WO2006020706A2 - Proteines de liaison au complexe tie - Google Patents

Proteines de liaison au complexe tie Download PDF

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Publication number
WO2006020706A2
WO2006020706A2 PCT/US2005/028413 US2005028413W WO2006020706A2 WO 2006020706 A2 WO2006020706 A2 WO 2006020706A2 US 2005028413 W US2005028413 W US 2005028413W WO 2006020706 A2 WO2006020706 A2 WO 2006020706A2
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WIPO (PCT)
Prior art keywords
tiel
protein
antibody
variable domain
binding
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PCT/US2005/028413
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English (en)
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WO2006020706A3 (fr
Inventor
Clive R. Wood
Daniel T. Dransfield
Henk Pieters
Rene Hoet
Simon E. Hufton
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Dyax Corp.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from PCT/US2004/026116 external-priority patent/WO2005019267A2/fr
Priority claimed from US11/049,536 external-priority patent/US7871610B2/en
Application filed by Dyax Corp. filed Critical Dyax Corp.
Priority to CA002576886A priority Critical patent/CA2576886A1/fr
Priority to EP05784935A priority patent/EP1789451A4/fr
Priority to AU2005272848A priority patent/AU2005272848A1/en
Priority to JP2007525760A priority patent/JP2008532476A/ja
Publication of WO2006020706A2 publication Critical patent/WO2006020706A2/fr
Publication of WO2006020706A3 publication Critical patent/WO2006020706A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • 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
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)

Definitions

  • the oxygen and nutrients supplied by the blood vessels are crucial for tissue development and function.
  • the cardiovascular system is the first organ system to develop in embryos.
  • organogenesis and the development of tissues or tumors the proximity of the growing cells to the circulatory system is ensured by the coordinated growth of blood vessels and organ parenchyma. It may be possible to prevent or treat diseases by modulating blood vessel development or angiogenesis.
  • Blood vessels are composed of an inner layer of endothelial cells and an outer layer of pericytes or smooth muscle cells.
  • the first tubular structures are formed by endothelial cells that subsequently recruit pericytes and smooth muscle cells to ensheath them.
  • vasculogenesis The de novo formation of blood vessels from a dispersed population of mesodermally derived endothelial precursor cells is termed vasculogenesis.
  • This primitive network undergoes successive morphogenetic events including sprouting, splitting, and remodeling to generate the hierarchical vascular network from large to branched small vessels. These successive morphogenetic events are collectively called angiogenesis.
  • RTKs endothelial cell specific receptor tyrosine kinases
  • VEGF vascular endothelial growth factor
  • Angs angiopoietins
  • Tie2 angiopoietins
  • Eph receptors ephrins
  • Tiel and Tie2 are RTKs that are expressed almost exclusively in endothelial cells and hematopoietic precursor cells. These two receptors are required for the normal development of vascular structures during embryogenesis.
  • the two Tie receptors form a RTK subfamily since, unlike other RTK family members, they include extracellular EGF-homology domains. See, e.g., Partanen (1992) MoI. Cell Biol. 12:1698 and WO 93/14124.
  • Targeted disruption of the Tiel gene in mice results in a lethal phenotype characterized by extensive hemorrhage and defective microvessel integrity. See, e.g., Puri et al. (1995) EMBO J. 14:5884.
  • Tie2 null embryos have defects in vascular remodeling and maturation, resulting from improper recruitment of periendothelial supporting cells.
  • Angiopoietins Ang, e.g., Angl, Ang2, Ang3, and Ang4 are proteins that interact with Tie2.
  • the invention features a method of modulating Tie complex formation, or interactions between Tie complex components, in a subject.
  • the method includes administering, to a subject, an agent that binds to Tiel.
  • the agent promotes Tiel self-association (e.g., homodimerization) or antagonizes an association between at least two of the following: Tiel, Tie2, and an angiopoietin (Ang; such as Angl, Ang2, Ang3, or Ang4).
  • Ang angiopoietin
  • the agent antagonizes formation of a heteromeric complex of Tiel , Tie2, and Ang.
  • the binding of the agent can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang.
  • the agent binds to Tiel .
  • the agent antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang.
  • the binding of the agent can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang.
  • the agent enhances Tiel self-association, e.g., homodimerization, and thereby associates Tiel with Tiel and prevents association of Tiel with Tie2 and/or Ang.
  • the agent can include at least two valencies for binding to Tiel. hi one embodiment, the agent increases phosphorylation of Tiel, e.g., Tiel "a ⁇ phosphorylation. This increase can, but need not, depend on Tiel self- association.
  • the agent includes a protein, such as an antibody, that binds to the extracellular domain of human Tiel .
  • the antibody can be one or more of the following: human, humanized, non-immunogenic, isolated, monoclonal, and recombinant.
  • the antibody can bind to the first Ig-like C2-type domain (Ig 1) or to the second Ig-like C2-type domain (Ig 2) of Tiel.
  • the antibody binds to an EGF-like domain of Tiel (e.g., first, second, or third EGF-like domain).
  • the antibody binds to the fibronectin type III repeats region of Tiel .
  • the antibody binds to amino acid residues 24-124, 74-174, 124-224, 174-274, 224-324, 274-374, 324-424, 374-474, 424-524, 474-574, 524-624, 574-674, 624-724, 674-759, or 724-759 of SEQ ID NO:2.
  • the agent includes a protein that binds to a Tiel ectodomain and includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence.
  • the protein can further include one or more of the following properties: (1) at least one of the variable domain sequences includes at least one CDR of the E3 or E3b antibody (e.g., one, two, or three CDRs of the E3 or E3b antibody); (2) at least one of the variable domain sequences includes CDR sequences at least 85% identical, in sum, to the CDRs of the corresponding variable domain of the E3 or E3b antibody, (3) at least one of the variable domains is at least 85% identical to the corresponding immunoglobulin variable domains of the E3 or E3b antibody, and (4) the protein competes with E3 or E3b for binding to Tiel or binds to an epitope that overlaps the epitope bound by E3 or E3b on Tiel .
  • Example of antibodies that include an antigen binding site that competes with E3 for binding to Tiel include M0044B08, M0056G08, M0045B03, M0053F04, M0055E10, M0060H01, M0054H10, M0058F03, and related antibodies.
  • the agent includes the HC and/or LC variable domain of the E3 or E3b antibody, or a sequence at least 70, 80, 85, 90, 95, 98, 99% identical to the HC and/or LC variable domains of the E3 or E3b antibody.
  • the amino acid sequences of the HC variable domain sequence include CDRl, CDR2, and CDR3 sequences from the E3 or E3b clone and the LC variable domain sequence includes CDRl, CDR2, and CDR3 sequences from the E3 or E3b clone.
  • the agent comprises the E3 or E3b antibody.
  • the LC variable domain sequence can include SEQ ID NO: 116.
  • the HC variable domain sequence can include SEQ ID NO: 114.
  • the HC and LC framework regions are human. In one embodiment, that agent includes SEQ ID NO:723 and SEQ ID NO:724.
  • the agent binds to Tie2.
  • the agent antagonizes formation of a heteromeric complex of Tie 1, Tie2, and Ang.
  • the binding of the agent can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang.
  • the agent enhances Tie2 self-association, e.g., homodimerization, and, thereby associates Tie2 with Tie2 and prevents association of Tie2 with Tiel and/or Ang.
  • the agent includes a protein, e.g., an antibody that binds to the extracellular domain of human Tie2.
  • the antibody can be one or more of the following: human, humanized, non-immunogenic, isolated, monoclonal, and recombinant.
  • the antibody can bind to the first Ig-like C2- type domain (Ig 1) or to the second Ig-like C2-type domain (Ig 2) of Tie2.
  • the antibody binds to an EGF-like domain of Tie2 (e.g., first, second, or third EGF-like domain).
  • the antibody binds to the fibronectin type III repeats region of Tie2.
  • the antibody binds to amino acid residues 19-119, 69-169, 119-229, 169-269, 229-329, 269-369, 329-429, 369-469, 429-529, 469-569, 529-629, 569-669, 629-729, 669-745, 729-745 of SEQ ID NO:162.
  • the agent binds to Ang (e.g., Angl, Ang2, Ang3, or Ang4).
  • the agent antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang.
  • the binding of the agent can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang.
  • the agent includes a protein, e.g., an antibody that binds to Ang.
  • the antibody can be one or more of the following: human, humanized, non-immunogenic, isolated, monoclonal, and recombinant.
  • the antibody binds to the N-terminal domain of Angl (e.g., the N- terminal 50 amino acids of Angl).
  • the antibody binds to the coiled-coil domain of Angl.
  • the antibody binds to the fibrinogen-like domain of Angl.
  • the antibody binds to amino acid residues 1-100, 50-150, 100-200, 150-250, 200-300, 250-350, 300-400, 350-450, 400-497, or 450-497 of SEQ ID NO: 163.
  • the agent includes a protein that contains a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence, hi one embodiment, the HC and LC framework regions are human, hi one embodiment, the agent also includes an Fc domain.
  • the agent includes the constant domains of a human IgGl, IgG2, IgG3, or IgG4. hi one embodiment, the constant domains of the heavy chain are f allotype, (a,z) allotype, or any other allotype.
  • the agent is administered in an amount effective to decrease vascular development or angiogenesis.
  • the subject has an angiogenesis-related disorder
  • the subject has for example: a neoplastic disorder, metastatic cancer, an angiogenesis-dependent cancer or tumor, an inflammatory disorder, rheumatoid arthritis, or psoriasis
  • the protein is delivered systemically.
  • the protein is administered in an amount effective to reduce one or more of the following activities: sprouting, splitting, remodeling of blood vessels, vasculogenesis, and tubule formation.
  • the method can include other features described herein.
  • the invention includes a method of decreasing or inhibiting endothelial cell activity in the subject, the method includes administering an agent that decreases or inhibits Tie complex formation in an amount effective to decrease or inhibit endothelial cell activity in the subject.
  • the method can include other features described herein.
  • the invention includes a method of decreasing endothelial cell activity by administering an agent that causes Tiel phosphorylation, hi one embodiment, the phosphorylation decreases endothelial cell differentiation, e.g., sprouting, splitting, and tube formation.
  • the invention includes a method of decreasing endothelial cell activity, the method by administering an agent that activates a signaling pathway.
  • the signaling pathway decreases endothelial cell differentiation, e.g., sprouting, splitting, and tube formation.
  • the agent increases Tiel autophosphorylation.
  • the method can include other features described herein.
  • the invention includes an antibody for modulating Tie complex formation in a subject, wherein the antibody antagonizes an association between at least two of the following: Tiel, Tie2, and an angiopoietin (Ang).
  • the antibody binds to a Tie complex component or to one or more of Tiel, Tie2, and an Ang.
  • the antibody antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang.
  • the antibody can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang.
  • the antibody binds to Tiel.
  • the antibody antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang.
  • the binding of the antibody can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang.
  • the antibody enhances Tiel self-association, e.g., homodimerization, and thereby associates Tiel with Tiel and prevents association of Tiel with Tie2 or Ang.
  • the antibody increases Tiel phosphorylation and/or prevents association of Tie 1 with Tie2 or Ang.
  • the antibody includes an antibody that binds to the extracellular domain of human Tiel.
  • the antibody can be one or more of the following: human, humanized, non- immunogenic, isolated, monoclonal, and recombinant.
  • the antibody can bind to the first Ig-like C2-type domain (Ig 1) or to the second Ig-like C2-type domain (Ig 2) of Tiel.
  • the antibody binds to an EGF- like domain of Tiel (e.g., first, second, or third EGF-like domain).
  • the antibody binds to the fibronectin type III repeats region of Tiel.
  • the antibody binds to amino acid residues 24-124, 1 A-Il A, 124-224, 174-274, 224-324, 274-374, 324-424, 374-474, 424-524, 474-574, 524-624, 574-674, 624-724, 674-759, or 724-759 of SEQ ID NO:2.
  • the antibody binds to a Tiel ectodomain and includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence
  • the protein further includes one or more of the following properties: (1) at least one of the variable domain sequences includes at least one CDR of the E3 or E3b antibody; (2) at least one of the variable domain sequences includes CDR sequences at least 85% identical, in sum, to the CDRs of the corresponding variable domain of the E3 or E3b antibody; (3) at least one of the variable domains is at least 85% identical to the corresponding immunoglobulin variable domains of the E3 or E3b antibody, and (4) the protein competes with E3 or E3b for binding to Tiel or binds to an epitope that overlaps the epitope bound by E3 or E3b on Tiel.
  • the antibody is at least bivalent, e.g., with at least two antigen binding sites that bind to Tie
  • the antibody includes one or more variable domains from the E3 or E3b antibody or a variable domain sequence that is at least 70, 75, 80, 85, 90, 95, 98, or 995 identical to such a variable domain.
  • the amino acid sequences of the HC variable domain sequence include CDRl, CDR2, and CDR3 sequences from the E3 or E3b clone
  • the LC variable domain sequence includes CDRl, CDR2, and CDR3 sequences from the E3 or E3b clone.
  • the LC variable domain sequence includes SEQ ID NO:116.
  • the HC variable domain sequence includes SEQ ID NO:114.
  • the HC and LC framework regions are human.
  • the antibody binds to Tie2.
  • the antibody antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang.
  • the binding of the antibody can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang.
  • the antibody enhances Tie2 self-association, e.g., homodimerization, and thereby associates Tie2 with Tie2 and prevents association of Tie2 with Tiel or Ang.
  • the antibody causes Tiel phosphorylation.
  • the antibody prevents association of Tiel with Tie2 or Ang.
  • the antibody includes an antibody that binds to the extracellular domain of human Tie2.
  • the antibody may have one or more of these properties, e.g., the antibody may cause Tiel phosphorylation and prevent association of Tiel with Tie2 or Ang, etc.
  • the antibody can be one or more of the following: human, humanized, non-immunogenic, isolated, monoclonal, and recombinant.
  • the antibody can bind to the first Ig-like C2-type domain (Ig 1) or to the second Ig-like C2-type domain (Ig 2) of Tie2.
  • the antibody binds to an EGF-like domain of Tie2 (e.g., first, second, or third EGF-like domain).
  • the antibody binds to the fibronectin type III repeats region of Tie2.
  • the antibody binds to amino acid residues 19-119, 69-169, 119-229, 169-269, 229-329, 269-369, 329-429, 369-469, 429-529, 469-569, 529-629, 569-669, 629-729, 669-745, 729-745 of SEQ ID NO: 162.
  • the antibody binds to Ang.
  • the antibody antagonizes formation of a heteromeric complex of Tiel, Tie2, and Ang.
  • the binding of the antibody can antagonize the association between Tiel and Tie2, between Tiel and Ang, or between Tie2 and Ang.
  • the antibody can be one or more of the following: human, humanized, non- immunogenic, isolated, monoclonal, and recombinant, hi one embodiment, the antibody binds to the N-terminal domain of Angl (i.e., the N-teraiinal 50 amino acids of Angl). Li one embodiment, the antibody binds to the coiled-coil domain of Angl.
  • the antibody binds to the fibrinogen-like domain of Angl. hi one embodiment, the antibody binds to amino acid residues 1-100, 50-150, 100-200, 150- 250, 200-300, 250-350, 300-400, 350-450, 400-497, or 450-497 of SEQ ID NO.163.
  • the antibody includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence.
  • HC heavy chain
  • LC light chain
  • the HC and LC framework regions are human, hi one embodiment, the antibody also includes an Fc domain, hi one embodiment, the antibody includes the constant domains of a human IgGl, IgG2, IgG3, or IgG4.
  • the antibody is administered in an amount effective to decrease vascular development and angiogenesis.
  • the antibody is delivered systemically.
  • antibody is administered in an amount effective to reduce one or more of the following activities: sprouting, splitting, remodeling of blood vessels, vasculogenesis, and tubule formation.
  • the invention includes an isolated protein that includes one or more variable domains of an antibody described herein.
  • the invention includes a nucleic acid that includes a coding sequence that encodes a polypeptide that includes a variable domain of an antibody described herein.
  • the invention includes a pharmaceutical composition that includes an antibody described herein. The composition and antibody can include other features described herein.
  • the invention includes an antibody described herein for treatment of an angiogenesis-related disorder.
  • the antibody and treatment can include other features described herein.
  • the invention includes an antibody described herein for the manufacture of a medicament for treating an angiogenesis-related disorder.
  • the medicament and antibody can include other features described herein.
  • the invention includes a method of providing a first therapy that includes administering a first agent in combination with a second therapy, e.g., an anti-cancer therapy.
  • the first agent is an agent that decreases Tie complex formation or an agent that increases Tiel homodimerization.
  • the first agent is a Tiel binding protein
  • the second therapy includes radiation therapy or surgery.
  • the second therapy includes administering a second agent.
  • the second agent antagonizes or decreases Tie complex formation or increases Tiel homodimerization.
  • the second agent is an agent that antagonizes signaling through a VEGF pathway, e.g., a VEGF antagonist antibody, e.g., bevacizumab; VEGF-Receptor tyrosine kinase inhibitor, or another agent that antagonizes VEGF pathway signalling. See also "Combination Therapies" below.
  • the invention includes a composition that includes an agent that decreases Tie complex formation and an anti-cancer agent.
  • the anti-cancer agent can be a second agent that antagonizes Tie complex formation or a second agent that antagonizes a VEGF pathway.
  • the invention features an antibody that decreases endothelial cell activity by causing Tiel phosphorylation.
  • the antibody may decrease endothelial cell differentiation, e.g., sprouting, splitting, and tube formation.
  • the invention features a protein (e.g., an isolated protein) that includes a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence and binds to Tiel ectodomain.
  • the binding protein binds to Tiel ectodomain.
  • the protein binds with an affinity K 0 of less than 10 "8 M, 5-10 "9 M, 10 "9 M 5 10 "10 M, 10 "11 M, or 1(T 12 M.
  • one or more of the CDRs of the heavy and/or light chain variable domain sequence are human, primate, non-rodent (e.g., non-mouse or non-rat), or synthetic.
  • one or more of the framework regions of the heavy and/or light chain variable domain sequence are human, primate, or non- rodent (e.g., non-mouse or non-rat).
  • the heavy chain variable domain sequence includes one or more of the following properties: i) a HC CDRl that includes an amino acid sequence as follows:
  • X-I-Y-P-S-G-G-(WPS)-T-(YVH)-Y-A-D (SEQ ID NO : 722), wherein X is any amino acid; iii) a HC CDR3 that includes an amino acid sequence as follows:
  • VNYYDSSGYGPIAPGLDY (SEQ ID NO:128), or
  • the light chain variable domain sequence includes one or more of the following properties: i) a LC CDRl that includes an amino acid sequence as follows: R-A-S-Q-S-(IV)-S-(SR)-Xl-Y-L-(AN) (SEQ ID NO: 129), R-A-S-Q-S-(IV)-S-S-(YS)-L-(ALN) (SEQ ID NO:706), T-G-T-(SN)-S-D-V-G-(GS)-Y (SEQ ID NO:707),
  • LC CDR2 that includes an amino acid sequence as follows: X-A-S-X-R-A-T (SEQ ID NO: 133), wherein X can be any amino acid, (AGD)-A-S-(STN)-R-A-T (SEQ ID NO: 134), (DG)-(AV)-S-N-(RL)-(AP)-ST) (SEQ ID NO:709), (AGD)-A-S-(STN)-(LR)-(AEQ)-(ST) (SEQ ID NO:135), or (AGTKDEH)-A-S-(STN)-(LR)-(AVEQ)-(ST) (SEQ ID NO: 136); and iii) a LC CDR3 that includes an amino acid sequence as follows: Q-Q-(SYFR)-(GSYN)-S-(STYW)
  • the light chain variable domain sequence includes one or more of the following properties: i) a LC CDRl that includes an amino acid sequence as follows:
  • Xl is any amino acid (e.g., G or R)
  • X2 is any amino acid (e.g., Y or N)
  • X3 is any amino acid (e.g., F, N, or K)
  • X4 is any amino acid (e.g., aliphatic, e.g., V or A);
  • a LC CDR2 that includes an amino acid sequence as follows:
  • the HC CDR2 includes an amino acid sequence as follows: (GSVW)-I-(SY)-P-SG-G-(AGVMYWPQH)-T-(AGSTLVMYFKH)-Y-(AT)- D-S-V-K-G (SEQ ID NO: 147) or (GSV)-I-(SY)-P-SG-G-(WQ)-T-(GY)-Y-(AT)-D-S- V-K-G (SEQ ID NO:148).
  • the protein includes HC CDRl and HC CDR2 sequences that are related to the corresponding CDR sequences of p-F3, E3 or E3b.
  • the protein includes the sequence MYGM (SEQ ID NO: 149), at a position corresponding to HC CDRl.
  • the sequence can be followed by a small amino acid, e.g., glycine, alanine, valine, or serine.
  • the protein the sequence VISPSGGX 1 TX 2 YADSAVKG (SEQ ID NO: 150), at a position corresponding to HC CDR2.
  • X 1 can be a hydrophilic amino acid, e.g., glutamine or asparagine.
  • X 2 can be a small amino acid, e.g., glycine, alanine, valine, or serine.
  • the heavy chain variable domain sequence can have one or more of the following features: the amino acid residue at Kabat position 31 is A, H, K, N, Q, R, S, or T, e.g., H, N, R, or S; the amino acid residue at Kabat position 32 is Y; the amino acid residue at Kabat position 33 is G, K, P, R, or V, e.g., K or V; the amino acid residue at Kabat position 34 is M; the amino acid residue at Kabat position 35 is A, G, H, I, L, M, S, or V, e.g., G, H, M, or V; the amino acid residue at Kabat position 50 is G, R, S, or V, e.g., S or V; the amino acid residue at Kabat position 51 is I; the amino acid residue at Kabat position 52 is S or Y, e.g., Y; the amino acid residue at Kabat position 52a is P or S, e.g., P; the amino acid residue at Kabat position 31 is
  • two or three of the CDRs of the HC variable domain sequence match motifs that also match a HC variable domain of an antibody described herein.
  • two or three of the CDRs of the LC variable domain sequence match motifs that also match a LC variable domain of an antibody described herein.
  • the matched motifs for the CDRs are based on a HC and a LC that are paired in an antibody described herein.
  • the Hl and H2 hypervariable loops have the same canonical structure as an antibody described herein, hi one embodiment, the Ll and L2 hypervariable loops have the same canonical structure as an antibody described herein.
  • the HC CDRl amino acid sequences have a length of at least 5 amino acids of which at least 3, 4, or 5 amino acids are identical to the CDRl sequence of the HC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, ⁇ -C6, p- D12, p-F3, ⁇ -F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s- El 1, s-GlO, s-H4, or another antibody described herein, hi one embodiment, the HC CDR2 amino acid sequences have a length of at least 15, 16, or 17 amino acids of which at least 10, 12, 14, 15, 16, or 17 amino acids are identical to the CDR2 sequence of the HC of clone E3, E3b, G2, p-
  • the HC CDR2 amino acid sequences have a length of at least 17 amino acids of which at least 14, 15, 16, or 17 amino acids are identical to the CDR2 sequence of the HC of clone E3, E3b, G2, p-Al, p-AlO, p-Bl, p-B3, ⁇ -C6, p-D12, p-F3, p-F4, ⁇ -G3, s-AlO, s-Hl, s-A2, s- B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, S-GlO, s-H4, or another antibody described herein.
  • the HC CDR3 amino acid sequences have a length of at least of at least 7 or 8 amino acids of which at least 5, 6, 7, or 8 amino acids are identical to the CDR3 sequence of the HC of clone E3, E3b, G2, p-Al, p- AlO, p-Bl, p-B3, p-C6, ⁇ -D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s- ClO, s-C2, s-C7, s-Dll, s-El 1, s-GlO, s-H4, or another antibody described herein.
  • two or three of the CDRs of the HC variable domain sequence match motifs described herein such that the motifs are a set of motifs that match a HC variable domain of a clone described herein, e.g., E3, E3b, G2, p-Al, p-AlO, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s- B2, s-B9, s-ClO, s-C2, s-C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein.
  • the protein may include SEQ ID NO:118 and SEQ ID NO: 160, e.g., motifs that match the E3 HC variable domain.
  • the LC CDRl amino acid sequences have a length of at least 10, 11, or 12 amino acids of which at least 7, 8, 9, 10, or 11 amino acids are identical to the CDRl sequence of the LC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, ⁇ -D12, p-F3, ⁇ -F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s- C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein.
  • the LC CDR2 amino acid sequences have a length of at least 6 or 7 amino acids of which at least 5, 6, or 7 amino acids are identical to the CDR2 sequence of the LC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p- F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dl l, s-El 1, s- GlO, s-H4, or another antibody described herein.
  • the LC CDR3 amino acid sequences have a length of at least of at least 8, 9, or 10 amino acids of which at least 7, 8, 9, or 10 amino acids are identical to the CDR3 sequence of the LC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, ⁇ -F3, p-F4, p-G3, s- AlO, s-Hl, S-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein.
  • two or three of the CDRs of the LC variable domain sequence match motifs described herein such that the motifs are a set of motifs that match a LC variable domain of a clone described herein, e.g., E3, E3b, G2, p-Al, p-AlO, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s- B9, s-CIO, s-C2, s-C7, s-Dl 1, s-El 1, s-GlO, s-H4, or another antibody described herein.
  • the protein may include SEQ ID NO:132, SEQ ID NO:136, and SEQ ID NO:161, e.g., motifs that match the E3 LC variable domain.
  • the amino acid sequence of the HC variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of the HC variable domain of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s- C2, s-C7, s-Dl 1, s-El 1, S-GlO, s-H4, or another antibody described herein.
  • the amino acid sequence of the LC variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of the LC variable domain of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3 5 p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s- C2, s-C7, s-Dll, s-El 1, S-GlO, s-H4, or another antibody described herein.
  • the amino acid sequences of the HC and LC variable domain sequences are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequences of the HC and LC variable domains of a clone selected from the group consisting of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p- D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s- EIl, s-GlO, s-H4, and any other antibody described herein.
