:40.
124. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises SEQ ID NO:34 and the VH comprises SEQ ID NO:40.
125. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 85% identity with SEQ ID NO:36 and the VH comprises an amino acid sequence with at least 85% identity with SEQ ID NO:42.
126. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 90% identity with SEQ ID NO:36 and the VH comprises an amino acid sequence with at least 90% identity with SEQ ID NO:42.
127. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 95% identity with
202424720.3 198 SEQ ID NO:36 and the VH comprises an amino acid sequence with at least 95% identity with SEQ ID NO:42.
128. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises SEQ ID NO:36 and the VH comprises SEQ ID NO:42.
129. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 85% identity with SEQ ID NO:38 and the VH comprises an amino acid sequence with at least 85% identity with SEQ ID NO:42.
130. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 90% identity with SEQ ID NO:38 and the VH comprises an amino acid sequence with at least 90% identity with SEQ ID NO:42.
131. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 95% identity with SEQ ID NO:38 and the VH comprises an amino acid sequence with at least 95% identity with SEQ ID NO:42.
132. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises SEQ ID NO:38 and the VH comprises SEQ ID NO:42.
133. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 85% identity with SEQ ID NO:79 and the VH comprises an amino acid sequence with at least 85% identity with SEQ ID NO:81.
134. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 90% identity with SEQ ID NO:79 and the VH comprises an amino acid sequence with at least 90% identity with SEQ ID NO:81.
135. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises an amino acid sequence with at least 95% identity with
202424720.3 199 SEQ ID NO:79 and the VH comprises an amino acid sequence with at least 95% identity with SEQ ID NO:81.
136. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-116, wherein the VL comprises SEQ ID NO:79 and the VH comprises SEQ ID NO:81.
137. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 109-136, wherein the monoclonal antibody or antigen-binding fragment thereof binds to human BMP-7.
138. An antibody comprising: a) a VL comprising a CDR-L1 comprising SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:50, or SEQ ID NO:67; a CDR-L2 comprising SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:53, or SEQ ID NO:69; and a CDR-L3 comprising SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:57, SEQ ID NO:71; and b) a VH comprising a CDR-H1 comprising SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:59, or SEQ ID NO:73; a CDR-H2 comprising SEQ ID NO:10, SEQ ID NO:28, SEQ ID NO:62, or SEQ ID NO:75; and a CDR-H3 comprising SEQ ID NO:12, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:65, or SEQ ID NO:77.
139. An antibody comprising: a) a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and b) a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:12.
140. An antibody comprising: a) a VL comprising a CDR-L1 comprising SEQ ID NO:2, a CDR-L2 comprising SEQ ID NO:4, and a CDR-L3 comprising SEQ ID NO:6; and b) a VH comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:10, and a CDR-H3 comprising SEQ ID NO:32.
141. An antibody comprising: c) a VL comprising a CDR-L1 comprising SEQ ID NO:20, a CDR-L2 comprising SEQ ID NO:22, and a CDR-L3 comprising SEQ ID NO:24; and
202424720.3 200 d) a VH comprising a CDR-H1 comprising SEQ ID NO:26, a CDR-H2 comprising SEQ ID NO:28, and a CDR-H3 comprising SEQ ID NO:30.
142. The antibody of any one of claims 138-141, wherein the VL comprises SEQ ID NO:14.
143. The antibody of any one of claims 138-141, wherein the VH comprises SEQ ID NO:16.
144. An antibody comprising: e) a VL comprising a CDR-L1 comprising SEQ ID NO:50, a CDR-L2 comprising SEQ ID NO:53, and a CDR-L3 comprising SEQ ID NO:57; and f) a VH comprising a CDR-H1 comprising SEQ ID NO:59, a CDR-H2 comprising SEQ ID NO:62, and a CDR-H3 comprising SEQ ID NO:65.
145. The antibody of claim 144, wherein the VL comprises SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38.
146. The antibody of claim 144 or 145, wherein the VH comprises SEQ ID NO:40 or SEQ ID NO:42.
147. The antibody of any one of claims 144-146, wherein the VL comprises SEQ ID NO:34 and the VH comprises SEQ ID NO:40.
148. The antibody of any one of claims 144-146, wherein the VL comprises SEQ ID NO:36 and the VH comprises SEQ ID NO:42.
149. The antibody of any one of claims 144-146, wherein the VL comprises SEQ ID NO:38 and the VH comprises SEQ ID NO:42.
150. A method of increasing the efficacy of a cancer therapy administered to a subject for treatment of cancer, the method comprising administering to the subject a therapeutically effective amount of the cancer therapy and the BMP-7-binding protein of any of claims 1-35, the nucleic acid of any of claims 51-54, the expression vector of any of claims 55-84, or the cell of any of claims 85-94.
151. The method of claim 150, wherein the cancer therapy and the BMP-7-binding protein of any of claims 1-35, the nucleic acid of any of claims 51-54, the expression vector of any of claims 55-84, or the cell of any of claims 85-94 are comprised in the same composition.
202424720.3 201
152. The method of claim 150, wherein the cancer therapy and the BMP-7-binding protein of any of claims 1-35, the nucleic acid of any of claims 51-54, the expression vector of any of claims 55-84, or the cell of any of claims 85-94 are comprised in the different compositions.
153. The method of any one of claims 150-152, wherein the cancer therapy comprises an immunotherapy.
154. The method of claim 153, wherein the immunotherapy comprises a CAR-T cell therapy.
155. The method of claim 153 or 154, wherein the immunotherapy comprises EGFR- targeted CAR-T cells.
156. The method of claim 153 or 154, wherein the immunotherapy comprises CAR-T cells genetically modified to have disrupted or reduced expression of one or more Bone Morphogenetic Protein (BMP) receptors.
157. The method of any one of claims 150-156, wherein the cancer cells overexpress BMP- 7 compared to non-cancerous cells.
158. The method of any one of claims 150-157, wherein the cancer is pancreatic cancer, osteosarcoma, prostate cancer, colorectal cancer, malignant melanoma, breast cancer, ovarian cancer, esophageal cancer, gastric cancer, or renal cell cancer.
159. The method of any one of claims 150-158, wherein the cancer is pancreatic cancer.
202424720.3 202
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