  • a clone selected from the group consisting of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B
  • the amino acid sequences of one or more framework regions (e.g., FRl, FR2, FR3, and/or FR4) of the HC and/or LC variable domain are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to corresponding framework regions of the HC and LC variable domains of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s- A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s-G10, s-H4, or another antibody described herein.
  • FRl, FR2, FR3, and/or FR4 are at least 70, 80, 85, 90,
  • the amino acid sequences of the HC and LC variable domain sequences comprise a sequence encoded by a nucleic acid that hybridizes (e.g., under high stringency) to a nucleic acid encoding a variable domain of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s- Hl, S-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-El I 5 s-GlO, s-H4, or another antibody described herein.
  • the light chain variable domain sequence is human or non-immunogenic in a human.
  • the heavy chain variable domain sequence is human or non-immunogenic in a human.
  • the protein can bind to cells that express Tiel, e.g., endothelial cells.
  • the protein does not substantially bind (e.g., does not detectably bind) to platelets (e.g., resting and/or activated platelets).
  • the protein inhibits tube formation by HUVECs in vitro.
  • the E3 antibody inhibits tube formation by HUVECs in vitro (e.g., under conditions described in Example 18).
  • the protein inhibits angiogenesis in an in vivo MATRIGELTM plug assay.
  • the E3 antibody can inhibit angiogenesis in an exemplary assay (see, e.g., an exemplary assay described in Example 21).
  • the protein recognizes melanoma-associated structures in a histological section, e.g., not only melanoma tissue, but antigen in surrounding structures. In one embodiment, the protein does not stain blood vessels in normal skin in a histological section.
  • the protein specifically binds to Tiel, e.g., it binds with at least a 10, 50, 100, 10 3 , or 10 4 fold preference for Tiel relative to another human protein, e.g., Tie2, a natural protein other than Tiel that has a Ig-like domain, an EGF-like domain, or fibronectin Type III repeat, or human serum albumin.
  • the protein binds to a domain of Tiel described herein.
  • the invention features a protein (e.g., an isolated protein) that modulates activity of Tiel, e.g., the Tiel receptor.
  • the protein is not naturally occurring.
  • the protein includes a HC and LC immunoglobulin variable domain sequence.
  • one or more of the CDRs of the heavy and/or light chain variable domain sequence are human, primate, non-rodent (e.g., non-mouse or non-rat), or synthetic.
  • one or more of the framework regions of the heavy and/or light chain variable domain sequence are human, primate, or non-rodent (e.g., non-mouse or non-rat).
  • the protein is substantially free of an immunoglobulin variable domain, e.g., the protein includes a peptide that independently interacts with Tiel or a polypeptide that does not include a immunoglobulin variable domain.
  • the protein activates an activity of the Tiel protein, e.g., an activity in the Tiel/EpoR chimeric BaF3 cell assay described in Example 2.
  • a protein that activates in this assay can behave as antagonists in other conditions, for example, in vivo.
  • the protein includes the HC and LC immunoglobulin variable domains of the E3, E3b, or other antibody, HC and/or LC immunoglobulin variable domain sequences that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical in the CDR regions to the respective CDRs of the E3, E3b or other antibody described herein.
  • the protein competes with E3, E3b, or other antibody described herein for binding to Tiel or binds to an epitope that overlaps an epitope that is recognized by E3, E3b, or other antibody described herein, or that has at least one, two or three residues in common with an epitope that is recognized by E3, E3b, or other antibody described herein.
  • the activating protein enables IL-3 dependent cells that express a chimeric receptor including the Tiel extracellular domain and the EpoR intracellular domain to survive in the absence of IL-3.
  • the protein can cause dimerization of Tiel .
  • the protein can cause auto-phosphorylation of the RTK domain of Tiel.
  • the protein synergizes with the E3 or E3b antibody to activate an activity of Tie, e.g., in the Tiel/EpoR chimeric BaF3 cell assay.
  • the protein includes the HC and LC immunoglobulin variable domains of the G2 or C7 antibody or domains that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical in the CDR regions.
  • the protein competes with G2 or C7 for binding to Tiel or binds to an epitope that overlaps an epitope that is recognized by G2 or C7 or that has at least one, two or three residues in common with an epitope that is recognized by G2 or C7.
  • the protein antagonizes an activity of the Tiel protein.
  • the protein can at least partially inhibit the ability of the E3 or E3b antibody to agonize the Tie protein.
  • the protein can at least partially inhibit the ability of the E3 or E3b antibody to enable IL-3 dependent cells that express a chimeric receptor including the Tiel extracellular domain and the EpoR intracellular domain to survive in the absence of IL-3.
  • the HC and LC immunoglobulin variable domain sequences of the protein include the amino acid sequences that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequences of respective immunoglobulin variable domains of B2 or Dl 1.
  • the Tiel binding protein includes the HC and LC immunoglobulin variable domains of an antibody selected from the group consisting of: B2, Dl 1, A2, AlO, P-Bl, P-B3, and P-C6 or immunoglobulin domains that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical in the CDR regions to the CDR regions of the respective antibodies.
  • the protein binds with an affinity K D of less than 10 "8 M, 5-10 "9 M, 10 "9 M, 10 '10 M, 10 ' ⁇ M, or 10 '12 M.
  • the protein can at least partially inhibit the ability of a naturally occurring Tiel binding protein from interacting with the Tie protein.
  • the protein can include other features described herein.
  • the invention features an antibody (e.g., an isolated antibody) that binds to the Tiel ectodomain, but does not substantially bind to platelets, e.g., as detected by fluorescence activated cell sorting.
  • the antibody does not substantially bind to activated platelets and/or resting platelets.
  • the antibody binds to endothelial cells, hi one embodiment, the protein is a monoclonal antibody.
  • the antibody can be provided in a preparation that is free of other Tiel-binding antibodies that have other specificities, e.g., free of Tiel binding antibodies that bind to platelets.
  • the antibody can include other features described herein.
  • the invention features a protein (e.g., an isolated protein) that preferentially binds to a Tiel protein in a conformation stabilized by the E3 or E3b antibody relative to an endogenous Tiel protein in an unstimulated state, hi one embodiment, the protein includes immunoglobulin HC and LC domains, hi another embodiment, the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Tiel.
  • the peptide can include one, two, or three disulfide bonds.
  • the protein can include other features described herein.
  • the invention features a protein (e.g., an isolated protein) that preferentially binds to a Tiel protein in a dimeric conformation relative to a monomelic Tiel protein.
  • the protein includes immunoglobulin HC and LC domains.
  • the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Tiel .
  • the peptide can include one, two, or three disulfide bonds.
  • the protein can include other features described herein.
  • the invention features a protein (e.g., an isolated protein) that preferentially binds to a Tie2 protein in a conformation that is biased against interaction with Ang or Tiel .
  • the protein includes immunoglobulin HC and LC domains
  • the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Tie2.
  • the peptide can include one, two, or three disulfide bonds.
  • the protein can include other features described herein.
  • the invention also features nucleic acid aptamers that have one or more of these properties.
  • the invention features a protein (e.g., an isolated protein) that preferentially binds to an Ang protein, and modulates (e.g., inhibits) interaction with Tiel and Tie2.
  • the protein includes immunoglobulin HC and LC domains
  • the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Ang.
  • the peptide can include one, two, or three disulfide bonds.
  • the protein can include other features described herein.
  • the invention also features nucleic acid aptamers that have one or more of these properties.
  • the invention features a protein (e.g., an isolated protein) that binds to an epitope of Tiel ectodomain with a K D of less than 2 X 10 "7 M.
  • the epitope overlaps, is within, or includes an epitope bound by E3, E3b, G2, p- Al, p-AlO, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s- B9, s-ClO, s-C2, s-C7, s-Dll, s-Ell, s-G10, s-H4, or another antibody described herein or that includes at least one, two, or three residues in common.
  • the protein binds with an affinity K D of less than 10 "8 M, 5-10 "9 M, 10 “9 M, 10 '10 M, 10 "n M, or 10 "12 M.
  • the protein includes immunoglobulin HC and LC domains.
  • the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Tiel .
  • the peptide can include one, two, or three disulfide bonds.
  • the protein can include other features described herein.
  • the invention also features nucleic acid aptamers that have one or more of these properties.
  • the invention features a protein (e.g., an isolated protein) that competitively inhibits binding of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s- Dl 1, s-El 1, s-GlO, s-H4, or another antibody described herein to a Tiel ectodomain.
  • a protein e.g., an isolated protein
  • the protein includes immunoglobulin HC and LC domains, hi another embodiment, the protein includes a peptide (e.g., of length less than 30, 28, 25, 22, 20, 18, 16, or 14 amino acids) that independently binds to Tiel.
  • the peptide can include one, two, or three disulfide bonds.
  • the protein can include other features described herein.
  • the invention features a protein (e.g., an isolated protein) that includes a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence and that antagonizes an activity of the Tiel ectodomain.
  • CDRl of the light chain variable domain sequence includes: Q-S-X-S-S (SEQ ID NO:151) or R-A-S-Q-S-X- S-S-Y-L-A (SEQ ID NO: 152), wherein X is any amino acid or optionally aliphatic, e.g., isoleucine or valine.
  • CDR2 of the light chain variable domain sequence includes: A-S-X 1 -R-X 2 -T (SEQ ID NO:153) or D-A-S-X 1 -R-X 2 -T (SEQ ID NO: 154) , wherein X 1 is any amino acid or optionally a hydrophilic amino acid, e.g., serine or asparagine, and X 2 is any amino acid or optionally aliphatic or small aliphatic, e.g., alanine or valine.
  • CDR3 of the light chain variable domain sequence includes: Q-R-S-X 2 -W-P-R (SEQ ID NO: 155) or X 1 -Q-R- S-X 2 -W-P-R-T (SEQ ID NO: 156), wherein X 1 is any amino acid or optionally leucine or glutamine, and X 2 is any amino acid or optionally lysine or serine.
  • the protein competes with the B2 and/or DIl antibody for binding to Tiel or competitively inhibits binding of B2 and/or Dl 1 to Tiel.
  • the protein antagonizes a Tiel activity that is stimulated by the E3 or E3b antibody. In one embodiment, the protein inhibits dimerization of Tiel.
  • the protein can include other features described herein.
  • the invention features an isolated, mono-specific protein including a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to Tiel ectodomain and includes a human or non-mouse constant domain (e.g., a human IgGl, IgG2, IgG3, or IgG4 constant domain).
  • a human or non-mouse constant domain e.g., a human IgGl, IgG2, IgG3, or IgG4 constant domain.
  • the protein can include other features described herein.
  • the invention features an isolated, human antibody that binds to a Tiel ectodomain.
  • the protein can include other features described herein.
  • the invention features an isolated antibody (e.g., an isolated antibody) that binds to a Tiel ectodomain and contains less than 5, 4, 3, or 2 peptides (of between 6-9 amino acid length) that are non-human in origin or less than 5, 4, 3, or 2 peptides that are potential human T cell epitopes.
  • the antibody contains no peptide (of 6-9 amino acid length) that is non-human in origin or that is a potential human T cell epitope.
  • the antibody is obtained by a method that includes deimmunization.
  • the antibody is deimmunized, e.g., completely deimmunized.
  • the protein can include other features described herein.
  • the invention features an isolated antibody that binds to a Tiel ectodomain and that includes a modified Fc domain, e.g., a modified human Fc domain.
  • a modified Fc domain e.g., a modified human Fc domain.
  • antibodies may include modifications, e.g., that alter Fc function.
  • the human IgGl constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237, e.g., according to the number in US 5,648,260. Other exemplary modifications include those described in US 5,648,260.
  • the protein can include other features described herein.
  • the invention features an isolated protein that binds to the Tiel receptor with an affinity K 0 of less than 10 "7 M, 10 "8 M, 5-10 “9 M, 10 “9 M, 10 "10 M, 10 "11 M, or 10 '12 M.
  • the protein can include other features described herein.
  • the invention features an isolated protein including a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to Tiel ectodomain and, for example, includes at least one or more CDRs that are a non- primate CDR (e.g., a non-mouse or non-rabbit CDR) or a synthetic CDR.
  • the protein can include other features described herein.
  • the invention features an isolated nucleic acid including a coding sequence that encodes a polypeptide including an immunoglobulin HC variable domain of an antigen binding protein that binds to Tiel .
  • the nucleic acid or polypeptide can include one or more other features described herein.
  • the nucleic acid can include one or more altered codons.
  • the nucleic acid includes SEQ ID NOs:725 and/or 726. Also featured is a mammalian expression vector that includes SEQ ID NOs:725 and/or 726.
  • the nucleic acid further includes a second coding sequence that encodes a polypeptide including an immunoglobulin HC variable domain, e.g., an HC domain described herein.
  • the nucleic acid further includes a promoter operably linked to the coding sequence.
  • the invention features a nucleic acid that includes one or more coding sequence that encodes one or more polypeptide chains that collectively include an immunoglobulin HC or LC variable domain of an antigen binding protein that binds to Tiel.
  • the nucleic acid segment encoding at least one of the variable domains hybridizes to a nucleic acid described herein, e.g., under stringent conditions (e.g., high stringency conditions), e.g., it hybridizes to a region encoding a variable domain and is at least 80, 85, 90, 95, or 98% of the length of such a region.
  • the nucleic acid can include other features described herein.
  • the invention features a host cell that contains a first nucleic acid sequence encoding a polypeptide including a HC variable domain of an antigen binding protein and a second nucleic acid sequence encoding a polypeptide including a LC variable domain of the antigen binding protein, wherein the antigen binding protein binds to Tiel with a K D of less than 2 x 10 "7 M.
  • the HC or LC variable domain includes at least one human CDR.
  • the antigen binding protein can include other features described herein.
  • the invention features a host cell that contains a first nucleic acid encoding a polypeptide including a HC variable region and a second nucleic acid encoding a polypeptide including a LC variable region, wherein the HC and the LC variable regions each include at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to respective amino acid sequences of the HC and LC variable domains of a clone selected from the group consisting of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, ⁇ -C6, ⁇ -D12, ⁇ -F3, ⁇ -F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s- C2, s-C7, s-Dl 1, s-El 1, s-GlO, and s
  • the invention features a pharmaceutical composition including a protein described herein that interacts with Tiel and a pharmaceutically acceptable carrier.
  • the invention features a therapeutic composition including a protein described herein that interacts with Tiel wherein the composition is sterile and suitable for administration to a subject.
  • the invention features a method that includes: providing a signal-dependent or signal-responsive cell that expresses a chimeric receptor including the Tiel extracellular domain and a heterologous intracellular sequence that can produce a signal; contacting a candidate compound to the cell; and evaluating a property of the cell that is dependent on the signal.
  • the intracellular sequence includes at least a region of an intracellular sequence of the EpoR protein. The method can be used, e.g., to evaluate activity of a candidate compound, or a plurality of compounds.
  • the invention features a method that includes: providing an IL-3 dependent cell that expresses a chimeric receptor including the Tiel extracellular domain and the EpoR intracellular domain; contacting a candidate compound to the cell under conditions in which the concentration of IL-3 is not sufficient to sustain viability of the cell; and evaluating a property of the cell.
  • the method can be used, e.g., to evaluate activity of a candidate compound, or a plurality of compounds.
  • the property is viability.
  • the evaluating includes an MTT assay.
  • the method further includes administering the candidate compound to a subject.
  • the candidate compound includes a protein, e.g., a protein that includes an immunoglobulin variable domain.
  • the invention features method of identifying a compound that modulates Tiel activity.
  • the method includes: providing a plurality of candidate compounds; and evaluating each compound of the plurality using a method described herein.
  • the invention features a culture cell that expresses a chimeric transmembrane protein including a region of the Tiel extracellular domain and a heterologous intracellular sequence.
  • the intracellular sequence includes a region of the EpoR intracellular domain.
  • the cell requires IL-3 or Tiel for viability.
  • the cell is IL-3 dependent in the absence of the chimeric transmembrane protein, but is viable in the presence of the E3 or E3b antibody and the absence of IL-3.
  • the invention features a preparation that includes the isolated mammalian cells (e.g., cells that expresses a chimeric transmembrane protein including a region of the Tiel extracellular domain and a heterologous intracellular sequence) and a Tiel -binding protein, wherein the Tiel -binding protein is necessary to sustain viability of the cells.
  • isolated mammalian cells e.g., cells that expresses a chimeric transmembrane protein including a region of the Tiel extracellular domain and a heterologous intracellular sequence
  • Tiel -binding protein is necessary to sustain viability of the cells.
  • the invention features a kit including: a Tiel -binding protein and a culture cell that expresses a chimeric transmembrane protein including a region of the Tiel extracellular domain and a heterologous intracellular sequence.
  • the invention features a method of evaluating a candidate compound.
  • the method includes: providing a preparation that includes (i) a cell or membrane fraction that contains (a) an insoluble protein that includes a region of the Tiel extracellular domain and a kinase domain and (b) ATP; (ii) a ligand that alters activity of the kinase domain; and (iii) the candidate compound; and evaluating the phosphorylation state of the insoluble protein.
  • the invention features a method of evaluating a candidate compound.
  • the method includes: providing a preparation that includes (i) a cell or membrane fraction that includes a Tiel protein or a transmembrane protein that includes at least a region of the Tiel extracellular domain and ATP; (ii) a ligand that causes autophosphorylation of Tiel or the transmembrane protein; and (iii) the candidate compound; and evaluating phosphorylation state of the Tiel protein.
  • the ligand is an antibody.
  • the ligand includes the HC and LC immunoglobulin variable domains of the E3 or E3b antibody or domains that are at least 90% identical in the CDR regions.
  • the method further includes administering the candidate compound to a subject.
  • the invention features a method that includes: providing a preparation that includes (i) a cell or membrane fraction that includes a transmembrane protein that includes at least a region of the Tiel extracellular domain and ATP; and (ii) a ligand that causes autophosphorylation of Tiel or the transmembrane protein; and evaluating phosphorylation state of the transmembrane protein.
  • the invention features a method that includes: contacting a mammalian cell with a ligand that (i) can agonize Tiel autophosphorylation and/or (ii) can enable an IL-3 dependent cell that expresses a chimeric receptor including the Tiel extracellular domain and the EpoR intracellular domain to remain viable under conditions in which the concentration of IL-3 is not sufficient to sustain viability of the cell; and evaluating the mammalian cell, hi one embodiment, the cell expresses an endogenous Tiel protein.
  • the cell is an endothelial cell
  • the method further includes contacting the mammalian cell with a test compound, other than the ligand.
  • the ligand is an antibody.
  • the ligand includes the HC and LC immunoglobulin variable domains of the E3 or E3b antibody or domains that are at least 90% identical in the CDR regions.
  • the invention features a method that includes: contacting a mammalian cell or fraction thereof with an agent that can modulate the activity of Tiel; and evaluating the mammalian cell or fraction thereof.
  • the agent is contacted to the cell while the cell is living, and the evaluating includes isolating a fraction of the cell, hi one embodiment, the agent is a protein, e.g., an antibody or a peptide.
  • the agent includes the HC and LC immunoglobulin variable domains of the E3 or E3b antibody or domains that are at least 90% identical in the CDR regions to the E3 or E3b antibody.
  • the agent includes the HC and LC immunoglobulin variable domains of the B2 or Dl 1 antibody or domains that are at least 90% identical in the CDR regions to the B2 or Dl 1 antibody.
  • the agent includes the HC and LC immunoglobulin variable domains of the A2, AlO, P-Bl, P-B3, or P-C6 antibody or domains that are at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical% identical in the CDR regions to the A2, AlO, P-Bl, P-B3, or P-C6 antibody.
  • the agent includes the HC and LC immunoglobulin variable domains of the G2 or C7 antibody or domains that are at least 90% identical in the CDR regions to the G2, or C7 antibody.
  • the agent can include other features described herein.
  • the invention features a method of evaluating a test compound.
  • the method includes evaluating interaction between an agent that can modulate the activity of Tiel and a protein that includes at least a region of the Tiel extracellular domain in the presence of the test compound.
  • the agent is a test compound is a small organic compound with molecular weight less than 8000, 7000, 6000, 5000, or 3000 Daltons.
  • the evaluating includes contacting cells that include the protein that includes at least a region of the Tiel extracellular domain with the agent in the presence of the test compound.
  • the evaluating includes forming a cell-free preparation that includes the protein that includes at least a region of the Tiel extracellular domain, the agent, and the test compound.
  • the invention features an artificial protein complex that includes (i) a protein that includes a Tiel extracellular domain and (ii) a Tiel binding protein that can modulate (e.g., agonize or antagonize) an activity of Tiel.
  • the ligand is an antibody (e.g., an antibody described herein).
  • the ligand includes the HC and LC immunoglobulin variable domains of an antibody selected from the group consisting of: E3, E3b, B2, Dl 1, A2, AlO, P-Bl, P- B3, P-C6, G2 and C7, or immunoglobulin domains that are at least 90% identical in the CDR regions to the CDR regions of the respective antibody.
  • the complex is present in a membrane fraction, on a mammalian cell, and/or in a subject.
  • the invention features a method that includes: administering a composition that includes a protein that interacts with Tiel, Tie2, or Ang (e.g., a protein described herein) to a subject in an amount effective to reduce angiogenesis in the subject or otherwise treat or prevent a disorder in a subject.
  • the protein binds to Tiel, Tie2, or Ang with an affinity K D of less than 10 " M, 5-10- 9 M, 10 "9 M 3 10 "10 M, 10 "n M, or 10 "12 M.
  • the protein is a Tiel binding protein.
  • the protein can have at least two valencies, each of which binds to Tiel .
  • at least one, two, or all of the valencies can be binding sites that competes with E3 for binding to Tiel .
  • the protein competes with E3 for binding to Tiel or binds to an epitope that overlaps the epitope bound by E3 on Tiel.
  • the protein comprises a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence.
  • the protein further includes one or more of the following properties: (1) at least one of the variable domain sequences comprising at least one CDR of the E3 antibody; (2) at least one of the variable domain sequences comprising CDR sequences at least 85% identical, in sum, to the CDRs of the corresponding variable domain of the E3 antibody; (3) at least one of the variable domains is at least 85% identical to the corresponding immunoglobulin variable domains of the E3 antibody, and (4) the protein competes with E3 for binding to Tiel or binds to an epitope that overlaps the epitope bound by E3 on Tiel.
  • one or more of the CDRs of the heavy and/or light chain variable domain sequence are human, primate, non-rodent (e.g., non-mouse or non-rat), or synthetic.
  • one or more of the framework regions of the heavy and/or light chain variable domain sequence are human, primate, or non- rodent (e.g., non-mouse or non-rat).
  • the heavy chain includes one or more of the following properties: i) a HC CDRl that includes an amino acid sequence as follows: (AGSR)-Y-(GVK)-M-(GSVF), (SEQ ID NO: 117) (AGSIMRH)-Y-(GVMK)-M-(GSVMFH) (SEQ ID NO: 118), or (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID NO: 119); ii) a HC CDR2 that includes an amino acid sequence as follows:
  • X-I-Y-P-S-G-G-(WPS)-T-(YVH)-Y-A-D (SEQ ID NO:704), wherein X is any amino acid; iii) a HC CDR3 that includes an amino acid sequence as follows:
  • VNYYDSSGYGPIAPGLDY (SEQ ID NO: 128), or
  • the light chain includes one or more of the following properties: i) a light chain cdrl that includes an amino acid sequence as follows:
  • R-A-S-Q-S-(IV)-S-(SR)-Xl-Y-L-(AN) (SEQ ID NO: 129)
  • R-A-S-Q-S-(IV)-S-S-(YS)-L-(ALN) (SEQ ID NO:706)
  • T-G-T-(SN)-S-D-V-G-(GS)-Y (SEQ ID NO:707)
  • the heavy chain includes one or more of the following properties: i) a HC CDRl that includes an amino acid sequence as follows: (AGSIMRH)-Y-(GVMK)-M-(GSVMFH) (SEQ ID NO: 118), or (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID NO: 118), or (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID NO: 118), or (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID NO: 118), or (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVMYWFKH) (SEQ ID NO: 118), or (AGSIMRNH)-Y-(AGTVMKPQ)-M-(AGSTVM
  • a HC CDR2 that includes an amino acid sequence as follows: (GSV)-I-(SY)-P-S-G-G-(NWQ)-T-(GY) (SEQ ID NO: 160), (GSV)-I-(SY)-P-S-G-G-(NWQ)-T-(GY)-Y-A-D-S-V-K-G (SEQ ID NO:122), or
  • HC CDR3 that includes an amino acid sequence as follows: APRGYSYGYYY (SEQ ID NO:712).
  • the light chain includes one or more of the following properties: i) a LC CDRl that includes an amino acid sequence as follows: R-A-S-(REQ)-(GSTRISO-(IV)-(GSTIRN)-(STIRH)-XI-(SYWNH)-(LV)-(ASN) (SEQ ID NO: 132), wherein Xl can be serine or absent; ii) a LC CDR2 that includes an amino acid sequence as follows: (TAGD)-A-S-(STN)-(LR)-(AEQ)-(ST) (SEQ ID NO:713), or (AGTKDEH)-A-S-(STN)-(LR)-(AVEQ)-(ST) (SEQ ID NO:136); and iii) a LC CDR3 that includes an amino acid sequence as follows: Q-Q-(SYFR)- (GSYN)-S-(STYW)-(RP)-(LH
  • the light chain includes one or more of the following properties: i) a LC CDRl that includes an amino acid sequence as follows: S-X-(ND)-(IV)-(AG)-Xl -X2-X3 (SEQ ID NO:142), or T-(GR)-(ST)-S-X5-(ND)- (rV)-(AG)-Xl-X2-X3-Y-X4-S (SEQ ID NO: 143), wherein Xl is any amino acid (e.g., G or R), X2 is any amino acid (e.g., Y or N), X3 is any amino acid (e.g., F, N, or K), X4 is any amino acid (e.g., aliphatic, e.g., V or A); iii) a LC CDR2 that includes an amino acid sequence as follows: (DE)-V-N-N-R-P-S (SEQ ID NO: 144); (DE)-V-N-N
  • the HC CDR2 includes an amino acid sequence as follows: (GSVW)-I-(SY)-P-SG-G-(AGVMYWPQH)-T-(AGSTLVMYFKH)-Y-(AT)- D-S-V-K-G (SEQ ID NO:147)or (GSV)-I-(SY)-P-SG-G-(WQ)-T-(GY)-Y-(AT)-D-S- V-K-G (SEQ ID NO: 148).
  • the HC CDRl amino acid sequences have a length of at least 5 amino acids of which at least 3, 4, or 5 amino acids are identical to the CDRl sequence of the HC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, ⁇ -C6, p- D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s- EIl, S-GlO, s-H4, or another antibody described herein, hi one embodiment, the HC CDR2 amino acid sequences have a length of at least 15, 16, or 17 amino acids of which at least 10, 12, 14, 15, 16, or 17 amino acids are identical to the CDR2 sequence of the HC of clone E3, E3b, G2, p-Al, p-
  • the HC CDR2 amino acid sequences have a length of at least 17 amino acids of which at least 14, 15, 16, or 17 amino acids are identical to the CDR2 sequence of the HC of clone E3, E3b, G2, p-Al, p-AlO, p-Bl, p-B3, ⁇ -C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s- B2, s-B9, s-ClO, s-C2, s-C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein.
  • the HC CDR3 amino acid sequences have a length of at least of at least 7 or 8 amino acids of which at least 5, 6, 7, or 8 amino acids are identical to the CDR3 sequence of the HC of clone E3, E3b, G2, p-Al, p- AlO, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s- ClO, s-C2, s-C7, S-DIl 5 S-EIl, S-GlO 5 s-H4, or another antibody described herein.
  • the LC CDRl amino acid sequences have a length of at least 10, 11, or 12 amino acids of which at least 7, 8, 9, 10, or 11 amino acids are identical to the CDRl sequence of the LC of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s- C7, s-Dl 1, s-El 1, S-GlO 5 s-H4, or another antibody described herein.
  • the LC CDR2 amino acid sequences have a length of at least 6 or 7 amino acids of which at least 5, 6, or 7 amino acids are identical to the CDR2 sequence of the LC of clone E3, E3b, G2, p-Al, ⁇ -A10, p-Bl, p-B3, ⁇ -C6, p-D12, p- F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s- GlO 5 s-H4, or another antibody described herein.
  • the LC CDR3 amino acid sequences have a length of at least of at least 8, 9, or 10 amino acids of which at least 7, 8, 9, or 10 amino acids are identical to the CDR3 sequence of the LC of clone E3, E3b, G2, p-Al 5 ⁇ -A10, p-Bl, ⁇ -B3, ⁇ -C6, ⁇ -D12, ⁇ -F3, ⁇ -F4, p-G3, s- AlO, s-Hl, s- A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s-G10, s-H4, or another antibody described herein.
  • the amino acid sequence of the HC variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of the HC variable domain of clone E3, E3b, G2, p-Al, p-A10, p-Bl, ⁇ -B3, p-C6, p-D12, ⁇ -F3, ⁇ -F4, ⁇ -G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s- C2 5 s-C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein.
  • the amino acid sequence of the LC variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of the LC variable domain of clone E3, E3b 5 G2, p-Al, p-A10 5 p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9 5 s-CIO, s- C2, s-C7, s-Dll, s-Ell, S-GlO, s-H4, or another antibody described herein.
  • the amino acid sequences of the HC and LC variable domain sequences are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to the amino acid sequences of the HC and LC variable domains of a clone selected from the group consisting of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p- D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s- EIl, s-GlO, and s-H4.
  • a clone selected from the group consisting of E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p
  • the amino acid sequences of one or more framework regions (e.g., FRl, FR2, FR3, and/or FR4) of the HC and/or LC variable domain are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to corresponding framework regions of the HC and LC variable domains of clone E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, p-F3, p-F4, p-G3, S-AlO, s-Hl, s- A2, S-B2, s-B9, s-CIO, s-C2, s-C7, s-Dll, s-Ell, s-GlO, s-H4, or another antibody described herein.
  • FRl, FR2, FR3, and/or FR4 are at least 70, 80, 85, 90, 92
  • the light chain variable domain sequence is human or non-immunogenic in a human.
  • the heavy chain variable domain sequence is human or non-immunogenic in a human.
  • the protein can bind to cells that express Tiel, e.g., endothelial cells.
  • the protein does not substantially bind (e.g., does not detectably bind) to platelets.
  • the protein specifically binds to Tiel , e.g., it binds with at least a 10, 50, 100, 10 3 , or 10 4 fold preference for Tiel relative to another human protein, e.g., Tie2, a natural protein other than Tiel that has a Ig-like domain, an EGF-like domain, or fibronectin Type III repeat, or human serum albumin.
  • the protein binds to a domain of Tiel described herein.
  • the protein is delivered locally. In one embodiment, the protein is delivered systemically.
  • the subject is in need of reduced angiogenesis, or identified as such.
  • the subject has an angiogenesis-related disorder.
  • the subject has a neoplastic disorder, e.g., a metastatic cancer.
  • the subject has an angiogenesis-dependent cancer or tumor.
  • the tumor can be a solid tumor, e.g., a tumor at least 1, 2, 3, 5, 8 or 10 mm in diameter.
  • the solid tumor has a hypoxic core.
  • the method can further include administering an anti-metabolite (e.g., 5-FU, with leucovorin), irinotecan, (or other topoisomerase inhibitor), doxorubicin, bevacizumab, or all of these agents.
  • an anti-metabolite e.g., 5-FU, with leucovorin
  • irinotecan or other topoisomerase inhibitor
  • doxorubicin doxorubicin
  • bevacizumab or all of these agents.
  • the method can include, prior to administering the antagonist, evaluating the subject and detecting a solid tumor in the subject.
  • the subject has an inflammatory disorder, e.g., rheumatoid arthritis, psoriasis, rheumatoid or rheumatic inflammatory disease, or other chronic inflammatory disorders, such as chronic asthma, arterial or post- transplantational atherosclerosis, and endometriosis.
  • inflammatory disorders e.g., rheumatoid arthritis, psoriasis, rheumatoid or rheumatic inflammatory disease, or other chronic inflammatory disorders, such as chronic asthma, arterial or post- transplantational atherosclerosis, and endometriosis.
  • Other disorders that can be treated include those that have deregulated or undesired angiogenesis, such as ocular neovascularization, e.g., retinopathies (including diabetic retinopathy and age-related macular degeneration) hemangioblastoma, hemangioma, and arteriosclerosis.
  • the protein is administered in an amount effective to reduce one or more of the following activities: sprouting, splitting and remodeling of blood vessels. In one embodiment, the protein is administered in an amount effective to reduce vasculogenesis or tubule formation.
  • the method further includes, prior to the administering, identifying the subject as a subject in need of reduced angiogenesis. In one embodiment, the method further includes administering the protein continuously or in separate boluses. In one embodiment, the method further includes monitoring the subject during the course of administration. For example, the monitoring includes imaging blood vessels (locally or throughout) the subject. In another example, the monitoring include evaluating tumor size or tumor load in the subject.
  • the invention features a method that includes: administering a composition that includes a protein described herein (e.g., a protein that reduces a Tiel activity) to a subject in an amount effective to reduce a Tiel activity in the subject.
  • a composition that includes a protein described herein (e.g., a protein that reduces a Tiel activity) to a subject in an amount effective to reduce a Tiel activity in the subject.
  • the method can include other features described herein.
  • the invention features a method that includes: administering a composition that includes a protein described herein (e.g., a protein that can modulate an activity of Tiel) to a subject in an amount effective to modulate endothelial cell activity in the subject.
  • a protein described herein e.g., a protein that can modulate an activity of Tiel
  • the protein is delivered into the circulation.
  • the composition is effective for sensitizing endothelial cells to a treatment, and providing a treatment to the subject that inhibits, kills, ablates, or otherwise arrests the sensitized endothelial cells.
  • the invention features a method that includes: (i) contacting the sample (and optionally, a reference, e.g., control, sample) with a protein that binds to Tiel, e.g., a protein described herein, under conditions that allow interaction of the Tiel -binding protein and the Tiel protein to occur; and (ii) detecting formation of a complex between the Tiel -binding protein, and the sample (and optionally, the reference, e.g., control, sample).
  • a protein that binds to Tiel e.g., a protein described herein
  • the invention features a method that includes: (i) administering to a subject (and optionally a control subject) a Tiel -binding protein (e.g., an antibody or antigen binding fragment thereof), under conditions that allow interaction of the Tiel -binding protein and the Tiel protein to occur; and (ii) detecting formation of a complex between the Tiel -binding protein and a Tiel molecule of the subject or detecting distribution of Tiel -binding protein or at least one location of the Tiel-binding protein in the subject.
  • the Tiel-binding protein does not modulate the activity of Tiel.
  • the Tiel-binding protein can be a protein described herein.
  • the ligand detects activated Tiel.
  • An antibody that binds to Tiel is preferably monospecific, e.g., a monoclonal antibody, or antigen-binding fragment thereof.
  • the antibody can recognize Tiel on a living cell, e.g., an endogenous Tiel molecule or a Tiel molecule that is expressed from a heterologous nucleic acid.
  • the Tiel-binding protein interacts with primary endothelial cells.
  • the term "monospecific antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope.
  • a monoclonal antibody which refers to an antibody that is produced as a single molecular species, e.g., from a population of homogenous isolated cells.
  • a "monoclonal antibody composition” refers to a preparation of antibodies or fragments thereof of in a composition that includes a single molecular species of antibody.
  • a monoclonal antibody is produced by a mammalian cell.
  • One or more monoclonal antibody species may be combined.
  • the Tiel-binding antibodies can be foil-length (e.g., an IgG (e.g., an IgG (e.g., an IgG) (e.g., an IgG)
  • the antibody can include two heavy chain immunoglobulins and two light chain immunoglobulins, or can be a single chain antibody.
  • the antibodies can, optionally, include a constant region chosen from a kappa, lambda, alpha, gamma, delta, epsilon or a mu constant region gene.
  • a Tiel-binding antibody can include a heavy and light chain constant region substantially from a human antibody, e.g., a human IgGl constant region or a portion thereof.
  • the antibody is a recombinant or modified antibody, e.g., a chimeric, a humanized, a deimmunized, or an in vitro generated antibody.
  • the term "recombinant” or “modified” human antibody, as used herein, is intended to include all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant antibodies include humanized, CDR grafted, chimeric, deimmunized, in vitro generated antibodies, and may optionally include constant regions derived from human germline immunoglobulin sequence
  • the antibody binds to an epitope distinct from an epitope bound by known monoclonal antibodies that bind to Tiel, e.g., an antibody described in WO 95/26364, e.g., 3C4C7G6 and 10Fl 1G6.
  • the antibody does not compete with known monoclonal antibodies that bind to Tiel, e.g., 3C4C7G6 and 10F11G6.
  • the antibody does not compete with ligand described herein, e.g., the E3 antibody.
  • the antibodies or other agents bind overlapping epitopes of or competitively inhibit the binding of monospecific antibodies, e.g., E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, p-C6, p-D12, ⁇ -F3, p-F4, p- G3, S-AlO, s-Hl, S-A2, s-B2, s-B9, s-CIO, s-C2, s-C7, s-Dl l, s-Ell, S-GlO, s-H4, or another antibody described herein to Tiel, or vice versa (e.g., the monospecific antibodies competitively inhibiting binding of the ligands
  • Overlapping epitopes can include at least one amino acid in common.
  • Agents that competitively inhibit binding of one another do not necessarily bind to overlapping epitopes. For example, they may inhibit binding by steric interference or by altering the conformation of Tiel.
  • binding proteins are within the scope of the invention, e.g., two or more antibodies that bind to different regions of Tiel, Tie2, or Ang, e.g., antibodies that bind to two different epitopes on the extracellular domain of Tiel, Tie2, or Ang, e.g., abispecific antibody.
  • the Tiel-binding antibody or antigen-binding fragment thereof includes at least one light or heavy chain immunoglobulin (or preferably, at least one light chain immunoglobulin and at least one heavy chain immunoglobulin).
  • each immunoglobulin includes a light or a heavy chain variable region having at least one, two and, preferably, three complementarity determining regions (CDRs) substantially identical to a CDR from an anti-Tiel light or heavy chain variable region, respectively, i.e., from a variable region of an antibody described herein, e.g., E3, E3b, G2, p-Al, p-A10, p-Bl, p-B3, ⁇ -C6, p-D12, p-F3, p-F4, p-G3, s-AlO, s-Hl, s-A2, s-B2, s-B9, s-CIO, s-C2, s-C7,
  • CDRs complementarity
  • the invention features an agent (e.g., an antibody) that decreases endothelial cell activity by increasing Tiel phosphorylation, hi one embodiment, the agent decreases endothelial cell differentiation, e.g., sprouting, splitting, and tube formation.
  • an agent e.g., an antibody
  • the agent decreases endothelial cell differentiation, e.g., sprouting, splitting, and tube formation.
  • the invention features an agent (e.g., an antibody) that decreases endothelial cell activity by activating a signaling pathway.
  • an agent e.g., an antibody
  • the antibody decreases endothelial cell differentiation, e.g., sprouting, splitting, and tube formation. This agent-induced effect can be independent or dependent of Tiel self-association.
  • the invention features an isolated protein that includes a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to Tiel ectodomain and the heavy chain immunoglobulin variable domain sequence includes one or more of the following properties: i) a HC CDRl that includes an amino acid sequence of a clone from the group consisting of: M0044-A06; M0044-A11; M0044- B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0044-H09; M00
  • the protein also includes the light chain immunoglobulin variable domain sequence which includes one or more of the following properties: i) a LC CDRl that includes an amino acid sequence of a clone from the group consisting of: M0044-A06; M0044-A11; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-
  • the protein includes the amino acid sequence of the HC variable domain sequence which is at least 85, 90, 95, 98, or 99% identical to the amino acid sequence of the HC variable domain of clone M0044-A06; M0044- Al 1; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-
  • the protein includes the amino acid sequence of the LC variable domain sequence which is at least 85, 90, 95, 98, or 99% identical to the amino acid sequence of the LC variable domain of clone M0044-A06; M0044- AIl; M0044-B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06; M0044-F09; M0044-G06; M0044-G07; M0044-G11; M0044-H03; M0044-H05; M0044-H07; M0044-H09; M0045-A02; M0045-A04; M0045-B01; M0045-B03; M0045-B11; M0045-C02; M0045-C11; M0045-
  • An antibody or other binding protein e.g., a Tiel-binding protein
  • Tie2-binding protein, or Ang binding protein described herein can be administered to a subject or used in vitro in non-derivatized or unconjugated forms.
  • the binding protein can be derivatized, modified or linked to another functional molecule, e.g., another protein (e.g., HSA, an Fc domain, etc.), a polymer (e.g., PEG) isotope, cell, or insoluble support.
  • another functional molecule e.g., another protein (e.g., HSA, an Fc domain, etc.), a polymer (e.g., PEG) isotope, cell, or insoluble support.
  • the binding protein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as an antibody (e.g., if the protein is an antibody to form a bispecific or a multi-specific antibody), a toxin, a radioisotope, a therapeutic (e.g., a cytotoxic or cytostatic) agent or moiety, among others.
  • an antibody e.g., if the protein is an antibody to form a bispecific or a multi-specific antibody
  • a toxin e.g., if the protein is an antibody to form a bispecific or a multi-specific antibody
  • a therapeutic e.g., a cytotoxic or cytostatic agent or moiety, among others.
  • the binding protein can be coupled to a radioactive ion (e.g., an ⁇ -, ⁇ -, or ⁇ -emitter), e.g., iodine ( 131 I or 125 I), yttrium ( 90 Y), lutetium ( 177 Lu), actinium ( 225 Ac), rhenium ( 186 Re), or bismuth ( 212 Bi or 213 Bi).
  • a radioactive ion e.g., an ⁇ -, ⁇ -, or ⁇ -emitter
  • iodine 131 I or 125 I
  • yttrium 90 Y
  • lutetium 177 Lu
  • actinium 225 Ac
  • rhenium 186 Re
  • bismuth 212 Bi or 213 Bi
  • the invention features a nucleic acid that includes a coding sequence that encodes a polypeptide comprising an immunoglobulin heavy or light chain variable domain that binds to Tiel, e.g., an immunoglobulin heavy or light chain variable domain described herein.
  • the nucleic acid can include a particular nucleic acid sequence described herein, a nucleic acid that is at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to a nucleic acid sequence described herein (e.g., a particular nucleic acid sequence), or a nucleic acid that specifically hybridizes (e.g., under conditions described herein, e.g., high stringency conditions) to a nucleic acid sequence described herein (e.g., a particular nucleic acid sequence, e.g., a nucleic acid encoding one or more variable domains of M0044-A06; M0044-A11; M0044- B04; M0044-B05; M0044-B08; M0044-B09; M0044-B10; M0044-B12; M0044-C07; M0044-D01; M0044-E03; M0044-F03; M0044-F06;
  • a nucleic acid described herein can further include a promoter operably linked to the coding sequence.
  • a nucleic acid can include a first and second coding sequence, e.g., wherein the first coding sequence encodes a polypeptide that includes an immunoglobulin heavy chain variable domain and the second coding sequence encodes a polypeptide that includes an immunoglobulin light chain variable domain.
  • the invention features a host cell that contains a first nucleic acid encoding a polypeptide comprising a heavy chain variable region and a second nucleic acid encoding a polypeptide comprising a light chain variable region.
  • the heavy chain variable region and the light chain variable region can associate to form a Tiel binding protein.
  • These variable regions can have one or more properties described herein, e.g., at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to a sequence described herein.
  • the invention also includes a method of providing a Tiel- binding antibody.
  • the method can include providing a host cell described herein; and expressing said first and second nucleic acids in the host cell under conditions that allow assembly of said light and heavy chain variable regions to form an antigen binding protein that interacts with Tiel .
  • the invention provides compositions, e.g., pharmaceutical compositions, which include a pharmaceutically acceptable carrier, excipient or stabilizer, and at least one of the Tiel -binding proteins (e.g., antibodies or fragments thereof) described herein.
  • the compositions, e.g., the pharmaceutical compositions include a combination of two or more of the aforesaid Tiel -binding proteins.
  • the invention features a kit that includes a Tiel- binding antibody (or fragment thereof), e.g., a Tiel-binding antibody (or fragment thereof) as described herein, for use alone or in combination with other therapeutic modalities, e.g., a cytotoxic or labeling agent, e.g., a cytotoxic or labeling agent as described herein, along with instructions on how to use the Tiel antibody or the combination of such agents to treat, prevent or detect a Tiel-related disorder, e.g., an endothelial cell related disorder, e.g., rheumatoid arthritis or metastatic cancer.
  • a Tiel-related disorder e.g., an endothelial cell related disorder, e.g., rheumatoid arthritis or metastatic cancer.
  • the binding protein that binds to Tiel is a polypeptide that is not an immunoglobulin.
  • the polypeptide can be of variable length, e.g., 4 to 100 amino acid residues in length, preferably 5 to 75, 6 to 50, or 7 to 40 amino acid residues in length, or more preferably 8 to 30 or 10 to 25 amino acid residues in length, hi some embodiments, the polypeptide includes non- standard or synthetic amino acid residues, e.g., norleucine, selenocysteine, pyrrolysine, etc.
  • the polypeptide includes cross-linking groups, e.g., two cysteine residues that can form a disulfide bond or some other type of chemical cross-linking moieties that can be used to cyclize the peptide.
  • the polypeptide can be modified, e.g., using polyethylene glycol or fusion to a soluble protein, e.g., to increase the solubility or circulatory half- life of the polypeptide.
  • the target-binding protein can be physically associated with (e.g., fused to) another protein, e.g., a protein that does not bind to the target, e.g., to the amino or carboxy terminus.
  • the target-binding protein can be associated with (e.g., fused to) a protein that increases serum residence or alters stability, e.g., an albumin, e.g., a serum albumin, e.g., HSA (human serum albumin).
  • the target binding protein is physically associated with (e.g., fused to) a moiety that facilitates purification, e.g., a purification tag such as His, PEG, or to a functional moiety, e.g., Fc.
  • the invention features a method of identifying a protein that specifically binds to Tiel .
  • the invention includes: providing a Tiel antigen; providing a display library (e.g., a phage display library member); identifying a member present in the library, wherein the member expresses a protein that specifically binds to the Tiel antigen.
  • Tiel antigen refers to any antigenic fragment of Tiel that is at least 8 amino acids in length.
  • a Tiel antigen can include a fragment of the Tiel ectodomain, e.g., a fragment that includes a folded protein domain such as a fragment described herein.
  • the Tiel antigen is of human origin and includes, e.g., the extracellular domain of human Tiel or a fragment thereof (e.g., a fragment described herein.
  • the Tiel antigen can be a recombinant polypeptide optionally fused to another polypeptide, e.g., a Fc domain, or it can be a cell that expresses Tiel on its surface (e.g., an endothelial cell).
  • the Tiel antigen has an activated conformation, e.g., the Tiel antigen is a dimeric conformation or a conformation stabilized by the E3 or E3b antibody described herein.
  • the methods described here are, for example, applicable to libraries that are based on bacteriophage with a substantially complete genome (e.g., including a modified gene III) and to libraries that are based on bacteriophage particles that include a phagemid nucleic acid.
  • the terms "bacteriophage library member” and “phage” encompass members of both types of libraries.
  • the term "bacteriophage particle” refers to a particle formed of bacteriophage coat proteins that packages a nucleic acid.
  • the packaged nucleic acid can be a modified bacteriophage genome or a phagemid, e.g., a nucleic acid that includes a bacteriophage origin of replication but lacks essential phage genes and cannot propagate in E. coli without help from "helper phage” or phage genes supplied in trans.
  • the invention features a method of identifying a protein that specifically binds to Tiel .
  • the method includes: providing a Tiel antigen (e.g., an region of the Tiel ectodomain); immunizing a non-human animal with the Tiel antigen; and isolating a cell that produces a immunoglobulin that interacts with Tiel .
  • the method can include producing hybridoma cells from the spleen of the animal (e.g., an immunized mouse); and identifying individual hybridoma cell lines expressing an antibody that specifically binds to the Tiel antigen.
  • the Tiel antigen e.g., an region of the Tiel ectodomain
  • the Tiel antigen is of human origin and includes, e.g., the extracellular domain of human Tiel or some fragment thereof, e.g., the HA binding domain of Tiel .
  • the Tiel antigen can be a recombinant polypeptide optionally fused to another polypeptide, e.g., a purification handle, or it can be a cell that expresses Tiel (e.g., an endothelial cell) on its surface.
  • the Tiel antigen has an activated conformation, e.g., dimerized.
  • the methods further include isolating a nucleic acid molecule from the identified phage or hybridoma, wherein the nucleic acid molecule encodes the polypeptide or antibody that specifically binds to the Tiel antigen.
  • the isolated nucleic acid molecules can be used to produce therapeutic agents, as described herein.
  • the invention features nucleic acids that encode proteins identified by the methods described herein.
  • the nucleic acids include sequences encoding a heavy and light chain immunoglobulin or immunoglobulin fragment described herein.
  • the invention features, a first and second nucleic acid encoding a heavy and light chain variable region, respectively, of a Tiel -binding antibody molecule as described herein. Sequences encoding a heavy and light chain that function together can be present on separate nucleic acid molecules or on the same nucleic acid molecule.
  • the invention features host cells and vectors containing a nucleic acid described herein.
  • the invention features a method of producing a
  • the method includes: providing a host cell that contains a first nucleic acid encoding a polypeptide comprising a heavy chain variable region, e.g., a heavy chain variable region as described herein; providing a second nucleic acid encoding a polypeptide comprising a light chain variable region, e.g., a light chain variable region as described herein; and expressing said first and second nucleic acids in the host cell under conditions that allow assembly of said light and heavy chain variable regions to form an antigen binding protein that interacts with Tiel .
  • the first and second nucleic acids can be linked or unlinked, e.g., expressed on the same or different vector, respectively.
  • the ⁇ rst ana second nucleic acids can be components of the same molecule or can reside on different molecules (e.g., different chromosomes or plasmids).
  • the host cell can be a eukaryotic cell, e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E. coli.
  • the mammalian cell can be a cultured cell or a cell line.
  • Exemplary mammalian cells include lymphocytic cell lines (e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, HEK294, oocyte cells, and cells from a transgenic animal, e.g., mammary epithelial cell.
  • lymphocytic cell lines e.g., NSO
  • CHO Chinese hamster ovary cells
  • COS cells e.g., COS cells
  • HEK294 oocyte cells
  • cells from a transgenic animal e.g., mammary epithelial cell.
  • nucleic acids encoding the antibodies described herein can be expressed in a transgenic animal
  • the nucleic acids are placed under the control of a tissue-specific promoter (e.g., a mammary specific promoter) and the antibody is produced in the transgenic animal.
  • a tissue-specific promoter e.g., a mammary specific promoter
  • the antibody molecule is secreted into the milk of the transgenic animal, such as a transgenic cow, pig, horse, sheep, goat or rodent.
  • the nucleic acid is configured to encode a single polypeptide that comprises both the heavy and light chain variable domains.
  • Tiel has been found to be overexpressed in association with a wide range of cancers.
  • Targeting Tiel on the tumor vasculature with Tiel-binding proteins can be used to inhibit, destroy, or otherwise antagonize the vasculature so that tumor growth and metastasis is reduced.
  • the proteins can be, for example, associated with a toxic payload or can mediate direct functional inhibition. Proteins (e.g., proteins that have an Fc domain) that can cause ADCC can also be used.
  • the invention features a method of inhibiting an activity of a cell, e.g., an endothelial cell, e.g., proliferation, adhesion, growth or survival of a cell, e.g., an endothelial cell, e.g., an endothelial cell in the vicinity of a cancer, e.g., a tumor.
  • exemplary methods include contacting the cell with a Tiel binding protein, in an amount sufficient to inhibit the adhesion, migration, growth or proliferation of the cell.
  • Methods of administering a Tiel binding protein can be used, for example, to treat or prevent a disorder, e.g., an inflammatory disorder (e.g., rheumatoid arthritis, lupus, restenosis, psoriasis, graft v. host response, or multiple sclerosis), or a cancerous disorder (e.g., a malignant or metastatic disorder), by administering to a subject (e.g., an experimental animal or a human patient) a Tiel- binding protein in an amount effective to treat or prevent such disorder.
  • a Tiel-binding protein can be used to treat or prevent angiogenesis- related disorders, particularly angiogenesis-dependent cancers and tumors.
  • Angiogenesis-related disorders include, but are not limited to, solid tumors; tumor metastasis; benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis); psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neo vascular glaucoma, retrolental fibroplasia, rubeosis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation.
  • benign tumors e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheuma
  • Angiogenesis-dependent cancers and tumors are cancers tumors that require, for their growth (expansion in volume and/or mass), an increase in the number and density of the blood vessels supplying then with blood, hi one embodiment a Tiel-binding protein causes regression of such cancers and tumors.
  • regression refers to the reduction of tumor mass and size, e.g., a reduction of at least 2, 5, 10, or 25%.
  • Tiel and Tie2 are also expressed in hematopoietic cells.
  • a Tiel-binding protein is used to treat hematopoietic conditions, e.g., hematopoietic cancers.
  • hematopoietic cancers include: cancers derived from hyperplastic/neoplastic cells of hematopoietic origin, e.g., cells arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • Exemplary cancers include acute promyeloid leukemia (APML), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM), non-Hodgkin's lymphoma, peripheral T-cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T- cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), B cell chronic lymphocytic leukemia, myelodysplastic syndrome, and Hodgkin's disease.
  • APML acute promyeloid leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • ALL acute lymphoblastic leukemia
  • CLL chronic
  • the invention features a method of contacting a cell
  • the method can include providing an agent (e.g., a protein) that interacts with Tiel, e.g., a protein described herein, and contacting the cell with the protein, in an amount sufficient to form at least one detectable ligand-cell complex.
  • an agent e.g., a protein
  • the protein can include, for example, a label or cytotoxic entity, e.g., an immunoglobulin Fc domain or a cytotoxic drug.
  • the invention features administering the agent described herein as an adjuvant therapy, e.g., to a subject.
  • the adjuvant therapy can be a post-operative therapy that is administered to the subject after the subject has undergone surgery to remove all or part of a tumor (e.g., after surgery to treat glioblastoma or colorectal, breast, or lung cancer).
  • the agent is a protein that inhibits Tie complex formation, promotes Tiel homodimerization, or increases Tiel phosphorylation.
  • the agent is a protein that binds Tiel (e.g., an anti-Tiel antibody).
  • the agent is administered within 6, 12, 24, 48, or 100 hours of surgery. The agent can be administered before as well as after surgery.
  • An exemplary agent is a Tiel binding agent that includes (a) a heavy chain variable domain sequence that is at least 85, 90, 95, 98, 99%, or 100% identical to the heavy chain variable domain of the E3 antibody and a light chain variable domain sequence that is at least 85, 90, 95, 98, 99%, or 100% identical to the light chain variable domain of the E3 antibody; (b) a heavy chain variable domain sequence and a light chain variable domain sequence that form an antigen binding site that competes with E3 for binding to Tiel; or (c) one, two, or three, of the CDRs of the heavy chain variable domain of the E3 antibody, and one, two, or three of the CDRs of the light chain variable domain of the E3 antibody.
  • Tiel binding agents described herein can also be used, e.g., a Tiel binding agent that includes a heavy chain variable domain sequence that is at least 85, 90, 95, 98, 99%, or 100% identical to the heavy chain variable domain of M0059A02, M0045A02*, M0054G05, M0053F05, M0053G05, M0061C06, M0045B01, M0046G12, M0046H11, M0053A02, M0053A05, M0046B06, M0044B10, M0044B08, M0056G08, M0045B03, M0053F04, M0055E10, M0060H01, M0054H10, or M0058F03, and a light chain variable domain sequence that is at least 85, 90, 95, 98, 99%, or 100% identical to the light chain variable domain of M0059A02, M0045A02*, M0054G05, M0053F05, M0053G05,
  • the invention features a method of treating, e.g., inhibiting, ablating or killing, a cell or impairing at least one activity of the cell.
  • the method includes providing a Tiel -binding protein, e.g. a ligand described herein, and contacting the cell with the protein, in an amount sufficient to impair at least one activity of the cell, inhibit, ablate or kill the cell.
  • the contacting can be in vitro or in vivo.
  • the cell can be, e.g., an endothelial cell, e.g., an endothelial cell in the vicinity of a cancer, e.g., a tumor.
  • the protein can include a cytotoxic entity.
  • Methods of administering a Tiel binding protein or other agent described herein can be used, for example, to treat or prevent a disorder, e.g., a endothelial cell-based disorder, a blood vessel disorder, wound healing, or a cancerous disorder (e.g., a malignant or metastatic disorder), by administering to a subject (e.g., an experimental animal or a human patient) a Tiel -binding protein in an amount effective to treat or prevent such disorder.
  • a disorder e.g., a endothelial cell-based disorder, a blood vessel disorder, wound healing, or a cancerous disorder (e.g., a malignant or metastatic disorder)
  • a subject e.g., an experimental animal or a human patient
  • a Tiel -binding protein in an amount effective to treat or prevent such disorder.
  • a Tiel binding protein or other agent described herein can be used on cells in culture, e.g. in vitro or ex vivo.
  • an endothelial cell e.g., an endothelial cell in cancer biopsy
  • the contacting step can be effected by adding the Tiel -binding protein to the culture medium.
  • the method can be performed on cells (e.g., cancerous or metastatic cells) present in a subject, as part of an in vivo (e.g., therapeutic or prophylactic) protocol.
  • the contacting step is effected in a subject and includes administering the Tiel -binding protein to the subject under conditions effective to permit both binding of the protein to the cell, and the inhibition of adhesion, migration, growth or proliferation of the cell.
  • a Tiel binding protein or other agent described herein can be used to treat or prevent cancerous disorders, e.g., including hematopoietic cancers, solid tumors, soft tissue tumors, and metastatic lesions, particularly tumors that require a blood supply or angiogenesis.
  • cancerous disorders e.g., including hematopoietic cancers, solid tumors, soft tissue tumors, and metastatic lesions, particularly tumors that require a blood supply or angiogenesis.
  • solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary tract (e.g., renal, urothelial cells), pharynx, as well as adenocarcinomas which include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • malignancies e.g., sarcomas, adenocarcinomas, and carcinomas
  • adenocarcinomas which include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • the subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having, or at risk of, a disorder described herein, e.g., an endothelial cell-based disorder, e.g., cancer).
  • a primate preferably a higher primate
  • a human e.g., a patient having, or at risk of, a disorder described herein, e.g., an endothelial cell-based disorder, e.g., cancer.
  • Tiel-binding antibody or fragment thereof e.g., a Tiel-binding antibody or fragment thereof as described herein, can be administered to the subject systemically (e.g., orally, parenterally, subcutaiieously, intravenously, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation), topically, or by application to mucous membranes, such as the nose, throat and bronchial tubes.
  • the methods can further include the step of monitoring the subject, e.g., for a reduction in one or more of: a reduction in tumor size; reduction in cancer markers, e.g., levels of cancer specific antigen; reduction in the appearance of new lesions, e.g., in a bone scan; a reduction in the appearance of new disease-related symptoms; or decreased or stabilization of size of soft tissue mass; or any parameter related to improvement in clinical outcome.
  • the subject can be monitored in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Monitoring can be used to evaluate the need for further treatment with the same Tiel-binding protein or for additional treatment with additional agents. Generally, a decrease in one or more of the parameters described above is indicative of the improved condition of the subject. Information about the monitoring can be recorded, e.g., in electronic or digital form.
  • the Tiel-binding protein can be used alone in unconjugated form to thereby inhibit adhesion, migration, or extravasation or the Tiel -expressing cells, or ablate or kill the Tiel -expressing cells. If the Tiel-binding protein is an antibody, the ablation or killing can be mediated, e.g., by an antibody-dependent cell killing mechanisms such as complement-mediated cell lysis and/or effector cell-mediated cell killing.
  • the Tiel-binding protein can be bound (e.g., physically associated, either directly or indirectly, covalently or non-covalently) to a substance, e.g., a cytotoxic agent or moiety, effective to kill or ablate the Tiel- expressing cells.
  • a substance e.g., a cytotoxic agent or moiety
  • the Tiel-binding protein can be coupled to a radioactive ion (e.g., an ⁇ -, ⁇ -, or ⁇ -emitter), e.g., iodine ( 131 I or 125 I), yttrium ( 90 Y), lutetium ( 177 Lu), actinium ( 225 Ac), or bismuth ( 212 Bi or 213 Bi).
  • a radioactive ion e.g., an ⁇ -, ⁇ -, or ⁇ -emitter
  • iodine 131 I or 125 I
  • yttrium 90 Y
  • the methods and compositions described herein can be used in combination with other therapeutic modalities.
  • the methods include administering to the subject a Tiel-binding protein, e.g., a Tiel-binding antibody or fragment thereof, in combination with a cytotoxic agent, in an amount effective to treat or prevent the disorder.
  • a Tiel-binding protein and the cytotoxic agent can be administered simultaneously or sequentially.
  • a Tiel binding protein or other agent described herein is used in combination with surgical and/or radiation procedures.
  • the invention features methods for detecting the presence of a Tiel protein or a cell expressing Tiel (e.g., an endothelial cell) in a sample, in vitro (e.g., a biological sample, a tissue biopsy, e.g., a cancerous lesion).
  • a sample e.g., a biological sample, a tissue biopsy, e.g., a cancerous lesion.
  • the subject method can be used to evaluate, e.g., diagnose or stage a disorder described herein, e.g., a cancerous disorder.
  • the method includes: (i) contacting the sample (and optionally, a reference, e.g., control sample) with a Tiel-binding protein, as described herein, under conditions that allow interaction of the Tiel-binding protein and the Tiel protein to occur; and (ii) detecting formation of a complex between the Tiel-binding protein, and the sample (and optionally, the reference, e.g., control, sample). Formation of the complex is indicative of the presence of Tiel protein (e.g., activated Tiel protein), and can indicate the suitability or need for a treatment described herein. For example, a statistically significant change in the formation of the complex in the sample relative to the reference sample, e.g., the control sample, is indicative of the presence of Tiel (e.g., activated Tiel) in the sample.
  • Tiel e.g., activated Tiel
  • the invention provides a method for detecting the presence of Tiel (e.g., activated Tiel) in vivo (e.g., in vivo imaging in a subject).
  • Tiel e.g., activated Tiel
  • the subject method can be used to evaluate, e.g., diagnose, localize, or stage a disorder described herein, e.g., a cancerous disorder.
  • the method includes: (i) administering to a subject (and optionally a control subject) a Tiel-binding protein (e.g., an antibody or antigen binding fragment thereof), under conditions that allow interaction of the Tiel-binding protein and the Tiel protein to occur; and (ii) detecting formation of a complex between the Tiel-binding protein and Tiel, wherein a statistically significant change in the formation of the complex in the subject relative to the reference, e.g., the control subject or subject's baseline, is indicative of the presence of the Tiel.
  • a Tiel-binding protein e.g., an antibody or antigen binding fragment thereof
  • Tiel in particular locations within a subject can be indicative of an endothelial-cell related disorder, e.g., an angiogenesis-related disorder, e.g., a cancer, e.g., metastatic cancer, or other angiogenesis-related disorder described herein.
  • angiogenesis-related disorder e.g., a cancer, e.g., metastatic cancer, or other angiogenesis-related disorder described herein.
  • Tumor cells can express Tiel .
  • the invention features a method of providing a sample from a subject and evaluating the Tiel expression in cells in the sample.
  • the result of evaluating Tiel expression levels is compared to a reference, e.g., a reference value or reference quality.
  • a reference value or quality can be determined using a control sample, a statistical value (e.g., an average, median, etc.) or an arbitrary value.
  • the control sample can be a normal sample, e.g., a sample devoid of tumor cells from the same or different subject.
  • a change (e.g., an increase) relative to the reference can indicate that the sample includes tumor cells, e.g., the subject may be indicated as having a tumor.
  • a method of diagnosing or staging a disorder as described herein includes: (i) identifying a subject having, or at risk of having, the disorder; (ii) obtaining a sample of a tissue or cell affected with the disorder; (iii) contacting said sample or a control sample with a Tiel -binding protein, under conditions that allow interaction of the binding agent and the Tiel protein to occur, and (iv) detecting formation of a complex.
  • a statistically significant increase in the formation of the complex between the Tiel -binding protein with respect to a reference sample, e.g., a control sample, is indicative of the disorder or the stage of the disorder.
  • a reference sample e.g., a control sample
  • the finding of activated Tiel on tumor cells located in a solid tumor can indicate that the tumor is progressing into a metastatic tumor.
  • the Tiel-binding protein used in the in vivo and in vitro diagnostic methods is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound binding agent.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • the Tiel- binding protein is coupled to a radioactive ion, e.g., indium ( 111 Li), iodine ( 131 I or 125 I), yttrium ( 90 Y), actinium ( 225 Ac), bismuth ( 212 Bi or 213 Bi), sulfur ( 35 S), carbon ( 14 C), tritium ( 3 H), rhodium ( 188 Rh), or phosphorous ( 32 P).
  • a radioactive ion e.g., indium ( 111 Li), iodine ( 131 I or 125 I), yttrium ( 90 Y), actinium ( 225 Ac), bismuth ( 212 Bi or 213 Bi), sulfur ( 35 S), carbon ( 14 C), tritium ( 3 H), rhodium ( 188 Rh), or phosphorous ( 32 P).
  • the Tiel -binding protein is labeled with an NMR contrast agent.
  • the invention features a method of imaging tumor vasculature, the method includes: providing a protein that binds to Tiel, Tie2, or Ang, e.g., a protein described herein, wherein the protein is physically associated to an imaging agent; administering the protein to a patient, e.g., with a tumor; and imaging the patient, e.g., to detect tumor vasculature.
  • the invention features a method of treating a subject with a blood born neoplastic disorder, the method includes administering a protein that binds to Tiel, Tie2, or Ang, e.g., a protein described herein, to a subject with a blood born neoplastic disorder (e.g., a proliferative disorder of hematopoietic cells, e.g., leukemia), thereby treating the disorder.
  • a blood born neoplastic disorder e.g., a proliferative disorder of hematopoietic cells, e.g., leukemia
  • the invention features a method of diagnosing and treating a subject, the method includes evaluating a parameter associated with Tiel, Tie2, or Ang in a subject; and, if the parameter is altered relative to a reference, administering a protein described herein to the subject, thereby treating the subject.
  • the parameter includes a value indicative of protein or mRNA levels, e.g., in a tissue of a subject, hi one embodiment, the reference includes a value determined for a reference subject, e.g., an age/gender matched subject, e.g., a control or normal subject.
  • the invention features a method of treating a subject, the method includes: administering a protein described herein to a subject that has elevated Tiel, Tie2, or Ang biomolecules or activity relative to a reference.
  • the method can include evaluating the subject, e.g., to determine if the subject has elevated Tiel, Tie2, or Ang biomolecules or activity relative to a reference, hi one embodiment, the subject has elevated Tiel protein or mRNA levels.
  • the invention also provides polypeptides and nucleic acids that encompass a range of amino acid and nucleic acid sequences, e.g., sequences described herein or sequences related to those described herein.
  • the invention features nucleic acids that encodes each of the polypeptides described herein.
  • the nucleic acid can include the cognate codons or any set of codons that can be translated to produce the respective polypeptide.
  • Such polypeptides include individual subunits of a multi-chain protein, e.g., an antibody that includes a plurality of different polypeptide chains.
  • the nucleic acid may also be a nucleic acid fragment or vector that is not expressed, but includes a sequence encoding at least a part of an immunoglobulin variable region (e.g., including a CDR described herein) or a complement thereof.
  • Such nucleic acids can be used to prepare useful constructs, cells, and proteins.
  • the invention features a host cell that includes a nucleic acid described herein.
  • the cell can express a protein described herein, e.g., on its surface.
  • the invention also includes are proteins that include an amino acid sequence encoded by a nucleic acid described herein or that hybridize to a nucleic acid described herein.
  • an antibody refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence.
  • an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL).
  • an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • the term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F(ab') 2 , a Fd fragment, a Fv fragments, and dAb fragments) as well as complete antibodies.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed “framework regions” (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • the extent of the framework region and CDRs has been precisely defined (see, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. MoI. Biol. 196:901-917). Kabat definitions are used herein.
  • Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • Immunoglobulin domain refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two ⁇ -sheets formed of about seven ⁇ -strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay 1988 Ann. Rev Immunol. 6:381- 405). The canonical structures of hypervariable loops of an immunoglobulin variable can be inferred from its sequence, as described in Chothia et al. (1992) J. MoI. Biol. 227:799-817; Tomlinson et al. (1992) J. MoI. Biol. 227:776-798); and Tomlinson et al. (1995) EMBO J. 14(18):4628-38.
  • an "immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain.
  • the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain.
  • the sequence may omit one, two or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other alterations.
  • a polypeptide that includes immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form a target binding structure (or "antigen binding site"), e.g., a structure that interacts with Tiel, e.g., binds to or inhibits Tiel.
  • a target binding structure e.g., a structure that interacts with Tiel, e.g., binds to or inhibits Tiel.
  • the VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively.
  • the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds.
  • the heavy chain constant region includes three domains, CHl, CH2 and CH3.
  • the light chain constant region includes a CL domain.
  • the variable region of the heavy and light chains contains a binding domain that interacts with an antigen.
  • the constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.
  • the term "antibody” includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof).
  • the light chains of the immunoglobulin may be of types kappa or lambda, hi one embodiment, the antibody is glycosylated.
  • An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.
  • One or more regions of an antibody can be human or effectively human.
  • one or more of the variable regions can be human or effectively human.
  • one or more of the CDRs can be human, e.g., HC CDRl, HC CDR2, HC CDR3, LC CDKl, LC CDR2, and LC CDR3.
  • Each of the light chain CDRs can be human.
  • HC CDR3 can be human.
  • One or more of the framework regions can be human, e.g., FRl, FR2, FR3, and FR4 of the HC or LC.
  • all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non- hematopoietic cell.
  • the human sequences are germline sequences, e.g., encoded by a germline nucleic acid.
  • One or more of the constant regions can be human or effectively human.
  • at least 70, 75, 80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FRl, FR2, and FR3, collectively, or FRl, FR2, FR3, and FR4, collectively) or the entire antibody can be human or effectively human.
  • FRl, FR2, and FR3 collectively can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical to a human sequence encoded by a human germline V segment of a locus encoding a light or heavy chain sequence.
  • All or part of an antibody can be encoded by an immunoglobulin gene or a segment thereof.
  • exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Full-length immunoglobulin light chains (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2 -terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus.
  • Full-length immunoglobulin heavy chains (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).
  • a light chain refers to any polypeptide that includes a light chain variable domain.
  • a heavy chain refers to any polypeptide that a heavy chain variable domain.
  • antibody portion refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest.
  • binding fragments encompassed within the term "antigen-binding fragment" of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) aF(ab') 2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality.
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHl domains
  • aF(ab') 2 fragment a bivalent fragment including two Fab
  • 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 protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv).
  • scFv single chain Fv
  • Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those with skill in the art.
  • the term “monospecific antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope.
  • This term includes a "monoclonal antibody” or “monoclonal antibody composition,” which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition.
  • isotype refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.
  • the HC or LC of an antibody includes sequences that correspond to an amino acid sequence encoded by a human germline sequence, e.g., the framework regions and/or in the CDRs.
  • the antibody can include sequences from the human DP47 antibody.
  • one or more codons for the antibody are altered relative to the germline nucleic acid sequence, but are chosen to encode the same amino acid sequence. Codons can be selected, e.g., to optimize expression in a particular system, create restriction enzyme sites, create a silent fingerprint, etc.
  • CDR2 of the antibody HC includes at least 11, 12,
  • a "humanized” immunoglobulin variable region is an immunoglobulin variable region that includes sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human.
  • Descriptions of "humanized” immunoglobulins include, for example, US 6,407,213 and US 5,693,762.
  • An "effectively human” immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human.
  • An "effectively human” antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
  • Tie complex refers to a heteromeric complex that includes Tiel, Tie2, and an angiopoietin (Ang).
  • the Tie complex is formed in part by association of the extracellular domains of Tiel and Tie2 and also includes Ang.
  • complex members refers to the proteins that are included in a heteromeric Tie complex. Accordingly, Tiel, Tie2, and Ang are all complex members.
  • Ang includes all angiopoietins, such as Angl, Ang2, Ang3, and Ang4.
  • the heteromeric Tie complex can include other proteins in addition to Tiel, Tie2, and Ang.
  • a protein or ligand that antagonizes complex formation inhibits or decreases the association of Tiel, Tie2, or Ang with at least one other member of the complex and thereby decreases Tie2 signaling and downstream effects such as angiogenesis.
  • Angiogenesis includes all stages of vessel development (e.g., blood or lymphatic vessel development), including initial vessel formation and later vessel remodeling and morphological changes.
  • the terms "agonist” and "antagonist” describe properties in context of a particular activity or effect.
  • the E3 or E3b antibody can be an agonist in the context of promoting Tiel self-association (e.g., homodimerization), yet an antagonist in the context of decreasing or inhibiting Tie complex formation and tube formation by HUVECs.
  • an agent that is an agonist in the context of a Tiel signaling pathway can be an antagonist in the context of endothelial cell sprouting, splitting, and tube formation.
  • Tiel ectodomain refers to an extracellular region of a Tiel protein, e.g., a region that includes about amino acids 25-759 of SEQ DD NO:2.
  • Other exemplary regions are regions that include one or more EGF-like domains (e.g., 214- 256, 258-303, 303-345, 214-303, 258-345, or 214-345 of SEQ ID NO:2); one or more Ig-Like C2-type domains (e.g., 43-105, 43-426, 372-426); one or more Fibronectin Type HI repeats (e.g., 446-540, 543-639, 643-744, 446-639, 543-744, or 446-744 of SEQ ID NO:2); and combinations thereof.
  • EGF-like domains e.g., 214- 256, 258-303, 303-345, 214-303, 258-345, or 214-345 of SEQ ID NO:2
  • first Ig-like C2-type domain and “Ig 1” refer to the immunoglobulin-like domain in Tiel or Tie2 that is located closest to the amino terminus of the protein relative to the other Ig-like C2-type domain (the second such domain).
  • first Immunoglobulin-like C2-type domain is located at about residue 43 to about residue 105 and the second Ig-like C2-type domain is located at about residue 372 to about residue 426.
  • binding affinity refers to the apparent association constant or K a .
  • the K a is the reciprocal of the dissociation constant (K d ).
  • a ligand may, for example, have a binding affinity of at least 10 5 , 10 6 , 10 7 or 10 s M "1 for a particular target molecule.
  • Higher affinity binding of a ligand to a first target relative to a second target can be indicated by a higher K a (or a smaller numerical value Kd) for binding the first target than the K 3 (or numerical value K d ) for binding the second target. In such cases the ligand has specificity for the first target relative to the second target.
  • Differences in binding affinity can be at least 1.5, 2, 5, 10, 50, 100, or 1000-fold.
  • a Tiel-binding protein may preferentially bind to Tiel at least 1.5, 2, 5, 10, 50, 100, or 1000-fold better than to another antigen, e.g., Tie2, EGF, fibronectin, or human serum albumin.
  • a Tiel- binding protein may also be species-specific or species-general (e.g., can bind to a Tiel protein from more than one species).
  • Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). These techniques can be used to measure the concentration of bound and free ligand as a function of ligand (or target) concentration.
  • concentration of bound ligand [Bound]) is related to the concentration of free ligand ([Free]) and the concentration of binding sites for the ligand on the target where (N) is the number of binding sites per target molecule by the following equation:
  • [Bound] N • [Free ⁇ /((1/Ka) + [Free]) [0210]
  • Althougl ⁇ quantitative measurements of Ka are routine, it is not always necessary to make an exact determination of K 3 , though, since sometimes it is sufficient to obtain a qualitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K a , and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2, 5, 10, 20, or 50 fold higher than a reference.
  • Binding affinity is typically evaluated in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 niM EDTA and 0.005 % (v/v) surfactant P20.
  • compositions produced artificially or naturally can be "compositions of at least" a certain degree of purity if the species or population of species of interests is at least 5, 10, 25, 50, 75, 80, 90, 95, 98, or 99% pure on a weight-weight basis.
  • an epitope refers to the site on a target compound that is bound by a ligand, e.g., an antigen-binding protein (e.g., a Fab or antibody).
  • a ligand e.g., an antigen-binding protein (e.g., a Fab or antibody).
  • an epitope may refer to the amino acids that are bound by the ligand. Overlapping epitopes include at least one common amino acid residue.
  • the term “substantially identical” is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities, hi the case of antibodies, the second antibody has the same specificity and has at least 50% of the affinity of the same.
  • a sufficient number of identical or equivalent e.g., with a similar side chain, e.g., conserved amino acid substitutions
  • sequences similar or homologous are also part of this application.
  • sequence identity can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.
  • substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • sequence identity is calculated as follows.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J MoI. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSga ⁇ dna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • similarity and means that a sequence of interest differs from a reference sequence by the presence of one or more amino acid substitutions (although modest amino acid insertions or deletions) may also be present.
  • Presently preferred means of calculating degrees of homology or similarity to a reference sequence are through the use of BLAST algorithms (available from the National Center of Biotechnology Information (NCBI), National Institutes of Health, Bethesda MD), in each case, using the algorithm default or recommended parameters for determining significance of calculated sequence relatedness.
  • the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used.
  • Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 50 0 C (the temperature of the washes can be increased to 55 0 C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 45 0 C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 0 C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65 0 C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C.
  • proteins described herein may have mutations relative to a particular protein described herein (e.g., a conservative or non-essential ammo acid substitutions), which do not have a substantial effect on function. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect desired biological properties, such as binding activity can be determined as described in Bowie, et al. (1990) Science 247:1306-1310.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • framework and CDR amino acid residues to include one or more conservative substitutions.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of the binding agent, e.g., the antibody, without abolishing or more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change.
  • any amino acid e.g., any of the twenty naturally occurring amino acids
  • polypeptide or “peptide” (which may be used interchangeably) refer to a polymer of three or more amino acids linked by a peptide bond, e.g., between 3 and 30, 12 and 60, or 30 and 300, or over 300 amino acids in length.
  • the polypeptide may include one or more unnatural amino acids. Typically, the polypeptide includes only natural amino acids.
  • a “protein” can include one or more polypeptide chains. Accordingly, the term “protein” encompasses polypeptides.
  • a protein or polypeptide can also include one or more modifications, e.g., a glycosylation, amidation, phosphorylation, and so forth.
  • small peptide can be used to describe a polypeptide that is between 3 and 30 amino acids in length, e.g., between 8 and 24 amino acids in length.
  • Exemplary statistical tests include: the Students T-test, Mann Whitney U non- parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05, or 0.02. Particular ligands may show a difference, e.g., in specificity or binding, that are statistically significant (e.g., P value ⁇ 0.05 or 0.02).
  • Embodiments of the invention can include any combination of features described herein. In no case does the term "embodiment” necessarily exclude one or more other features disclosed herein, e.g., in another embodiment.
  • the contents of all references, patent applications and patents, cited throughout this application are hereby expressly incorporated by reference.
  • FIG. 1 illustrates a bivariant FACS plot showing labelling with the platelet specific marker CD42 with Tiel and labelling with the E3 antibody. Only a background number of CD42 positive cells are labeled by the E3 antibody.
  • FIGs. 2 A, 2B, 2C, and 2D are plots of the number of branching points versus antibody concentration comparing germlined E3 (2C and 2D) with parental E3 (2A and 2B).
  • FIG. 3 depicts a graph of blood vessel density in matrigels that were stained with fluorescein-lectin from an in vivo assay using MATRIGELTM and evaluating the germlined E3 antibody.
  • FIG. 4 depicts results of tube formation in HUVECs using the parental
  • FIG. 5 depicts graphically the results from animal studies in which nu/nu mice were implanted with SW-480 colorectal cancer cells and treated with DX- 2220 (10 mg/kg), cisplatin (4 mg/kg), or a control. Control conditions were: no treatment, PBS vehicle alone, or a non-specific, isotype-matched IgGl antibody (A2- SV) (10 mg/kg). Tumor weight is plotted on the y axis; days after tumor cell injection is plotted on the x axis. [0230] FIG.
  • FIG. 6 depicts graphically the results from animal studies in which nu/nu mice were implanted with LNM35 lung cancer cells and treated with DX-2220 (20 mg/kg) or a non-specific, isotype-matched IgGl antibody (A2-SV) (20 mg/kg).
  • Tumor volume (mm 3 ) is plotted on the y axis; days after tumor cell injection is plotted on the x axis.
  • FIG 7A and 7B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-Al, respectively.
  • FIG 8A and 8B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-A5, respectively.
  • FIG 9 A and 9B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-A6, respectively.
  • FIG 1OA and 1OB list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-A10, respectively.
  • FIG 11 A and HB list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-Bl, respectively.
  • FIG 12A and 12B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-B3, respectively.
  • FIG 13A and 13B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-C6, respectively.
  • FIG 14A and 14B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-D6, respectively.
  • FIG 15A and 15B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-D10, respectively.
  • FIG 16A and 16B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-D12, respectively.
  • FIG 17A and 17B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-F3, respectively.
  • FIG 18A and 18B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-F4, respectively.
  • IUZ4JJ *1G 19A and 19B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-G3, respectively.
  • FIG 2OA and 2OB list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-A2, respectively.
  • FIG 21 A and 21B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone S-AlO, respectively.
  • FIG 22 A and 22B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-B2, respectively.
  • FIG 23 A and 23B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-B9, respectively.
  • FIG 24A and 24B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-C2, respectively.
  • FIG 25A and 25B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-C7, respectively.
  • FIG 26A and 26B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-CIO, respectively.
  • FIG 27A and 27B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-Dll, respectively.
  • FIG 28 A and 28B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-Ell, respectively.
  • FIG 29A and 29B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-G4, respectively.
  • FIG 30 lists the amino acid sequence of the light chain variable domain of clone s-G9.
  • FIG 31 A and 31B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-G10, respectively.
  • FIG 32 A and 32B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-Hl, respectively.
  • FIG 33 A and 33B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone s-H4, respectively.
  • FIG 34 A and 34B list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone G2, respectively.
  • FIG 35 and 36 list the amino acid sequence of the heavy chain variable domain and the light chain variable domain of clone p-Al, respectively.
  • FIG 37 provides Table 5, a summary of heavy chain sequences.
  • FIG 38 provides Table 6, a summary of light chain sequences.
  • FIG 39 provides Table 9, characteristics of some exemplary Tiel binding antibodies.
  • agents also referred to as binding proteins and ligands
  • bind to components of a Tie complex e.g.,Tiel, Tie2, and Ang.
  • agents include proteins, for example, a small peptide (e.g., a cyclic or linear peptide, e.g., of between 7 and 25 amino acids), a polypeptide (e.g., a polypeptide of at least 20 amino acids), or a multi-chain protein (e.g., including at least two peptides or polypeptides).
  • a multi-chain protein is an IgG full-length antibody that has separate heavy and light chains.
  • An example of a polypeptide is a single chain antibody.
  • Agents can be selected that have particular properties, e.g., ability to antagonize Tiel/Tie2/Ang complex formation, ability to promote Tiel homodimerization, and ability to promote Tiel phosphorylation.
  • agents that bind to Tiel, Tie2, or Ang can be tested for their ability to antagonize formation of heteromeric Tie complexes.
  • Antagonism of this complex decreases Tie2 signaling and its downstream effects, such as promoting angiogenesis.
  • Tiel is a receptor tyrosine kinase protein that includes a transmembrane domain. Tiel is present almost exclusively on endothelial cells. Accordingly, a Tiel-binding protein can be used, e.g., to specifically recognize or target an endothelial cell.
  • Tiel-binding proteins can also be used to agonize or antagonize en ⁇ otneliai cells, in some embodiments, these Tiel-binding proteins have an affinity for particular structural features (e.g., a feature listed below), a combination of features listed below, and/or an epitope that includes at least one amino acid in a structural feature listed below (The sequence is relative to the amino acid sequence provided in SEQ DD NO:2, Example 1, below):
  • NP_BIND 845 853 9 ATP (BY SIMILARITY) .
  • BINDING 870 870 ATP (BY SIMILARITY) .
  • Tie2 is a receptor tyrosine kinase protein that includes a transmembrane domain. Tie2 is present almost exclusively on endothelial cells. Accordingly, a Tie2-binding protein can be used, e.g., to specifically recognize or target an endothelial cell. Some Tie2-binding proteins can also be used to modulate (e.g., agonize or antagonize) an activity of an endothelial cell. In some embodiments, these Tie2-binding proteins have an affinity for particular structural features, a combination of features, and/or an epitope that includes at least one amino acid in a structural feature. Exemplary structural features of Tie2 include: two Ig-like domains, three EGF-like domains, and three fibronectin type III domains.
  • the angiopoietins are a family of ligands that bind to Tie2.
  • Ang-binding proteins e.g., antibodies or artificial Ang-binding proteins
  • these Ang-binding proteins have an affinity for particular structural features, a combination of features, and/or an epitope that includes at least one amino acid in a structural feature.
  • Exemplary structural features include: the N-terminal region of about 50 amino acids, the coiled-coil domain, or the fibrinogen-like domain.
  • Ang-binding proteins include proteins that inhibit Ang multimerization (e.g., ability of Ang proteins to form tetramers), proteins that inhibit Ang-Tie2 interactions, and proteins that inhibit a ternary complex of Tiel-Tie2-Ang. Inhibitory proteins can function by disrupting existing interactions or by preventing interactions from occurring.
  • Tiel and Tie2 can associate through their extracellular domains and form a heteromeric complex with an angiopoietin (Ang), such as Angl, Ang2, Ang3, and Ang4.
  • Ang angiopoietin
  • This heteromeric complex activates the intracellular signaling cascade mediated by Tie2.
  • antagonizing formation of this heteromeric complex provides a novel approach to inhibiting Tie2 signaling and its downstream effects, such as angiogenesis.
  • Complex formation can be antagonized by proteins that bind to the extracellular domains of Tiel or Tie2 or that bind to Ang so as to prevent its recruitment into the complex or to prevent its multimerization.
  • One method for identifying proteins that bind to Tiel includes: providing a library and selecting from the library one or more members that encode a protein that binds to the Tiel antigen or a fragment thereof (e.g., the extracellular domain, an EGF domain, a fibronectin repeat, or an Ig-superfamily domain (e.g., a Ig- like C2-type 2 domain)).
  • the selection can be performed in a number of ways.
  • the library can be a display library.
  • the Tiel can be tagged and recombinantly expressed.
  • the Tiel is purified and attached to a support, e.g., to affinity beads, or paramagnetic beads or other magnetically responsive particles.
  • the Tiel can also be expressed on the surface of a cell.
  • Analogous procedures can be performed to identify proteins that bind to Tie2 or a fragment thereof (e.g., the extracellular domain, an EGF domain, a fibronectin repeat, or an Ig-superfamily domain (e.g., a Ig-like C2-type 2 domain)).
  • Analogous procedures can also be performed to identify proteins that bind to Ang or a fragment thereof (e.g., the N-terminal domain, the coiled-coil domain, or the fibrinogen-like domain).
  • Proteins identified as being capable of binding a Tie complex member can be tested for their ability to antagonize heteromeric complex formation, ability to promote Tiel phosphorylatoin, and/or ability to promote Tiel homodimerization, as described in the examples below. Proteins identified as antagonizing formation of the heteromeric complex can be used in pharmaceutical compositions to treat a subject in need of such treatment, for example, a subject with an angiogenesis-dependent cancer or tumor or other angiogenesis-related disorders.
  • a Tiel-binding protein can modulate a Tiel activity.
  • a Tiel-binding protein can function as a Tiel agonist or antagonist in the Tiel/EpoR chimeric BaF3 cell assay described in Example 2.
  • Tiel agonists in this Tiel/EpoR chimeric BaF3 cell assay can stimulate certain activity of an endothelial cell under particular conditions, e.g., the conditions of the Tiel/EpoR chimeric BaF3 cell assay.
  • Tiel binding proteins increase phosphatidyl inositol 3-kinase
  • PI3 kinase PI3 kinase activity in an endothelial cell and/or Akt kinase activity.
  • Kontos et al. suggest that the cytoplasmic domain of Tiel can associate with the p85 subunit of PI3 kinase and activate PD kinase activity.
  • Kontos et al. (2002) MoI. Cell Biol. 22:1704- 1713.
  • the Tiel cytoplasmic domain may also associate with a protein tyrosine phosphatase Sh ⁇ 2. See, e.g., Marron et al. (2000) Adv. Exp. Med. Biol. 476:35-46.
  • Tie binding proteins may increase dimerization, and/or tyrosine phosphorylation (e.g., as a result of auto-phosphorylation) of the Tiel cytoplasmic domain, e.g., the tyrosine in the motif YVN at about amino acid 1117.
  • Tiel-binding protein can be evaluated in a cell assay (e.g., in the
  • Tiel/EpoR chimeric BaF3 cell assay as described below in Example 2.
  • An exemplary cell assay uses a growth factor dependent cell in which a chimeric receptor that includes the Tiel ectodomain fused to the intracellular domain of the growth factor receptor is expressed. Cells are evaluated for ability to grow in the absence of the essential growth factor, but in the presence of a test compound, e.g., a Tiel- binding protein. If the Tiel-binding protein agonizes Tiel in the Tiel/EpoR chimeric BaF3 cell assay, a signalling activity of the Tiel chimera can substitute for stimulation by the required growth factor thorough its cognate receptor. Thus, survival of the cell in the absence of the required growth factor can be used as an indication that the Tiel-binding protein interacts with the Tiel ectodomain.
  • Tiel agonists in the Tiel/EpoR chimeric BaF3 cell assay may behave as inhibitors of Tiel activity under other conditions, e.g., in vivo, and, irrespective of in vitro properties, may be useful as inhibitors of angiogenesis in vivo.
  • Tiel binding proteins can be used, e.g., to reduce an activity of an endothelial cell.
  • some Tiel binding proteins can be used to decrease phosphatidyl inositol 3-kinase (PI3 kinase) activity in an endothelial cell, Shp2 activity, and/or Akt kinase activity.
  • Some Tiel binding proteins may also reduce dimerization, and/or tyrosine phosphorylation (e.g., as a result of auto- phosphorylation) of the Tiel cytoplasmic domain, e.g., the tyrosine in the motif YVN at about amino acid 1117.
  • Tiel-binding protein can be evaluated for activity in a cell assay.
  • the binding protein can be assayed for ability to prevent another ligand, e.g., the E3 antibody, from modulating a Tiel activity in a cell assay described herein (e.g., the Tiel/EpoR chimeric BaF3 cell assay as described below in Example 2).
  • a display library is used to identify such proteins.
  • a display library is a collection of entities; each entity includes an accessible protein component and a recoverable component that encodes or identifies the protein component.
  • the protein component can be of any length, e.g. from three amino acids to over 300 amino acids.
  • the protein component of each member of the library is probed with a target, e.g., Tiel protein, and if the protein component binds to the target, the display library member is identified, e.g., by retention on a support.
  • the method can be adapted for other targets, such as Tie2, Ang, fragments thereof, complexes that include one or more of these proteins or fragments thereof.
  • Retained display library members are recovered from the support and analyzed.
  • the analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated.
  • the analysis can also include determining the amino acid sequence of the protein component and purification of the protein component for detailed characterization.
  • a variety of formats can be used for display libraries. Examples include the following.
  • Phage Display One format utilizes viruses, particularly bacteriophages. This format is termed "phage display.”
  • the protein component is typically covalently linked to a bacteriophage coat protein.
  • the linkage results form translation of a nucleic acid encoding the protein component fused to the coat protein.
  • the linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon.
  • Phage display is described, for example, in Ladner et al, U.S. Patent No.
  • Phage display systems have been developed for filamentous phage
  • phage fl, fd, and Ml 3 as well as other bacteriophage (e.g. T7 bacteriophage and lambdoid phages; see, e.g., Santini (1998) J. MoI. Biol. 282:125-135; Rosenberg et al. (1996) Innovations 6:1-6; Houshmet al. (1999) Anal Biochem 268:363-370).
  • the filamentous phage display systems typically use fusions to a minor coat protein, such as gene III protein, and gene VIII protein, a major coat protein, but fusions to other coat proteins such as gene VI protein, gene VII protein, gene IX protein, or domains thereof can also been used (see, e.g., WO 00/71694).
  • the fusion is to a domain of the gene III protein, e.g., the anchor domain or "stump," (see, e.g., U.S. Patent No. 5,658,727 for a description of the gene III protein anchor domain).
  • a non-peptide linkage e.g., a non-covalent bond or a non-peptide covalent bond.
  • a disulfide bond and/or c-fos and c-jun coiled-coils can be used for physical associations (see, e.g., Crameri et al. (1993) Gene 137:69 and WO 01/05950).
  • Bacteriophage displaying the protein component can be grown and harvested using standard phage preparatory methods, e.g., PEG precipitation from growth media. After selection of individual display phages, the nucleic acid encoding the selected protein components, by infecting cells using the selected phages. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.
  • the library is a cell-display library.
  • Proteins are displayed on the surface of a cell, e.g., a eukaryotic or prokaryotic cell.
  • exemplary prokaryotic cells include E. coli cells, B. subtilis cells, and spores (see, e.g., Lu et al. (1995) Biotechnology 13:366).
  • Exemplary eukaryotic cells include yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hanseula, or Pichia pastoris).
  • yeast surface display is described, e.g., in Boder and Wittrup (1997) Nat. Biotechnol. 15:553-557 and WO 03/029456, which describes a yeast display system that can be used to display immunoglobulin proteins such as Fab fragments and the use of mating to generate combinations of heavy and light chains.
  • RNA and the polypeptide encoded by the RNA can be physically associated by stabilizing ribosomes that are translating the RNA and have the nascent polypeptide still attached. Typically, high divalent Mg 2+ concentrations and low temperature are used. See, e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sd. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; and Schaffitzel et al. (1999) J Immunol Methods. 231(l-2):119-35.
  • Polypeptide-Nucleic Acid Fusions Another format utilizes polypeptide-nucleic acid fusions.
  • Polypeptide-nucleic acid fusions can be generated by the in vitro translation of mRNA that include a covalently attached puromycin group, e.g., as described in Roberts and Szostak (1997) Proc. Natl. Acad. ScL USA 94:12297-12302, and U.S. Patent No. 6,207,446. The mRNA can then be reverse transcribed into DNA and crosslinked to the polypeptide.
  • Yet another display format is a non- biological display in which the protein component is attached to a non-nucleic acid tag that identifies the polypeptide.
  • the tag can be a chemical tag attached to a bead that displays the polypeptide or a radiofrequency tag (see, e.g., U.S. Patent No. 5,874,214).
  • Display technology can also be used to obtain binding proteins, e.g., antibodies that interact with particular epitopes of a target. This can be done, for example, by using competing non-target molecules that lack the particular epitope or are mutated within the epitope, e.g., with alanine. Such non-target molecules can be used in a negative selection procedure as described below, as competing molecules when binding a display library to the target, or as a pre-elution agent, e.g., to capture in a wash solution dissociating display library members that are not specific to the target.
  • binding proteins e.g., antibodies that interact with particular epitopes of a target. This can be done, for example, by using competing non-target molecules that lack the particular epitope or are mutated within the epitope, e.g., with alanine.
  • Such non-target molecules can be used in a negative selection procedure as described below, as competing molecules when binding a display library to the target, or as a pre-e
  • display library technology is used in an iterative mode.
  • a first display library is used to identify one or more binding proteins for a target. These proteins are then varied, e.g., using a mutagenesis method, to form a second display library. Higher affinity binding proteins are then selected from the second library, e.g., by using higher stringency or more competitive binding and washing conditions.
  • the mutagenesis is targeted to regions known or likely to be at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs, e.g., framework regions, particular within ten, five, or three amino acids of a CDR junction. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make precise step-wise improvements.
  • Some exemplary mutagenesis techniques include: error-prone PCR
  • the methods described herein are used to first identify a binding protein from a display library that binds a Tiel with at least a minimal binding specificity for a target or a minimal activity, e.g., an equilibrium dissociation constant for binding of greater than 1 nM, 10 nM, or 100 nM.
  • the nucleic acid sequence encoding the initial identified binding protein is used as a template nucleic acid for the introduction of variations, e.g., to identify a second binding protein that has enhanced properties (e.g., binding affinity, kinetics, or stability) relative to the initial binding protein.
  • the library is contacted to an immobilized target.
  • the immobilized target is then washed with a first solution that removes non-specifically or weakly bound biomolecules.
  • the immobilized target is eluted with a second solution that includes a saturation amount of free target, i.e., replicates of the target that are not attached to the particle.
  • the free target binds to biomolecules that dissociate from the target. Rebinding is effectively prevented by the saturating amount of free target relative to the much lower concentration of immobilized target.
  • the second solution can have solution conditions that are substantially physiological or that are stringent.
  • the solution conditions of the second solution are identical to the solution conditions of the first solution.
  • Fractions of the second solution are collected in temporal order to distinguish early from late fractions. Later fractions include biomolecules that dissociate at a slower rate from the target than biomolecules in the early fractions. It is also possible to recover display library members that remain bound to the target even after extended incubation. These can either be dissociated using chaotropic conditions or can be amplified while attached to the target. For example, phage bound to the target can be contacted to bacterial cells.
  • selection refers to a process in which many members of a display library are allowed to contact the target and those that bind are recovered and propagated. The selection can be from a library having numerous members, e.g., more than 10 10 members. “Screening” refers to a process in which isolated members of the library are tested singly for binding to the target. Through automation, thousands of candidates may be screened in a highly parallel process.
  • the display library selection methods described herein can include a selection process that discards display library members that bind to a non-target molecule.
  • non-target molecules include, e.g., extracellular domains of molecules that include an immunoglobulin super-family domain or an EGF domain and receptor tyrosine kinases other than Tiel, e.g., Tie2, or other than Tie2, e.g., Tiel, or other than Tiel and Tie2.
  • a so-called "negative selection" step is used to discriminate between the target and related non-target molecule and a related, but distinct non-target molecules.
  • the display library or a pool thereof is contacted to the non-target molecule.
  • Members of the sample that do not bind the non-target are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections.
  • the negative selection step can be prior to or after selecting library members that bind to the target molecule.
  • a screening step is used. After display library members are isolated for binding to the target molecule, each isolated library member is tested for its ability to bind to a non-target molecule (e.g., a non-target listed above). For example, a high-throughput ELISA screen can be used to obtain this data. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target. The non-target and target binding data are compared (e.g., using a computer and software) to identify library members that etal
  • the display library selection and screening methods described herein can include a selection or screening process that selects for display library members that bind to specific sites on the target molecule. For example, elution with high concentration of an antibody described herein can be used to select for phage that bind to an epitope that is near or overlaps with the epitope bound by the antibody used for elution. Accordingly, one can screen for a phage that binds to the E3-binding site of Tiel by performing ELISAs with and without E3 antibody in the buffer.
  • the following description provides one exemplary method for identifying antibodies that bind to Tiel using a phagemid Fab library.
  • three rounds of selection can be performed with decreasing amounts of target protein (e.g., 100, 50 and 50 ⁇ g for first, second, and third rounds, respectively).
  • the target is immobilized on streptavidin coated magnetic beads (Dynal).
  • the library is depleted against streptavidin coated magnetic beads prior to each round of selection and optionally against an unrelated protein which may include a common purification handle.
  • the target is produced as a fusion to a Fc domain
  • the library can be depleted against soluble Trail-Fc (a commercially available Fc fusion protein). The depletion process removes Fc binders.
  • Each round of selection can include, e.g., two cycles of streptavidin magnetic bead depletion, a cycle of binding of phage to Tiel -coated beads, ten cycles of washes, elution of bound phage, and propagation of enriched phage for the next round.
  • Phage bound to Tiel -coated beads after ten washes can be directly amplified or eluted before amplification.
  • individual clones may be grown in 96-well microtiter plates and individually screened for Tiel binding activity by phage ELISA.
  • ELISAs can include evaluations of binding to Tiel, specificity controls, and unrelated controls.
  • Isolates can be DNA fingerprinted to determine the diversity emerging from the selection process.
  • positive isolates can be PCR amplified with the oligonucleotide primers M13-reverse and genelll-forward (see, e.g., Marks (1991), J. MoI. Biol. 222:581).
  • the products can be analyzed by BstNI fingerprinting.
  • An exemplary method for performing ELISA's with phage that display a binding protein is as follows. Individual clones can be grown and rescued as described previously (Marks et al. (1991), J. MoI. Biol. 222:581).
  • 96- well Immulon 2 HB plates (Thermo Labsystems) are coated with 1 ⁇ g/well ImmunoPureTM streptavidin (Pierce) in PBS and incubated overnight at 4°C. After three washes with PBS, 100 ⁇ L of biotinylated Tiel protein is allowed to bind to the immobilized streptavidin for 30-60 minutes at room temperature. Then, Tiel -coated wells are blocked with 300 ⁇ L of 2% milk/lx PBS/0.05% Tween (2% MPBST) for two hours at 37°C.
  • 2% milk/lx PBS/0.05% Tween 2% MPBST
  • the wells are incubated with 100 ⁇ L of phage culture supernatant that had been blocked with 2% MPBST for one hour at room temperature.
  • the wells are washed five times with lxPBS/Tween 0.1% (PBST), and incubated with 100 ⁇ L of anti-M13-HRP secondary antibody at a 1 :5,000 dilution for one hour at room temperature.
  • the wells are washed five times with PBST before developing with TMB-solution and read at 630 nm.
  • cells are washed once in PBS and resuspended at a concentration of 1 x 10 6 to 2 x 10 6 cells/niL of PBS.
  • a final concentration of 1-2 x 10 5 cells per well of a 96-well tissue culture plate (Falcon, VWR) can be used.
  • the cells are fixed by adding an equal volume of 0.2% glutaraldehyde (Sigma- Aldrich) and incubating at 37 0 C for 12 minutes. They are then washed three times with PBS using an automated plate washer (Bio-Tek Instruments, Inc.) and blocked with 200 ⁇ L of 2% MPBST for one hour at room temperature.
  • the rest of the ELISA procedure can be performed as described above except that lxPBS/Tween 0.05% is used for the washes and incubations.
  • an antibody that binds Tiel , Tie2, or Ang e.g., an antibody described herein, in order to make the variable regions of the antibody more similar to one or more germline sequences.
  • an antibody can include one, two, three or more amino acid substitutions, e.g., in a framework or CDR region, to make it more similar to a reference germline sequence.
  • One exemplary germlining method can include: identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Then mutations (at the amino acid level) can be made in the isolated antibody, either incrementally, in combination, or both.
  • a nucleic acid library that includes sequences encoding some or all possible germline mutations is made.
  • the mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity).
  • as many germline residues are introduced into an isolated antibody as possible.
  • mutagenesis is used to substitute or insert one or more germline residues into a CDR region.
  • the germline CDR residue can be from a germline sequence that is similar (e.g., most similar) to the variable region being modified.
  • activity e.g., binding or other functional activity
  • Similar mutagenesis can be performed in the framework regions.
  • a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity.
  • the selection can be performed using at least 2, 3, 5, or 10 germline sequences.
  • identifying a similar germline sequence can include selecting one such sequence.
  • identifying a similar germline sequence can include selecting one such sequence, but may including using two germline sequences that separately contribute to the amino-terminal portion and the carboxy-terminal portion. In other implementations more than one or two germline sequences are used, e.g., to form a consensus sequence.
  • a related variable domain sequence has at at least 30, 40, 50, 60, 70, 80, 90, 95 or 100% of the CDR amino acid positions that are not identical to residues in the reference CDR sequences, residues that are identical to residues at corresponding positions in a human germline sequence (i.e., an amino acid sequence encoded by a human germline nucleic acid).
  • a related variable domain sequence has at at least 30, 50, 60, 70, 80, 90 or 100% of the FR regions are identical to FR sequence from a human germline sequence, e.g., a germline sequence related to the reference variable domain sequence.
  • an antibody which has similar activity to a given antibody of interest, but is more similar to one or more germline sequences, particularly one or more human germline sequences.
  • an antibody can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% identical to a germline sequence in a region outside the CDRs (e.g., framework regions).
  • an antibody can include at least 1, 2, 3, 4, or 5 germline residues in a CDR region, the germline residue being from a germline sequence of similar (e.g., most similar) to the variable region being modified.
  • Germline sequences of primary interest are human germline sequences.
  • the activity of the antibody e.g., the binding activity
  • Exemplary germline reference sequences for Vkappa include:
  • a germline reference sequence for the HC variable domain can be based on a sequence that has particular canonical structures, e.g., 1-3 structures in the Hl and H2 hypervariable loops.
  • the canonical structures of hypervariable loops of an immunoglobulin variable domain can be inferred from its sequence, as described in Chothia et al. (1992) J. MoI. Biol. 227:799-817; Tomlinson et al. (1992) J. MoI. Biol. 227:776-798); and Tomlinson et al. (1995) EMBO J. 14(18):4628-38.
  • Exemplary sequences with a 1-3 structure include: DP-I, DP-8, DP-12, DP-2, DP-25, DP-15, DP- 7, DP-4, DP-31, DP-32, DP-33, DP-35, DP-40, 7-2, hv3005, hv3OO5f3, DP-46, DP- 47, DP-58, DP-49, DP-50, DP-51, DP-53, and DP-54.
  • Display libraries and other libraries include variation at one or more positions in the displayed polypeptide.
  • the variation at a given position can be synthetic or natural.
  • both synthetic and natural diversity are included.
  • Synthetic Diversity Libraries can include regions of diverse nucleic acid sequence that originate from artificially synthesized sequences. Typically, these are formed from degenerate oligonucleotide populations that include a distribution of nucleotides at each given position. The inclusion of a given sequence is random with respect to the distribution.
  • One example of a degenerate source of synthetic diversity is an oligonucleotide that includes NNN wherein N is any of the four nucleotides in equal proportion.
  • Synthetic diversity can also be more constrained, e.g., to limit the number of codons in a nucleic acid sequence at a given trinucleotide to a distribution that is smaller than NNN. For example, such a distribution can be constructed using less than four nucleotides at some positions of the codon.
  • trinucleotide addition technology can be used to further constrain the distribution. So-called “trinucleotide addition technology” is described, e.g., in Wells et al. (1985) Gene 34:315-323, US 4,760,025 and US 5,869,644.
  • Natural Diversity Libraries can include regions of diverse nucleic acid sequence that originate (or are synthesized based on) from different naturally- occurring sequences.
  • An example of natural diversity that can be included in a display library is the sequence diversity present in immune cells (see also below). Nucleic acids are prepared from these immune cells and are manipulated into a format for polypeptide display.
  • the display library presents a diverse pool of proteins, each of which includes an immunoglobulin domain, e.g., an immunoglobulin variable domain.
  • Display libraries are particular useful, for example for identifying human or "humanized" antibodies that recognize human antigens. Such antibodies can be used as therapeutics to treat human disorders such as endothelial-related disorders, e.g., metastatic cancer. Since the constant and framework regions of the antibody are human, these therapeutic antibodies may avoid themselves being recognized and targeted as antigens. The constant regions are also optimized to recruit effector functions of the human immune system. The in vitro display selection process surmounts the inability of a normal human immune system to generate antibodies against self-antigens.
  • a typical antibody display library displays a polypeptide that includes a VH domain and a VL domain.
  • An "immunoglobulin domain” refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two ⁇ -sheets formed of about seven ⁇ -strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay 1988 Ann. Rev Immunol. 6:381-405). The canonical structures of hypervariable loops of an immunoglobulin variable can be inferred from its sequence, as described in Chothia et al. (1992) J. MoI. Biol.
  • the display library can display the antibody as a Fab fragment (e.g., using two polypeptide chains) or a single chain Fv (e.g., using a single polypeptide chain). Other formats can also be used.
  • the displayed antibody can include a constant region as part of a light or heavy chain.
  • each chain includes one constant region, e.g., as in the case of a Fab.
  • additional constant regions are displayed.
  • Antibody libraries can be constructed by a number of processes (see, e.g., de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20. and Hoogenboom et al. (2000) Immunol Today 21:371-8). Further, elements of each process can be combined with those of other processes. The processes can be used such that variation is introduced into a single immunoglobulin domain (e.g., VH or VL) or into multiple immunoglobulin domains (e.g., VH and VL).
  • a single immunoglobulin domain e.g., VH or VL
  • multiple immunoglobulin domains e.g., VH and VL
  • the variation can be introduced into an immunoglobulin variable domain, e.g., in the region of one or more of CDRl, CDR2, CDR3, FRl, FR2, FR3, and FR4, referring to such regions of either and both of heavy and light chain variable domains.
  • variation is introduced into all three CDRs of a given variable domain.
  • the variation is introduced into CDRl and CDR2, e.g., of a heavy chain variable domain. Any combination is feasible.
  • antibody libraries are constructed by inserting diverse oligonucleotides that encode CDRs into the corresponding regions of the nucleic acid.
  • the oligonucleotides can be synthesized using monomelic nucleotides or trinucleotides.
  • Knappik et al. (2000) J. MoI. Biol. 296:57-86 describe a method for constructing CDR encoding oligonucleotides using trinucleotide synthesis and a template with engineered restriction sites for accepting the oligonucleotides.
  • an animal e.g., a non-human animal, e.g., a rodent, is immunized with the Tiel .
  • the animal is optionally boosted with the antigen to further stimulate the response.
  • the non-human animal can include one or more human immunoglobulin gene sequences.
  • the animal can include a complete human immunoglobulin locus.
  • the animal may also have an inactivated endogenous immunoglobulin locus.
  • antibody libraries are constructed from nucleic acid amplified from na ⁇ ve germline immunoglobulin genes (e.g., human genes).
  • the amplified nucleic acid includes nucleic acid encoding the VH and/or VL domain. Sources of immunoglobulin-encoding nucleic acids are described below.
  • Amplification can include PCR, e.g., with primers that anneal to the conserved constant region, or another amplification method.
  • Nucleic acid encoding immunoglobulin domains or fragments thereof can be obtained from the immune cells of, e.g., a human, a primate, mouse, rabbit, camel, or rodent.
  • the cells are selected for a particular property.
  • B cells at various stages of maturity can be selected.
  • the B cells are na ⁇ ve.
  • fluorescent-activated cell sorting is used to sort B cells that express surface-bound IgM, IgD, or IgG molecules.
  • B cells expressing different isotypes of IgG can be isolated, hi another preferred embodiment, the B or T cell is cultured in vitro.
  • the cells can be stimulated in vitro, e.g., by culturing with feeder cells or by adding mitogens or other modulatory reagents, such as antibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin or pokeweed mitogen.
  • the cells are isolated from a subject that has an immunological disorder, e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, vasculitis, Sjogren syndrome, systemic sclerosis, or anti-phospholipid syndrome.
  • the subject can be a human, or an animal, e.g., an animal model for the human disease, or an animal having an analogous disorder.
  • me cells are isolated from a transgenic non-human animal that includes a human immunoglobulin locus.
  • the cells have activated a program of somatic hypermutation.
  • Cells can be stimulated to undergo somatic mutagenesis of immunoglobulin genes, for example, by treatment with anti-immunoglobulin, anti- CD40, and anti-CD38 antibodies (see, e.g., Bergthorsdottir et al (2001) J Immunol. 166:2228).
  • the cells are na ⁇ ve.
  • the nucleic acid encoding an immunoglobulin variable domain can be isolated from a natural repertoire by the following exemplary method.
  • the reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., de Haard et al. (1999) J. Biol. Chem 21 A: 18218-30.
  • the primer binding region can be constant among different immunoglobulins, e.g., in order to reverse transcribe different isotypes of immunoglobulin.
  • the primer binding region can also be specific to a particular isotype of immunoglobulin.
  • the primer is specific for a region that is 3' to a sequence encoding at least one CDR.
  • poly-dT primers may be used (and may be preferred for the heavy-chain genes).
  • a synthetic sequence can be ligated to the 3' end of the reverse transcribed strand.
  • the synthetic sequence can be used as a primer binding site for binding of the forward primer during PCR amplification after reverse transcription.
  • the use of the synthetic sequence can obviate the need to use a pool of different forward primers to fully capture the available diversity.
  • variable domain-encoding gene is then amplified, e.g., using one or more rounds. If multiple rounds are used, nested primers can be used for increased fidelity.
  • the amplified nucleic acid is then cloned into a display library vector.
  • Any method for amplifying nucleic acid sequences may be used for amplification. Methods that maximize and do not bias diversity are preferred. A variety of techniques can be used for nucleic acid amplification.
  • the polymerase chain reaction (PCR; U.S. Patent Nos. 4,683,195 and 4,683,202, Saiki, et al. (1985) Science 230, 1350-1354) utilizes cycles of varying temperature to drive rounds of nucleic acid synthesis.
  • Transcription-based methods utilize RNA synthesis by RNA polymerases to amplify nucleic acid (U.S. Pat. No 6,066,457; U.S. Pat. No 6,132,997; U.S. Pat. No 5,716,785; Sarkar et.
  • NASBA U.S. Patent Nos. 5,130,238; 5,409,818; and 5,554,517
  • Still other amplification methods include rolling circle amplification (RCA; U.S. Patent Nos. 5,854,033 and 6,143,495) and strand displacement amplification (SDA; U.S. Patent Nos. 5,455,166 and 5,624,825).
  • each candidate display library member can be further analyzed, e.g., to further characterize its binding properties for the target.
  • candidate binding proteins e.g., by immunization, etc.
  • Each candidate binding protein can be subjected to one or more secondary screening assays.
  • the assay can be for a binding property, a catalytic property, a physiological property (e.g., cytotoxicity, renal clearance, irnmunogenicity), a structural property (e.g., stability, conformation, oligomerization state) or another functional property.
  • the same assay can be used repeatedly, but with varying conditions, e.g., to determine pH, ionic, or thermal sensitivities.
  • the assays can use the display library member directly, a recombinant polypeptide produced from the nucleic acid encoding a displayed polypeptide, or a synthetic peptide synthesized based on the sequence of a displayed polypeptide.
  • Exemplary assays for binding properties include the following.
  • Proteins encoded by a display library can also be screened for a binding property using an ELISA assay. For example, each protein is contacted to a microtitre plate whose bottom surface has been coated with the target, e.g., a limiting amount of the target. The plate is washed with buffer to remove non-specifically bound polypeptides. Then the amount of the protein bound to the plate is determined by probing the plate with an antibody that can recognize the polypeptide, e.g., a tag or constant portion of the polypeptide. The antibody is linked to an enzyme such as alkaline phosphatase, which produces a colorimetric product when appropriate substrates are provided.
  • an enzyme such as alkaline phosphatase
  • the protein can be purified from cells or assayed in a display library format, e.g., as a fusion to a filamentous bacteriophage coat.
  • cells e.g., live or fixed
  • the target molecule e.g., Tiel, Tie2, or Ang
  • Binding proteins e.g., Tiel, Tie2, or Ang binding proteins
  • Binding proteins can be evaluated for their ability to interact with one or more cell types, e.g., endothelial cells or platelets.
  • Fluorescent activated cell sorting FLC is one exemplary method for testing an interaction between a protein and a cell.
  • the binding protein is labeled directly or indirectly with a fluorophore, before or after, binding to the cells, and then cells are counted in a FACS sorter.
  • Tiel binding protein interacts with platelets or other cell types.
  • Human blood can be obtained from informed healthy volunteers. For example, venous blood is collected into one-sixth volume of ACD (2.5 g of sodium citrate, 1.5 g citric acid, and 2.5 g glucose in 100 ml dH 2 O). The blood is centrifuged at 800 Xg for 15 min at room temperature and the platelet- rich plasma is removed and incubated for 60 min at 37°C. in the presence of 1 mM acetylsalicylic acid followed by centrifugation at 1000 x g for 10 min at room temperature.
  • ACD 2.5 g of sodium citrate, 1.5 g citric acid, and 2.5 g glucose in 100 ml dH 2 O.
  • the blood is centrifuged at 800 Xg for 15 min at room temperature and the platelet- rich plasma is removed and incubated for 60 min at 37°C. in the presence of 1 mM acetylsalicylic acid followed by centrifugation at 1000 x g for 10 min at room temperature.
  • the platelet pellet can be resuspended at a density of 2 x 10 8 cells/ml with HEPES-buffered Tyrode's solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 3 mM NaH 2 PO 4 , 5 mM glucose, 10 mM HEPES pH 7.4, 0.2% bovine serum albumin, and 0.05 U/mL apyrase). See also, e.g., Kornecki et at. (1990) J Biol Chem. 265:10,042-10,048 and Naik et al. (1995) Biochem J. 310:155-162).
  • FACS FACS analysis of platelets
  • cells can be resuspended in 0.1% BSA/PBS (4 x 10 5 cells/sample) in the presence of PGEl (1 mg/mL) and incubated with a candidate Tiel binding protein (e.g., at about 5 ⁇ g/mL) or with a control. After a 1-hour incubation at 22°C, the cells are washed with 0.1% BSA/PBS, treated with 50 ⁇ L 1/100 diluted FITC-labeled secondary antibody , incubated for 30 minutes on ice, washed, and resuspended in 0.1% BSA/PBS.
  • the samples are analyzed using an Immunocytometry Systems flow cytometer (FACSORTTM, Becton Dickinson, San Jose, CA). See also, e.g., Malgorzata et al. (2000) Blood, Vol. 95 No. 8 (April 15 pp. 2600-2609.
  • FACSORTTM Immunocytometry Systems flow cytometer
  • SDS-page separated proteins from isolated platelets and by immunoprecipitation involve binding cells to surfaces to which the Tie 1 -binding protein is attached (e.g., coated to).
  • FRET fluorescence resonance energy transfer
  • a fiuorophore label on the first molecule is selected such that its emitted fluorescent energy can be absorbed by a fluorescent label on a second molecule (e.g., the target) if the second molecule is in proximity to the first molecule.
  • the fluorescent label on the second molecule fluoresces when it absorbs to the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal.
  • a binding event that is configured for monitoring by FRET can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fl ⁇ orimeter). By titrating the amount of the first or second binding molecule, a binding curve can be generated to estimate the equilibrium binding constant.
  • SPR Surface Plasmon Resonance
  • the binding interaction of a molecule isolated from a display library and a target can be analyzed using SPR.
  • SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules.
  • Methods for using SPR are described, for example, in U.S. Patent No.
  • Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (Ka), and kinetic parameters, including K 0n and K off , for the binding of a biomolecule to a target.
  • Ka equilibrium dissociation constant
  • kinetic parameters including K 0n and K off
  • Such data can be used to compare different biomolecules. For example, different proteins can be compared to identify individuals that have high affinity for the target or that have a slow K off .
  • This information can also be used to develop structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of matured versions of a parent protein can be compared to the parameters of the parent protein. Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity and slow K off .
  • This information can be combined with structural modeling (e.g., using homology modeling, energy minimization, or structure determination by crystallography or NMR). As a result, an understanding of the physical interaction between the protein and its target can be
  • Proteins identified from the display library can be immobilized on a solid support, for example, on a bead or an array.
  • a protein array each of the polypeptides is immobilized at a unique address on a support.
  • the address is a two-dimensional address. Protein arrays are described below (see, e.g., Diagnostics). It is also possible to use a protein array to evaluate any plurality of proteins, e.g., for interaction with Tiel, Tie2, or Ang.
  • Candidate proteins can be selected from a library by transforming the library into a host cell; the library could have been previously identified from a display library.
  • the library can include vector nucleic acid sequences that include segments that encode the polypeptides and that direct expression, e.g., such that the proteins are produced within the cell, secreted from the cell, or attached to the cell surface.
  • the cells can be screened or selected for proteins that bind to the Tiel, Tie2, or Ang, e.g., as detected by a change in a cellular phenotype or a cell-mediated activity.
  • the activity maybe autophosphorylation, activation of PB Kinase, activation of AKT, or a change in endothelial cell activity (e.g., proliferation).
  • the library of cells is in the form of a cellular array.
  • the cellular array can likewise be screened for any appropriate detectable activity.
  • competition binding assays are used to identify proteins that are compete with a reference protein for binding to Tiel .
  • epitope mapping can be used to identify proteins that bind to a particular epitope of Tie. Fragments and mutants of Tiel can be also be used in the binding protein- identification process, e.g., in one or more of characterization, screening, or immunization.
  • Tiel ectodomain or a region thereof can be used as an antigen in a non-human animal, e.g., a rodent.
  • Tie2 or Ang, or a region thereof can be used as an antigen in a non-human animal, e.g., a rodent.
  • the non-human animal includes at least a part of a human immunoglobulin gene.
  • a human immunoglobulin gene For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci.
  • antigen-specific Mabs derived from the genes with the desired specificity may be produced and selected. See, e.g., XenoMouseTM, Green et al. Nature Genetics 7:13-21 (1994), U.S. 20030070185, WO 96/34096, published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996.
  • a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized or deimmunized.
  • Winter describes a CDR-grafting method that may be used to prepare the humanized antibodies (UK Patent Application GB 2188638A, filed on March 26, 1987; Winter US 5,225,539.
  • AU of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-Human UDKS. it is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.
  • Humanized antibodies can be generated by replacing sequences of the
  • Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions.
  • General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain.
  • Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a predetermined target, as described above.
  • the recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.
  • a target-binding antibody may also be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317, the contents of which are specifically incorporated by reference herein. Briefly, the heavy and light chain variable regions of an antibody can be analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317).
  • peptide threading For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the V H and V L sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes.
  • Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable regions, or preferably, by single amino acid substitutions. As far as possible conservative substitutions are made, often but not exclusively, an amino acid common at this position in human germline antibody sequences may be used.
  • nucleic acids encoding V H and V L can be constructed by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, and so forth).
  • Mutagenized variable sequence can, optionally, be fused to a human constant region, e.g., human IgGl or K constant regions.
  • a potential T cell epitope will include residues which are known or predicted to be important for antibody function. For example, potential T cell epitopes are usually biased towards the CDRs. In addition, potential T cell epitopes can occur in framework residues important for antibody structure and binding. Changes to eliminate these potential epitopes will in some cases require more scrutiny, e.g., by making and testing chains with and without the change. Where possible, potential T cell epitopes that overlap the CDRs were eliminated by substitutions outside the CDRs.
  • a target-binding protein or a candidate binding protein can be characterized using a cellular assay, e.g., to evaluate a change in a cellular phenotype or other activity when the binding protein is contacted to the cell.
  • the cell is expresses a protein that includes at least part of the ectodomain of Tie.
  • the cell expresses liel, e.g., a full-length, mature Tiel protein, Tie2, and/or is contacted with Ang..
  • a candidate target-binding protein can be tested for endothelial proliferation inhibiting activity using a biological activity assay such as the bovine capillary endothelial cell proliferation assay, the chick CAM assay, the mouse corneal assay, and evaluating the effect of the binding protein on implanted tumors.
  • a biological activity assay such as the bovine capillary endothelial cell proliferation assay, the chick CAM assay, the mouse corneal assay, and evaluating the effect of the binding protein on implanted tumors.
  • the chick CAM assay is described, e.g., by O'Reilly, et al. in "Angiogenic Regulation of Metastatic Growth" Cell, vol. 79 (2), Oct. 21, 1994, pp. 315-328. Briefly, three day old chicken embryos with intact yolks are separated from the egg and placed in a petri dish.
  • the mouse corneal assay involves implanting a growth factor-containing pellet, along with another pellet containing the suspected endothelial growth inhibitor, in the cornea of a mouse and observing the pattern of capillaries that are elaborated in the cornea.
  • Angiogenesis may be assayed , e.g., using various human endothelial cell systems, such as umbilical vein, coronary artery, or dermal cells. Suitable assays include Alamar Blue based assays (available from Biosource International) to measure proliferation; migration assays using fluorescent molecules, such as the use of Becton Dickinson Falcon HTS FLUOROBLOCKTM cell culture inserts to measure migration of cells through membranes in presence or absence of angiogenesis enhancer or suppressors; and tubule formation assays based on the formation of tubular structures by endothelial cells on MATRIGELTM (Becton Dickinson) or collagen I.
  • Alamar Blue based assays available from Biosource International
  • migration assays using fluorescent molecules such as the use of Becton Dickinson Falcon HTS FLUOROBLOCKTM cell culture inserts to measure migration of cells through membranes in presence or absence of angiogenesis enhancer or suppressors
  • tubule formation assays
  • Cell adhesion assays measure adhesion of cells to purified adhesion proteins or adhesion of cells to each other, in presence or absence of candidate target-binding proteins.
  • Cell-protein adhesion assays measure the ability of agents to modulate the adhesion of cells to purified proteins. For example, recombinant proteins are produced, diluted to 2.5 g/mL in PBS, and used to coat the wells of a microtiter plate. The wells used for negative control are not coated. Coated wells are then washed, blocked with 1% BSA, and washed again. Compounds are diluted to 2 x final test concentration and added to the blocked, coated wells.
  • Cell-cell adhesion assays can be used to measure the ability of candidate target-binding proteins to modulate binding of cells to each other. These assays can use cells that naturally or recombinantly express an adhesion protein of choice, hi an exemplary assay, cells expressing the cell adhesion protein are plated in wells of a multiwell plate together with other cells (either more of the same cell type, or another type of cell to which the cells adhere). The cells that can adhere are labeled with a membrane-permeable fluorescent dye, such as BCECF, and allowed to adhere to the monolayers in the presence of candidate binding proteins. Unbound cells are washed off, and bound cells are detected using a fluorescence plate reader. High-throughput cell adhesion assays have also been described. See, e.g., Falsey J R et al., Bioconjug Chem. May-June 2001;12(3):346-53.
  • Tubulogenesis assays can be used to monitor the ability of cultured cells, generally endothelial cells, to form tubular structures on a matrix substrate, which generally simulates the environment of the extracellular matrix.
  • exemplary substrates include MATRIGELTM (Becton Dickinson), an extract of basement membrane proteins containing laminin, collagen IV, and heparin sulfate proteoglycan, which is liquid at 4°C. and forms a solid gel at 37°C.
  • Other suitable matrices comprise extracellular components such as collagen, fibronectin, and/or fibrin. Cells are stimulated with a pro-angiogenic stimulant, and their ability to form tubules is detected by imaging.
  • Tubules can generally be detected after an overnight incubation with stimuli, but longer or shorter time frames may also be used.
  • Tube formation assays are well known in the art (e.g., Jones M K et al., 1999, Nature Medicine 5:1418-1423). These assays have traditionally involved stimulation with serum or with the growth factors FGF or VEGF.
  • the assay is performed with cells cultured in serum free medium.
  • the assay is performed in the presence of one or more pro-angiogenic agents, e.g., inflammatory angiogenic factors such as TNF- ⁇ , or FGF, VEGF, phorbol myristate acetate (PMA), TNF-alpha, ephrin, etc.
  • pro-angiogenic agents e.g., inflammatory angiogenic factors such as TNF- ⁇ , or FGF, VEGF, phorbol myristate acetate (PMA), TNF-alpha, ephrin, etc.
  • HMVEC human microvascular endothelial
  • Migration assays are known in the art (e.g., Paik J H et al., 2001, J Biol Chem 276:11830-11837).
  • cultured endothelial cells are seeded onto a matrix-coated porous lamina, with pore sizes generally smaller than typical cell size.
  • the lamina is typically a membrane, such as the transwell polycarbonate membrane (Corning Costar Corporation, Cambridge, Mass.), and is generally part of an upper chamber that is in fluid contact with a lower chamber containing pro-angiogenic stimuli. Migration is generally assayed after an overnight incubation with stimuli, but longer or shorter time frames may also be used. Migration is assessed as the number of cells that crossed the lamina, and may be detected by staining cells with hemotoxylin solution (VWR Scientific), or by any other method for determining cell number. In another exemplary set up, cells are fluorescently labeled and migration is detected using fluorescent readings, for instance using the Falcon HTS FLUOROBLOKTM (Becton Dickinson). While some migration is observed in the absence of stimulus, migration is greatly increased in response to pro- angiogenic factors. The assay can be used to test the effect of a target-binding protein on endothelial cell migration.
  • An exemplary sprouting assay is a three-dimensional in vitro angiogenesis assay that uses a cell-number defined spheroid aggregation of endothelial cells ("spheroid"), embedded in a collagen gel-based matrix.
  • the spheroid can serve as a starting point for the sprouting of capillary-like structures by invasion into the extracellular matrix (termed "cell sprouting") and the subsequent formation of complex anastomosing networks (Korff and Augustin, 1999, J Cell Sci 112:3249-58).
  • spheroids are prepared by pipetting 400 human umbilical vein endothelial cells into individual wells of a nonadhesive 96-well plates to allow overnight spheroidal aggregation (Korff and Augustin: J Cell Biol 143: 1341- 52, 1998). Spheroids are harvested and seeded in 900 ⁇ l of methocel-collagen solution and pipetted into individual wells of a 24 well plate to allow collagen gel polymerization. Test agents are added after 30 min by pipetting 100 ⁇ l of 10-fold concentrated working dilution of the test substances on top of the gel. Plates are incubated at 37 0 C for 24 h.
  • a target-binding protein has a statistically significant effect on an assay described herein, e.g., a cellular assay desr ⁇ bed herein.
  • Standard recombinant nucleic acid methods can be used to express a binding proteinthat binds to Tiel, Tie2, or Ang. See, for example, the techniques described in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3 rd Edition, Cold Spring Harbor Laboratory, N.Y. (2001) and Ausubel et al, Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley Interscience, N.Y. (1989). Generally, a nucleic acid sequence encoding the binding proteinis cloned into a nucleic acid expression vector. If the protein includes multiple polypeptide chains, each chain can be cloned into an expression vector, e.g., the same or different vectors, that are expressed in the same or different cells. Methods for producing antibodies are also provided below.
  • Some antibodies can be produced in bacterial cells, e.g., E. coli cells.
  • the Fab is encoded by sequences in a phage display vector that includes a suppressible stop codon between the display entity and a bacteriophage protein (or fragment thereof)
  • the vector nucleic acid can be shuffled into a bacterial cell that cannot suppress a stop codon.
  • the Fab is not fused to the gene III protein and is secreted into the media.
  • Antibodies can also be produced in eukaryotic cells.
  • the antibodies e.g., scFv's
  • the antibodies are expressed in a yeast cell such as Pichia (see, e.g., Powers et al. (2001) J Immunol Methods. 251:123-35), Hanseula, or Saccharomyces.
  • antibodies are produced in mammalian cells.
  • Preferred mammalian host cells for expressing the clone antibodies or antigen-binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. ScL USA 77:4216- 4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) MoI. Biol. 159:601-621), lymphocytic cell lines, e.g., NSO myeloma cells, SP2 cells, COS cells, HEK 293T cells, and a cell from a transgenic animal, e.g., a transgenic mammal.
  • Chinese Hamster Ovary CHO cells
  • SP2 cells including dhfr- CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. ScL USA 77:4216- 4220, used with a DHFR selectable
  • the cell is a mammary epithelial cell.
  • the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patents Nos. 4,399,216, 4,634,665 and 5,179,017).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhff host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • GS glutamine synthase
  • a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection.
  • the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/ AdMLP promoter regulatory element or an SV40 enhancer/ AdMLP promoter regulatory element) to drive high levels of transcription of the genes.
  • enhancer/promoter regulatory elements e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/ AdMLP promoter regulatory element or an SV40 enhancer/ AdMLP promoter regulatory element
  • the recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.
  • the selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G.
  • the codon usage can adapted to the codon bias of the host cell, e.g., for
  • CHO cells it can be adapted for the codon bias Cricetulus griseus genes.
  • regions of very high (> 80%) or very low ( ⁇ 30%) GC content can be avoid avoided where possible.
  • cis-acting sequence motifs were avoided: internal TATA-boxes; chi-sites and ribosomal entry sites; AT-rich or GC-rich sequence stretches; ARE, BSfS, CRS sequence elements; repeat sequences and RNA secondary structures; and (cryptic) splice donor and acceptor sites, branch points.
  • Two STOP codons can be used to ensure efficient termination.
  • the codon optimization of the sequence can be evaluated according to Sharp, P.M., Li, W.H., Nucleic Acids Res. 15 (3), 1987).
  • the standard codon adaptation index (CAI) can be used. Rare codons include those with a quality class between 0-40.
  • the invention features isolated nucleic acid molecules that are altered relative to a sequence described herein, e.g., to include improved codons or sequence features, include an isolated nucleic acid molecule that comprises a heavy or light chain coding sequence.
  • a heavy or light chain coding sequence For example, at least 30, 40, 45, 50, 60, 65, 70, 75, or 80% of the codons in the heavy or light chain coding sequence are non-rare or frequent codons in a mammalian cell or the heavy or light chain coding sequence includes fewer than 50, 45, 40, 35, 30, 25, 20, 15, 10% rare codons in a mammalian cell, e.g., a Chinese hamster cell ⁇ Cricetulus griseus).
  • the codon adaptation index is greater than 0.6, 0.7, 0.8, 0.85, 0.90, 0.92, 0.94, 0.95, 0.96, 0.97, or 0.98.
  • the heavy chain coding sequence encodes (i) a polypeptide comprising an antibody heavy chain described herein (e.g., an E3 heavy chain as set forth in SEQ ID NO:723), (ii) a polypeptide at least 85, 90, 95, 96, 97, 98, or 99% identical to an antibody heavy chain coding sequence described herein (e.g., SEQ ID NO:723), or (iii) a polypeptide that comprises a heavy chain variable domain sequence having the CDRs of an antibody heavy chain variable domain described herein (e.g., an E3 heavy chain variable domain).
  • the heavy chain coding sequence differs from SEQ E) NO:703 at at least 2, 3, 5, 6, 8, 9, 10, or 15 codons.
  • the light chain coding sequence encodes (i) a polypeptide comprising an antibody light chain described herein (e.g., an E3 light chain as set forth in SEQ ID NO:724), (ii) a polypeptide at least 85, 90, 95, 96, 97, 98, or 99% identical to an antibody light chain coding sequence described herein (e.g., SEQ ID NO:724), or (iii) a polypeptide that comprises a light chain variable domain sequence having the CDRs of an antibody light chain variable domain described herein (e.g., an E3 light chain variable domain).
  • the light chain coding sequence differs from SEQ ID NO:702 at at least 3, 5, 6, 8, 9, 10, or 15 codons.
  • one or more of the ala-GCG codons can be changed to GCC; one or more of the arg-CGT codons are changed to CGC; one or more of the pro-CCG codons are changed to CCC, CCT, or CCA; one or more of the ser-TCG codons are changed to TCC; and/or one or more of the thr-ACG codons are changed to ACC.
  • Codon-altered (e.g., codon-optimized) sequences can be used to produce an antibody.
  • An exemplary method includes providing a mammalian cell that includes an antibody-coding nucleic acid and expressing the nucleic acid in the cell, e.g., maintaining the cell under conditions in which the protein is expressed.
  • the antibody-coding nucleic acid can be providing in a mammalian expression vector, e.g., a vector that is introduced into the cell.
  • the cell can be a non-human mammalian cell, e.g., a CHO cell.
  • the antibody production system preferably synthesizes antibodies in which the Fc region is glycosylated.
  • the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain.
  • This asparagine is the site for modification with biantennary-type oligosaccharides. It has been demonstrated that this glycosylation is required for effector functions mediated by Fc ⁇ receptors and complement CIq (Burton and Woof (1992) Adv. Immunol. 51:1-84; Jefferis et al. (1998) Immunol. Rev. 163:59-76).
  • the Fc domain is produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297.
  • the Fc domain can also include other eukaryotic post-translational modifications.
  • Antibodies can also be produced by a transgenic animal. For example,
  • U.S 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal.
  • a transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion.
  • the milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest.
  • the antibody can be purified from the milk, or for some applications, used directly.
  • Non-human antibodies can also be modified to include substitutions for human immunoglobulin sequences, e.g., consensus human amino acid residues at particular positions, e.g., at one or more of the following positions (preferably at least five, ten, twelve, or all): (in the FR of the variable domain of the light chain) 4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 7OL, 71L, 73L, 85L, 87L, 98L, and/or (in the FR of the variable domain of the heavy chain) 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 6OH, 67H, 68H,
  • Tiel protein, or Tiel liposomes are known in the art. See, e.g., WO 93/14124. Methods for producing Tie2 and Ang are similarly known. See e.g., U.S. Patent Nos. 6,521,424, 6,376,653; WO 96/11269; WO 96/31598 .
  • Biotinylation Methods A variety of methods are available to biotinylate proteins, e.g., an immunoglobulin protein or a target protein.
  • the protein can be incubated with a 5-fold molar excess of sulfo-NHS-SS-biotin in 50 mM HEPES, pH 8.0, 100 mM NaCl overnight at 4 0 C.
  • Free biotin is removed by buffer exchange into PBS, 0.01% Tween 20, e.g., using a BIOMAX® device with a 10 kDa molecular weight cut-off membrane or by dialysis.
  • the number of biotin molecules incorporated per mole of protein can be determined using the HABA assay as described by the manufacturer (Pierce).
  • compositions e.g., pharmaceutically acceptable compositions, which include an agent that binds to Tiel, Tie2, or Ang, e.g., an antibody molecule, other polypeptide or peptide identified as binding to Tiel, Tie2, or Ang, or described herein, formulated with a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions encompass labeled binding proteins (e.g., for in vivo imaging) as well as therapeutic compositions.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the binding protein may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • a "pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., etal. (1977) J. Pharm. ScL 66:1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions tablets, pills, powders, liposomes and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of humans with antibodies.
  • the preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular), hi a preferred embodiment, the target-binding protein is administered by intravenous infusion or injection.
  • the target-binding protein is administered by intramuscular or subcutaneous injection.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the binding protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution 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.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the binding proteins described herein can be administered by a variety of methods known in the art, although for many applications, the preferred route/mode of administration is intravenous injection or infusion.
  • the target-binding protein can be administered by intravenous infusion, e.g., at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m 2 or 7 to 25 mg/m 2 .
  • the route and/or mode of administration will vary depending upon the desired results.
  • the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • the binding protein may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
  • the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • compositions can be administered with medical devices known in the art.
  • a pharmaceutical can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • implants and modules include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4.,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No.
  • a binding protein described herein can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier (BBB) excludes many highly hydrophilic compounds.
  • BBB blood-brain barrier
  • it can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes may include one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J. CHn. Pharmacol 29:685).
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms can be dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody described herein is 0.1-20 mg/kg, more preferably 1-10 mg/kg.
  • the target-binding antibody can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m 2 or about 5 to 30 mg/m 2 .
  • appropriate amounts can be proportionally less. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated.
  • compositions may be prepared using a
  • therapeutically effective amount or a “prophylactically effective amount” of an target-binding protein described herein.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the binding protein to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
  • a "therapeutically effective dosage” preferably inhibits a measurable parameter, e.g., inflammation or tumor growth rate by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
  • a measurable parameter e.g., inflammation or tumor growth rate
  • the ability of a compound to inhibit a measurable parameter, e.g., cancer, can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • kits including the binding protein that binds to Tiel, Tie2, or Ang and instructions for use, e.g., treatment, prophylactic, or diagnostic use.
  • the instructions for diagnostic applications include the use of the target-binding protein (e.g., antibody or antigen- binding fragment thereof, or other polypeptide or peptide) to detect Tiel, Tie2, or Ang, in vitro, e.g., in a sample, e.g., a biopsy or cells from a patient having an inflammatory disorder or a cancer or neoplastic disorder, or in vivo.
  • the target-binding protein e.g., antibody or antigen- binding fragment thereof, or other polypeptide or peptide
  • the instructions for therapeutic applications include suggested dosages and/or modes of administration in a patient with a cancer or neoplastic disorder.
  • the kit can further contain at least one additional reagent, such as a diagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agent as described herein, and/or one or more additional target-binding proteins, formulated as appropriate, in one or more separate pharmaceutical preparations.
  • target binding proteins (such as the Tiel antibodies described herein) can be produced from gene-based vectors, such as transgenes or via adenoviral delivery.
  • a target-binding agent e.g., a Tiel -binding protein, polypeptide, antibody, or aptamer described herein
  • a target-binding agent is physically associated with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, lymph, or other tissues.
  • a target-binding agent can be associated with a polymer, e.g., a substantially non-antigenic polymers, such as polyalkylene oxides or polyethylene oxides.
  • a polymer e.g., a substantially non-antigenic polymers, such as polyalkylene oxides or polyethylene oxides.
  • Suitable polymers will vary substantially by weight.
  • Exemplary polymers include polymers having molecular number average weights ranging from about 200 to about 35,000, from about 1,000 to about 15,000, and 2,000 to about 12,500.
  • an target-binding agent can be conjugated to a water soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone.
  • a water soluble polymer e.g., hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone.
  • a non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
  • Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; branched or unbranched polysaccharides which comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g.
  • polymannuronic acid or alginic acid
  • D-glucosamine D- galactosamine
  • D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparon.
  • PAO's Mono-activated, alkoxy-terminated polyalkylene oxides
  • mPEG's monomethoxy-terminated polyethylene glycols
  • C 1-4 alkyl-terminated polymers C 1-4 alkyl-terminated polymers
  • bis-activated polyethylene oxides Glycols
  • poly(ethylene glycol) is a linear or branched polyether terminated with hydroxyl groups and having the general structure:
  • PEG can be synthesized by anionic ring opening polymerization of ethylene oxide initiated by nucleophilic attack of a hydroxide ion on the epoxide ring.
  • Particularly useful for polypeptide modification is monomethoxy PEG, mPEG, having the general structure:
  • the polymer prior to cross-linking need not be, but preferably is, water soluble.
  • the product is water soluble, e.g., exhibits a water solubility of at least about 0.01 mg/ml, and more preferably at least about 0.1 mg/ml, and still more preferably at least about 1 mg/ml.
  • the polymer should not be highly immunogenic in the conjugate form, nor should it possess viscosity that is incompatible with intravenous infusion or injection if the conjugate is intended to be administered by such routes.
  • the polymer contains only a single group which is reactive. This helps to avoid cross-linking of protein molecules.
  • the polymer contains two or more reactive groups for the purpose of linking multiple agents to the polymer backbone.
  • gel filtration or ion exchange chromatography can be used to recover the desired derivative in substantially homogeneous form.
  • the molecular weight of the polymer can range up to about 500,000 D, and preferably is at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. The molecular weight chosen can depend upon the effective size of the conjugate to be achieved, the nature (e.g. structure, such as linear or branched) of the polymer, and the degree of derivatization.
  • the covalent crosslink can be used to attach a target-binding agent
  • polymer e.g., a protein
  • a polymer for example, crosslinking to the N-terminal amino group and epsilon amino groups found on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl or other hydrophilic groups.
  • the polymer may be covalently bonded directly to the target-binding protein without the use of a multifunctional (ordinarily bifunctional) crosslinking agent.
  • Covalent binding to amino groups is accomplished by known chemistries based upon cyanuric chloride, carbonyl diimidazole, aldehyde reactive groups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde; PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde, activated succinimidyl esters, activated dithiocarbonate PEG, 2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate activated PEG.)
  • Carboxyl groups can be derivatized by coupling PEG-amine using carbodiimide.
  • Sulfhydryl groups can be derivatized by coupling to maleimido- substituted PEG (e.g. alkoxy-PEG amine plus sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate) WO 97/10847 or PEG-maleimide commercially available from Shearwater Polymers, Inc., Huntsville, Ala.).
  • maleimido- substituted PEG e.g. alkoxy-PEG amine plus sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate
  • WO 97/10847 or PEG-maleimide commercially available from Shearwater Polymers, Inc., Huntsville, Ala.
  • free amino groups on the binding protein e.g.
  • epsilon amino groups on lysine residues can be thiolated with 2-imino-thiolane (Traut's reagent) and then coupled to maleimide-containing derivatives of PEG, e.g., as described in Pedley et al., Br. J. Cancer, 70: 1126-1130 (1994).
  • PEG polymers that can be attached to a target-binding agent (e.g., protein) are available, e.g., from Shearwater Polymers, Inc. (Huntsville, Ala.).
  • a target-binding agent e.g., protein
  • PEG derivatives include, e.g., amino-PEG, PEG amino acid esters, PEG-hydrazide, PEG-thiol, PEG-succinate, carboxymethylated PEG, PEG-propionic acid, PEG amino acids, PEG succinimidyl succinate, PEG succinimidyl propionate, succinimidyl ester of carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidyl esters of amino acid PEGs, PEG- oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether, PEG-aldehyde, PEG vinylsulf
  • the reaction conditions for coupling these PEG derivatives may vary depending on the Tie 1 -binding protein, the desired degree of PEGylation, and the PEG derivative utilized. Some factors involved in the choice of PEG derivatives include: the desired point of attachment (such as lysine or cysteine R-groups), hydrolytic stability and reactivity of the derivatives, stability, toxicity and antigenicity of the linkage, suitability for analysis, etc. Specific instructions for the use of any particular derivative are available from the manufacturer.
  • conjugates of an target-binding agent e.g., a Tiel binding protein
  • a polymer can be separated from the unreacted starting materials, e.g., by gel filtration or ion exchange chromatography, e.g., HPLC.
  • Heterologous species of the conjugates are purified from one another in the same fashion. Resolution of different species (e.g., containing one or two PEG residues) is also possible, e.g., due to the difference in the ionic properties of unreacted amino acids. See, e.g., WO 96/34015.
  • a target binding protein can also be physically associated with a protein that provides a stabilizing or retention function, e.g., an albumin, e.g., human serum albumin.
  • an albumin e.g., human serum albumin.
  • US 20040171794 describes exemplary methods for physically associating a protein with serum albumin.
  • human albumin sequences or fragments thereof see EP 201 239, EP 322 094 WO 97/24445, WO95/23857 especially the mature form of human albumin as shown in SEQ ID NO:18 of US 20040171794 and WO 01/79480 or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.
  • exemplary human serum albumin proteins can include one or both of the following sets of point mutations Leu-407 to Ala, Leu-408 to VaI, Val-409 to Ala, and Arg-410 to Ala; or Arg-410 to Ala, Lys-413 to GIn, and Lys-414 to GIn (see, e.g., International Publication No. WO95/23857, with reference to SEQ ID NO:18 of US 20040171794).
  • the invention also features target protein-binding agents such as aptamers.
  • nucleic acid "aptamer,” as used herein, refers to a nucleic acid molecule which has a conformation that includes an internal non-duplex nucleic acid structure of at least 5 nucleotides.
  • An aptamer can be a single-stranded nucleic acid molecule which has regions of self-complementarity.
  • Exemplary aptamers include nucleic acid molecules that bind to a target molecule other than a nucleic acid, e.g., to Tiel, Tie2, or Ang.
  • Particular aptamers may also modulate formation of a Tie complex or have one or more properties of a target binding agent described herein and can be used in place of a target binding protein.
  • Aptamers can be screened in vitro since a selected aptamer can be recovered by standard nucleic acid amplification procedures.
  • the method can be enhanced, e.g., in later rounds of selection, by splitting selected aptamers into pools and modifying each aptamer in the pool with a detectable label such as a fluorophore. Pools having aptamers that functionally alter the properties of the label can be identified. Such pools can be repeatedly split and reanalyzed to identify the individual aptamers with the desired properties (see, e.g., Jhaveri et al. Nature Biotechnol. 18:1293).
  • aptamers can be screened for activity in vivo.
  • shuffled nucleic acids can be cloned into an expression vector that is introduced into cells.
  • RNA aptamers resulting from the expressed shuffled nucleic acids can be screened for a biological activity.
  • Cells having the activity can be isolated and the expression vector for the selected RNA aptamer recovered.
  • oligomers e.g., aptamers
  • the backbone contains internucleoside linkages that are stable in vivo and is structured such that the oligomer is resistant to endogenous nucleases, such as nucleases that attack the phosphodiester linkage.
  • the oligomer retains its ability to hybridize to the target DNA or RNA (Agarwal, K. L. et al. (1979) Nucleic Acids Res. 3:109; Agarwal, S. et al. (1988) Proc. Natl Acad. Sd USA 85:7079).
  • Modified oligonucleotides can be constructed using alternate internucleoside linkages.
  • exemplary linkages are described in Uhhnann, E. and Peyman, A. (1990) Chemical Reviews 90:543-584.
  • methylphosphonates wherein one of the phosphorus-linked oxygens has been replaced by methyl
  • phosphorothioates wherein sulphur replaces one of these oxygens
  • various amidates wherein NH 2 or an organic amine derivative, such as morpholidates or piperazidates, replace an oxygen.
  • WO 91/15500 teaches various oligonucleotide analogs in which one or more of the internucleotide linkages are replaced by a sulfur based linkage, typically sulfamate diesters, which are isosteric and isoelectric with the phosphodiester.
  • WO 89/12060 similarly discloses linkages containing sulfides, sulfoxides, and sulfones.
  • WO 86/05518 suggests a variant of stereoregular polymeric 3',5'linkages.
  • U.S. Pat. No. 5,079,151 discloses a msDNA molecule of branched RNA linked to a single strand DNA via a 2',5' phosphodiester linkage.
  • No. 5,264,562 describes modified linkages of the formula -Y'CX' 2 Y' ⁇ wherein Y 1 is independently O or S and wherein each X' is a stabilizing substituent and independently chosen. Morpholino-type internucleotide linkages are described in U.S. Pat. No. 5,034,506 and in some cases give rise to an increased affinity of the oligomer for complementary target sequences. U.S. Pat. Nos. 5,264,562 5,596,086 disclose modified oligonucleotides having modified nucleoside linkages which are capable of strong hybridization to target RNA and DNA.
  • Binding agents that bind to Tiel , Tie2, or Ang have therapeutic and prophylactic utilities.
  • these binding agents can be administered to cells in culture, e.g. in vitro or ex vivo, or can be administered to a subject, e.g., in vivo, to treat, prevent, and/or diagnose a variety of disorders, such as endothelial cell disorders, blood vessel development disorders, wound healing, inflammatory diseases and cancers, particularly metastatic cancers.
  • treat refers to the application or administration of an agent, alone or in combination with one or more other agents (e.g., a second agent) to a subject, e.g., a patient, e.g., a patient who has a disorder (e.g., a disorder as described herein), a symptom of a disorder or a predisposition for a disorder, e.g., to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder.
  • Treating a cell refers to a reduction in an activity of a cell, e.g., ability of an endothelial cell to form tubes or vessels. A reduction does not necessarily require a total elimination of activity, but a reduction, e.g., a statistically significant reduction, in the activity or the number of the cell.
  • an amount of a target binding agent effective to treat a disorder refers to an amount of the binding agent which is effective, upon single or multiple-dose administration to a subject, in treating a cell, e.g., an endothelial cell (e.g., a Tiel -expressing endothelial cell) or cancer cell (particularly a metastatic cell thereof), or in prolonging curing, alleviating, relieving or improving a subject with a disorder as described herein beyond that expected in the absence of such treatment, hi some cases, a therapeutically effective amount can be ascertained by evaluating the ability of the binding agent to reduce tumor size of a xenograft in a nude mouse model relative to an untreated control mouse.
  • inhibiting the growth" of a tumor or other neoplasm refers to slowing, interrupting, arresting or stopping its growth and metastases and does not necessarily indicate a total elimination of the neoplastic
  • an amount of an target-binding agent effective to prevent a disorder refers to an amount of a target binding agent, e.g., a Tiel-binding protein, e.g., a Tiel- binding antibody described herein, which is effective, upon single- or multiple-dose administration to the subject, for preventing or delaying the occurrence of the onset or recurrence of a disorder, e.g., an endothelial cell-related disorder, a blood vessel development disorder, an inflammatory disease or a cancer.
  • a target binding agent e.g., a Tiel-binding protein, e.g., a Tiel- binding antibody described herein
  • Subjects that can be treated include human and non-human animals.
  • the human can be a human patient having a disorder characterized by abnormal cell proliferation or cell differentiation.
  • non-human animals includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, sheep, dog, cow, pig, etc.
  • a binding agent described herein can be used to reduce angiogenesis in a subject, e.g., to treat a cancer (e.g., a solid tumor) or an angiogenesis-associated disorder.
  • the method includes administering the binding to the subject, e.g., in an amount effective to modulate angiogenesis, a symptom of the disorder, or progression of the disorder.
  • the agent e.g., a Tiel-binding protein, e.g., an anti-Tiel antibody, e.g., E3
  • the binding agent e.g., a Tiel binding protein
  • a Tiel binding protein can be used to treat or prevent cancer
  • reduction in Tiel activity by a Tiel-binding protein can reduce or prevent angiogenesis near and around the tumor, thereby reducing or preventing tumor growth
  • the neoplasia includes endothelial or hematopoietic cells that are proliferating abnormally.
  • a Tiel-binding protein can be used to modulate the cells of a cancer themselves, e.g., to kill or ablate a neoplastic cell that expresses Tiel.
  • the cell is a hematopoietic cell.
  • cancers that can be treated include, but are not limited to, solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary tract (e.g., renal, urothelial cells), pharynx, prostate, ovary as well as adenocarcinomas which include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and so forth. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the Tiel binding proteins and other agents described herein.
  • solid tumors that can be treated include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastrointestinal system carcinomas, colon carcinoma, pancreatic cancer, breast cancer, genitourinary system carcinomas, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bil
  • a Tiel-binding protein can also be used to inhibit the proliferation of hyperplastic/neoplastic cells of hematopoietic origin, e.g., cells arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof, particularly such cells that express Tiel .
  • the binding proteins described herein can be used for the treatment of various myeloid disorders including, but not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) CritRev. in Oncol./Hemotol. 11 :261-91).
  • APML acute promyeloid leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • Lymphoid malignancies which may be treated include, but are not limited to acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • HLL hairy cell leukemia
  • WM Waldenstrom's macroglobulinemia
  • Additional forms of malignant lymphomas include non-Hodgkin's lymphoma and variants thereof, peripheral T-cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF) and Hodgkin's disease.
  • Tiel has been shown to be upregulated in acute myelogenous leukemia and myelodysplastic syndrome (Verstovsek et al., 2001, Leuk, Lymphoma), B cell chronic lymphocytic leukemia (Aguayo et al, 2001. Leukemia Research 25(4):279-85.) 5 binding proteins that interact with Tiel can be used to detect, treat, or prevent these diseases.
  • a subject having or at risk for a hematopoietic disorder can be treated by administering a Tiel binding protein, e.g. a Tiel binding protein that increases Tiel homodimerization, or a binding protein that antagonizes Tie complex formation.
  • a Tiel binding protein e.g. a Tiel binding protein that increases Tiel homodimerization, or a binding protein that antagonizes Tie complex formation.
  • the Tiel binding protein can be an anti-Tiel antibody, e.g., an antibody described herein.
  • the administration of the binding protein can include multiple administrations, e.g., to achieve a therapeutic concentration using more than one dose. For example, the administrations can be about once a week, every second or third day, etc.
  • Tie 1 -binding proteins and other agents are also described in "Pharmaceutical Compositions". Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used.
  • the binding proteins can be used as competitive agents to inhibit, reduce an undesirable interaction, e.g., between a natural or pathological agent and the Tiel.
  • the Tiel-binding proteins are used to inhibit (e.g., inhibit at least one activity of, reduce proliferation, migration, growth or viability) of a cell, e.g., an endothelial cell in vivo.
  • the binding proteins can be used by themselves or conjugated to an agent, e.g., a cytotoxic drug, cytotoxin enzyme, or radioisotope. This method includes: administering the binding protein alone or attached to a cytotoxic drug, to a subject requiring such treatment.
  • Tiel-binding proteins recognize Tie 1 -expressing endothelial cells and can bind to endothelial cells that are associated with (e.g., in proximity of or intermingled with) cancer cells, e.g., cancerous lung, liver, colon, breast, ovarian, epidermal, laryngeal, and cartilage cells, and particularly metastatic cells thereof, Tiel-binding proteins can be used to inhibit (e.g., inhibit at least one activity, reduce growth and proliferation, or kill) any such cells and inhibit angiogenesis. Reducing endothelial cell activity near a cancer can indirectly inhibit (e.g., inhibit at least one activity, reduce growth and proliferation, or kill) the cancer cells which may be dependent on the endothelial cells for nutrients, growth signals and so forth.
  • cancer cells e.g., cancerous lung, liver, colon, breast, ovarian, epidermal, laryngeal, and cartilage cells, and particularly metastatic cells thereof.
  • Tiel-binding proteins can
  • the binding proteins bind to cells in the vicinity of the cancerous cells, but are sufficiently close to the cancerous cells to directly or indirectly inhibit (e.g., inhibit at least one activity, reduce growth and proliferation, or kill) the cancers cells.
  • the Tiel-binding proteins e.g., modified with a toxin, e.g., a cytotoxin
  • the binding proteins may be used to deliver a variety of cytotoxic drugs including therapeutic drugs, a compound emitting radiation, molecules of plants, fungal, or bacterial origin, biological proteins, and mixtures thereof.
  • the cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as toxins short-range radiation emitters, e.g., short-range, high-energy ⁇ -emitters.
  • a first binding protein is conjugated with a prodrug which is activated only when in close proximity with a prodrug activator.
  • the prodrug activator is conjugated with a second binding protein, preferably one which binds to a non-competing site on the target molecule. Whether two binding proteins bind to competing or non-competing binding sites can be determined by conventional competitive binding assays. Exemplary drug-prodrug pairs are described in Blakely et al., (1996) Cancer Research, 56:3287-3292.
  • the Tiel-binding proteins can be used directly in vivo to eliminate antigen-expressing cells via natural complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).
  • the binding proteins described herein can include complement binding effector domain, such as the Fc portions from IgGl , -2, or -3 or corresponding portions of IgM which bind complement.
  • a population of target cells is ex vivo treated with a binding agent described herein and appropriate effector cells. The treatment can be supplemented by the addition of complement or serum containing complement.
  • phagocytosis of target cells coated with a binding protein described herein can be improved by binding of complement proteins.
  • cells coated with the binding protein which includes a complement binding effector domain are lysed by complement.
  • Tiel expression may be induced in response to hypoxic signals that can arise within the interior of a tumor to stimulate changes in vasculature, including blood and lymphatic vessels so as to increase nutrient and oxygen supply to the tumor.
  • Certain Tiel -binding antibodies e.g., E3 and related antibodies
  • E3 and related antibodies may be particularly effective because they can inhibit changes to tumor vasculature and may cause a decrease in intra-tumor pressure.
  • These agents may also be well suited as therapeutics in situations in which conventional agents have difficulty in penetrating into a tumor.
  • Tiel binding proteins may leave hematopoiesis unaffected. Treatment can be effectively monitored with clinical parameters. Alternatively, these parameters can be used to indicate when such treatment should be employed.
  • a Tiel binding protein e.g. a Tiel binding protein that increases Tiel homodimerization, or a binding protein that antagonizes Tie complex formation can be administered to a subject to treat or prevent an inflammatory disorder, e.g., psoriasis or rheumatoid arthritis.
  • an inflammatory disorder e.g., psoriasis or rheumatoid arthritis.
  • Psoriasis is a chronic skin disease, characterized by scaling and inflammation. When psoriasis develops, typically patches of skin thicken, redden, and become covered with silvery scales, referred to as plaques. Psoriasis most often occurs on the elbows, knees, scalp, lower back, face, palms, and soles of the feet. The disease also may affect the fingernails, toenails, and the soft tissues inside the mouth and genitalia. About 10 percent of people with psoriasis have joint inflammation that produces symptoms of arthritis. Patients can be evaluated using a static Physician Global Assessment (sPGA), and receive a category score ranging from six categories between clear and very severe. The score is based on plaque, scaling, and erythema. The therapeutic methods herein can be used to achieve an improvement for at least one of these indicia.
  • sPGA Physician Global Assessment
  • RA Rheumatoid arthritis
  • RA Rheumatoid arthritis
  • RA is a chronic inflammatory disease that causes pain, swelling, stiffness, and loss of function, primarily the joints.
  • RA frequently begins in the synovium, the membrane that surrounds a joint creating a protective sac.
  • leukocytes infiltrate from the circulation into the synovium causing continuous abnormal inflammation (e.g., synovitis). Consequently, the synovium becomes inflamed, causing warmth, redness, swelling, and pain.
  • the collagen in the cartilage is gradually destroyed, narrowing the joint space and eventually damaging bone. The inflammation causes erosive bone damage in the affected area.
  • RA can be assessed by a variety of clinical measures.
  • Some exemplary indicia include the total Sharp score (TSS), Sharp erosion score, and the HAQ disability index.
  • TSS total Sharp score
  • Sharp erosion score the HAQ disability index.
  • the therapeutic methods herein can be used to achieve an improvement for at least one of these indicia.
  • a Tiel binding protein e.g. a Tiel binding protein that increases Tiel homodimerization
  • a binding protein that antagonizes Tie complex formation can be administered to a subject to treat or prevent a retinal disorder, e.g., a proliferative retinopathy, such as diabetic retinopathy, ischemic retinopathy, or retinopathy of prematurity; choroidal neovascularization; lens neovasculation; corneal neovascularization; iridial neovascularization; or conjunctival neovascularization.
  • the binding protein can be used to reduce the risk of retinal detachment associated with pathological ocular neovascularization, hi some cases, the binding protein is administered by subconjunctival administration.
  • Binding proteins described herein can be administered in combination with one or more of the other therapies for treating cancers, including, but not limited to: surgery; radiation therapy, and chemotherapy.
  • proteins that antagonize Tie complex formation or that modulate Tie signalling activity can also be used in combination with other anti-cancer therapies, such as radiation therapy, chemotherapy, surgery, or administration of a second agent.
  • the second agent can be one that targets or negatively regulates the VEGF signaling pathway.
  • VEGF antagonists e.g., anti-VEGF antibodies such as bevacizumab
  • VEGF receptor antagonists e.g., anti-VEGF receptor antibodies
  • One particularly combination includes bevacizumab.
  • the combination can further include 5 -FU and leucovorin, and/or irinotecan.
  • the term "combination” refers to the use of the two or more agents or therapies to treat the same patient, wherein the use or action of the agents or therapies overlap in time.
  • the agents or therapies can be administered at the same time (e.g., as a single formulation that is administered to a patient or as two separate formulations administered concurrently) or sequentially in any order. Sequential administrations are administrations that are given at different times.
  • the time between administration of the one agent and another agent can be minutes, hours, days, or weeks.
  • Tiel binding protein described herein can also be used to reduce the dosage of another therapy, e.g., to reduce the side-effects associated with another agent that is being administered, e.g., to reduce the side-effects of an anti-VEGF antibody such as bevacizumab.
  • a combination can include administering a second agent at a dosage at least 10, 20, 30, or 50% lower than would be used in the absence of the Tiel binding protein.
  • a subject can be treated for an angiogenesis-associated disorder by administering to the subject a first and second agent.
  • the first agent modulates early stage angiogenesis and the second agent modulates a subsequent stage of angiogenesis or also modulates early stage angiogenesis.
  • the first and second agents can be administered using a single pharmaceutical composition or can be administered separately.
  • the first agent is a VEGF pathway antagonist (e.g., an inhibitor of a VEGF (e.g., VEGF-A, -B, or -C) or a VEGF receptor (e.g., KDR or VEGF receptor III (Flt4)) or a bFGF pathway antagonist (e.g., an antibody that binds to bFGF or a bFGF receptor).
  • VEGF pathway antagonists are also described, herein and elsewhere.
  • the second agent inhibits or decreases assembly and stabilization of the blood vessels, disrupts maintenance of blood or lymphatic vessels, or alters distribution of lymphatic vessels in tumors.
  • the second agent comprises inhibits a Tie complex formation or promotes Tiel homodimerization.
  • the second agent is a Tiel binding protein described herein.
  • An early stage of tumor angiogenesis can include a signal from the tumor, e.g., secretion of VEGF, to stimulate the growth of new blood vessels from the host and infiltration of the tumor by the vessels.
  • VEGF can, for example, stimulate proliferation of endothelial cells that are then assembled into blood vessels.
  • a late stage of tumor angiogenesis can include a signal that leads to the assembly and stabilization of the blood vessels. This assembly and stabilization may involve interaction between the endothelial cells and the pericytes that surround the endothelial cells of the vessels.
  • Tiel may play a role in the assembly and stabilization of the vessels and in maintaining the association between the pericytes and endothelial cells.
  • an effective therapy to treat angiogenesis-related disorders can involve a combination of an agent that modulates an early stage angiogenesis (e.g., VEGF pathway antagonists, e.g., anti- VEGF (e.g., bevacizumab) or anti-VEGF receptor (e.g., anti-KDR) antibodies; or antagonists of other pro-angiogenic pathways, e.g., anti-bFGF antibodies or anti- bFGF receptor (e.g., anti-bFGF receptor-1, -2, -3) antibodies) and an agent that modulates a late stage of tumor angiogenesis (e.g., antagonists of Tiel (e.g., anti-Tiel antibodies (e.g., an antibody disclosed herein, e.g., an E3 antibody)), of Tie2 (e.g., anti-
  • Exemplary VEGF receptor antagonists include inhibitors of VEGF receptor tyrosine kinase activity.
  • 4-[4-(l-Amino-l-methylethyl)phenyl]-2-[4-(2- mo ⁇ holin-4-yl-ethyl)phenylamino]pyrimidine-5-carbonitrile JNJ-17029259 is one of a structural class of 5-cyanopyrimidines that are orally available, selective, nanomolar inhibitors of the vascular endothelial growth factor receptor-2 (VEGF-R2).
  • Additional examples include: PTK-787/ZK222584( Astra-Zeneca), SU5416, SUl 1248 (Pfizer), and ZD6474 ([N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(l- methylpiperidm-4-yl)methoxy]quinazohn-4-amme]).
  • Still other agents that can be used in combination with Tiel -binding proteins are broad specificity tyrosine kinase inhibitors, e.g., SU6668. See, e.g., Bergers, B. et al. (2003) J. Clin. Invest. Ill, 1287-1295.
  • the second agent or therapy can also be another anti-cancer agent or therapy.
  • anti-cancer agents include, e.g., anti-microtubule agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis, radiation, and antibodies against other tumor- associated antigens (including naked antibodies, immunotoxins and radioconjugates).
  • anti-cancer agents examples include antitubulin/antimicrotubule, e.g., paclitaxel, vincristine, vinblastine, vindesine, vinorelbin, taxotere; topoisomerase I inhibitors, e.g., irinotecan, topotecan, camptothecin, doxorubicin, etoposide, mitoxantrone, daunorubicin, idarubicin, teniposide, amsacrine, epirubicin, merbarone, piroxantrone hydrochloride; antimetabolites, e.g., 5-fluorouracil (5-FU), methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, cytarabine/Ara-C, trimetrexate, gemcitabine, acivicin, alanosine, pyrazofurin, N-Phosphor
  • a combination therapy can include administering an agent that reduces the side effects of other therapies.
  • the agent can be an agent that reduces the side effects of anti-cancer treatments.
  • the agent can be leucovorin.
  • Combination therapies that include administering a Tiel binding protein or other binding protein described herein can also be used to treat a subject having or at risk for another angiogenesis related disorder (e.g., a disorder other than cancer, e.g., disorders that include undesired endothelial cell proliferation or undesirable inflammation, e.g., rheumatoid arthritis.
  • another angiogenesis related disorder e.g., a disorder other than cancer, e.g., disorders that include undesired endothelial cell proliferation or undesirable inflammation, e.g., rheumatoid arthritis.
  • Binding proteins that bind to Tiel have in vitro and in vivo diagnostic, therapeutic and prophylactic utilities.
  • the invention provides a diagnostic method for detecting the presence of a Tiel, in vitro (e.g., a biological sample, such as tissue, biopsy, e.g., a cancerous tissue) or in vivo (e.g., in vivo imaging in a subject).
  • the method includes: (i) contacting a sample with Tiel -binding protein; and (ii) detecting formation of a complex between the Tiel -binding protein and the sample.
  • the method can also include contacting a reference sample (e.g., a control sample) with the binding protein, and determining the extent of formation of the complex between the binding protein and the sample relative to the same for the reference sample.
  • a change, e.g., a statistically significant change, in the formation of the complex in the sample or subject relative to the control sample or subject can be indicative of the presence of Tiel in the sample.
  • the Tiel -binding protein can be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • Tiel -binding protein and Tiel can be detected by measuring or visualizing either the binding protein bound to the Tiel or unbound binding protein.
  • Conventional detection assays can be used, e.g., an enzyme-linked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry.
  • ELISA enzyme-linked immunosorbent assays
  • RIA radioimmunoassay
  • tissue immunohistochemistry e.g., tissue immunohistochemistry.
  • the presence of Tiel can be assayed in a sample by a competition immunoassay utilizing standards labeled with a detectable substance and an unlabeled Tiel -binding protein.
  • the biological sample, the labeled standards and the Tiel binding agent are combined and the amount of labeled standard bound to the unlabeled binding protein is determined.
  • the amount of Tiel in the sample is inversely proportional to the amount of labeled standard bound to the Tiel binding agent.
  • Fluorophore and chromophore labeled binding proteins can be prepared. Since antibodies and other proteins absorb light having wavelengths up to about 310 nm, the fluorescent moieties should be selected to have substantial absorption at wavelengths above 310 nm and preferably above 400 nm. A variety of suitable fluorescers and chromophores are described by Stryer (1968) Science, 162:526 and Brand, L. et al. (1972) Annual Review of Biochemistry, 41:843-868. The binding proteins can be labeled with fluorescent chromophore groups by conventional procedures such as those disclosed in U.S. Patent Nos. 3,940,475, 4,289,747, and 4,376,110.
  • the binding protein can be used to detect the presence or localization of the Tiel in a sample, e.g., using fluorescent microscopy (such as confocal or deconvolution microscopy).
  • Immunohistochemistry can be performed using the binding proteins described herein.
  • the antibody in the case of an antibody, the antibody can synthesized with a label (such as a purification or epitope tag), or can be detectably labeled, e.g., by conjugating a label or label-binding group.
  • a chelator can be attached to the antibody.
  • the antibody is then contacted to a histological preparation, e.g., a fixed section of tissue that is on a microscope slide. After an incubation for binding, the preparation is washed to remove unbound antibody.
  • the preparation is then analyzed, e.g., using microscopy, to identify if the antibody bound to the preparation.
  • the method can be used to evaluate an endothelial cell or tissue formed by endothelial cells, e.g., blood vessels.
  • the antibody (or other polypeptide or peptide) can be unlabeled at the time of binding. After binding and washing, the antibody is labeled in order to render it detectable.
  • the Tiel -binding protein can also be immobilized on a protein array.
  • the protein array can be used as a diagnostic tool, e.g., to screen medical samples (such as isolated cells, blood, sera, biopsies, and the like).
  • the protein array can also include other binding proteins, e.g., that bind to Tiel or to other target molecules, such as hyaluronic acid.
  • Methods of producing polypeptide arrays are described, e.g., in De
  • Polypeptides for the array can be spotted at high speed, e.g., using commercially available robotic apparati.
  • the array substrate can be, for example, nitrocellulose, plastic, glass, e.g., surface-modified glass.
  • the array can also include a porous matrix, e.g., acrylamide, agarose, or another polymer.
  • the array can be an array of antibodies, e.g., as described in De Wildt, supra.
  • Cells that produce the binding proteins can be grown on a filter in an arrayed format. Polypeptide production is induced, and the expressed polypeptides are immobilized to the filter at the location of the cell.
  • a protein array can be contacted with a labeled target to determine the extent of binding of the target to each immobilized polypeptide. If the target is unlabeled, a sandwich method can be used, e.g., using a labeled probed, to detect binding of the unlabeled target.
  • Information about the extent of binding at each address of the array can be stored as a profile, e.g., in a computer database.
  • the protein array can be produced in replicates and used to compare binding profiles, e.g., of a target and a non-target.
  • FACS Fluorescent Activated Cell Sorting
  • the target-binding protein can be used to label cells, e.g., cells in a sample (e.g., a patient sample).
  • the binding protein can also be attached (or attachable) to a fluorescent compound.
  • the cells can then be sorted using fluorescent activated cell sorted (e.g., using a sorter available from Becton Dickinson Immunocytometry Systems, San Jose CA; see also U.S. 5,627,037; 5,030,002; and 5,137,809).
  • a laser beam excites the fluorescent compound while a detector counts cells that pass through and determines whether a fluorescent compound is attached to the cell by detecting fluorescence.
  • the amount of label bound to each cell can be quantified and analyzed to characterize the sample.
  • the sorter can also deflect the cell and separate cells bound by the binding protein from those cells not bound.
  • the separated cells can be cultured and/or characterized.
  • the invention provides a method for detecting the presence of a Tiel-expressing cancerous tissues in vivo.
  • the method includes: administering the Tie 1 -binding protein to a subject; and detecting the Tie 1 -binding protein in the subject.
  • the detecting can include determining location or time of formation of the complex.
  • the method can include scanning or otherwise imaging the subject, e.g., a region of the subject's body.
  • Another method includes (i) administering to a subject (e.g., a patient having a cancer or neoplastic disorder) a Tiel-binding antibody, conjugated to a detectable marker; (ii) exposing the subject to a means for detecting said detectable marker to the Tiel- expressing tissues or cells.
  • a subject e.g., a patient having a cancer or neoplastic disorder
  • exposing the subject a means for detecting said detectable marker to the Tiel- expressing tissues or cells.
  • the method can be used visualize blood vessels or the location of endothelial cells, e.g., Tiel-expressing endothelial cells.
  • the subject can be imaged, e.g., by NMR or other tomographic means.
  • labels useful for diagnostic imaging include radiolabels such as 131 1, 111 In, 123 1, 99m Tc, 32 P, 125 1, 3 H, 14 C, and 188 Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography (“PET") scanner, chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase.
  • Short-range radiation emitters, such as isotopes detectable by short-range detector probes can also be employed.
  • the binding protein can be labeled with such reagents using known techniques.
  • a radiolabeled binding protein can also be used for in vitro diagnostic tests.
  • the specific activity of an isotopically-labeled protein depends upon the half-life, the isotopic purity of the radioactive label, and how the label is incorporated into the protein.
  • Effective imaging agents for tumor-associated neo-vasculature are needed. Tiel is up regulated on tumor-associated vasculature.
  • the binding proteins described herein can be used to image such vasculature.
  • binding proteins described herein can be used for imaging in several ways.
  • a binding protein can be physically associated, e.g., coupled to a chelator for imaging agents such as 99m Tc, 186 Re, or 188 Re.
  • 99m Tc and 188 Re emit gamma rays suitable for single photon emission computer tomography (SPECT) imaging.
  • Radioactive fluorine ( 18 F), indium ( 111 In), iodine ( 123 1, 131 I), gallium ( 68 Ga, 67 Ga), carbon ( 11 C), thallium ( 201 Tl), and other elements may be used as imaging agents.
  • the binding proteins can also be attached, covalently or non- covalently, to a particle, e.g., a nano-particle, that includes a radionuclide or spin labels suitable for use as an imaging agent.
  • the binding proteins can be linked to a spin label that would allow imaging through MRI.
  • Botnar et al. ⁇ Circulation. (2004) 109:2023-2029.) describe MRI imaging using an exemplary gadolinium-labeled peptide.
  • the binding proteins described herein can be similarly labeled for imaging.
  • Positron Emission Tomography can be used with imaging agents such as positron emitters such as 64 Cu and 18 F. These isotopes are becoming more readily available. 6 Cu can be captured in the chelator DOTA.
  • DOTA derivatives can be covalently linked to proteins. In one embodiment, one or more DOTA derivatives are attached to a binding protein (e.g., a Fab) through a lysine side group.
  • Fabs are useful binding agents for imaging because they: a) clear from the system fairly raipdly, allowing imaging within a few hours of injection, and b) penetrate tumors efficiently.
  • Fabs that bind to Tiel , Tie2, or Ang can be produced, e.g., in E. coli or in eukaryotic cells.
  • the Fabs can be purified by chromatography over protein A. Ion exchange chromatography can also be used.
  • covalent attachment of a chelating group suitable to the desired radionuclide or other imaging agent allows the Fab to be labeled at the time of use.
  • the Fabs can also have spin labels attached to allow MRI imaging.
  • Fabs can also be attached to particles (e.g., nano-particles) that include a radionuclide or spin label suitable for imaging.
  • Fabs may be coupled to PEG molecules to adjust the rate and pathway of clearance. In other embodiments, the Fabs are not coupled to PEG, e.g., to maintain their rapid clearance properties.
  • the binding protein is administered to the patient, is localized to the tumor bearing the antigen with which the binding protein reacts, and is detected or "imaged" in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. See e.g., A.R. Bradwell et al., "Developments in Antibody Imaging", Monoclonal Antibodies for Cancer Detection and Therapy, R.W. Baldwin et al., (eds.), pp 65-85 (Academic Press 1985).
  • positron emission transaxial tomography scanner such as designated Pet VI located at Brookhaven National Laboratory, can be used where the radiolabel emits positrons (e.g., 1 C, F, 5 O, and 13 N).
  • positrons e.g., 1 C, F, 5 O, and 13 N.
  • NMR magnetic resonance to visualize internal features of living subject
  • MRI magnetic resonance
  • Some MRI techniques are summarized in EP-A-O 502 814.
  • Tl and T2 of water protons in different environments are used to generate an image.
  • these differences can be insufficient to provide sharp high resolution images.
  • contrast agents include a number of magnetic agents paramagnetic agents (which primarily alter Tl) and ferromagnetic or superparamagnetic (which primarily alter T2 response).
  • Chelates e.g., EDTA, DTPA and NTA chelates
  • Some paramagnetic substances e.g., Fe +3 , Mn +2 , Gd +3 .
  • Other agents can be in the form of particles, e.g., less than 10 ⁇ m to about 10 nM in diameter).
  • Particles can have ferromagnetic, antiferromagnetic or superparamagnetic properties.
  • Magnetic particles can include, e.g., magnetite (Fe 3 O 4 ), ⁇ -Fe 2 O 3 , ferrites, and other magnetic mineral compounds of transition elements.
  • Magnetic particles may include: one or more magnetic crystals with and without nonmagnetic material.
  • the nonmagnetic material can include synthetic or natural polymers (such as sepharose, dextran, dextrin, starch and the like
  • the target-binding proteins can also be labeled with an indicating group containing of the NMR-active 19 F atom, or a plurality of such atoms inasmuch as (i) substantially all of naturally abundant fluorine atoms are the 19 F isotope and, thus, substantially all fluorine-containing compounds are NMR-active; (ii) many chemically active polyfluorinated compounds such as trifluoracetic anhydride are commercially available at relatively low cost, and (iii) many fluorinated compounds have been found medically acceptable for use in humans such as the perfluorinated polyethers utilized to carry oxygen as hemoglobin replacements. After permitting such time for incubation, a whole body MRI is carried out using an apparatus such as one of those described by Pykett (1982) Scientific American, 246:78-88 to locate and image cancerous tissues.
  • Information obtained from evaluating an target-binding protein can be recorded on machine-compatible media, e.g., computer readable or computer accessible media.
  • the information can be stored as a computer representation, e.g., in a database (e.g., in the case of imaging using a binding protein, a database of images for one or a plurality of subjects).
  • computer representation refers to information which is in a form that can be manipulated by a computer.
  • the act of storing a computer representation refers to the act of placing the information in a form suitable for manipulation by a computer.
  • kits including the binding protein that binds to Tiel and instructions for diagnostic use, e.g., the use of the target-binding protein (e.g., antibody or antigen-binding fragment thereof, or other polypeptide or peptide) to detect Tiel, in vitro, e.g., in a sample, e.g., a biopsy or cells from a patient having a cancer or neoplastic disorder, or in vivo, e.g., by imaging a subject.
  • the kit can further contain a least one additional reagent, such as a label or additional diagnostic agent.
  • the binding protein can be formulated as a pharmaceutical composition.
  • SEQ ID NO:2 An exemplary Tiel amino acid sequence (SEQ ID NO:2) is as follows:
  • phage display to select Tiel-specific antibodies from a very large phage library that displays immunoglobulins as Fab fragments.
  • a phage displayed Fab antibody library was selected against the Tiel extracellular domain fused to human Fc or to a histidine purification tag.
  • Round 2 (I x IO 7 Tiel expressing cells/Kingfisher), Round 3 (I x IO 7 Tiel expressing cells/Kingfisher)
  • Strategy 2 Round 1 (50OmM biotin labelled Tie 1 /magnetic beads), Round 2 (1 x 10 7 Tiel expressing cells/KINGFISHERTM), (30OmM biotin labelled Tiel/magnetic beads)
  • the specificity of the Tiel specific IgG's can be determined in a whole cell ELISA on mouse lung microvascular endothelial cells (LEII) and LEII-Tiel cells transfected with a Tiel expression construct.
  • LII mouse lung microvascular endothelial cells
  • LEII-Tiel cells transfected with a Tiel expression construct.
  • Cells are seeded into 96 well plates at a density of 10,000 cells/well and were fixed using 4% paraformaldehyde. Staining and detection of binding of IgGl to LEII cells are detected using standard labelling with a HRP conjugated rabbit anti human HRP and TMB staining. Binding of purified IgGl to LEII-Tiel transfected cells can also be corrected for Tiel protein that is expressed endogenously. Alternatively cells that have little or no endogenous Tiel can be used for the analysis.
  • At least one of the binding antibodies - E3 - functions as a Tiel activating antibody in the BaF3 cell bioassay.
  • the BaF3 cell bioassay (also referred to as the "Tiel/EpoR chimericBAF cell assay" may provide an indication of a ligand's ability to cross-link the Tiel receptor. Because the assay is artificial, crosslinking of the non-naturally occurring Tie-Epo fusion proteins may or may not be predictive of a ligand's ability to modulate in vivo function.
  • E3 can be used, instead of possible natural ligands to characterize several functions of Tiel in vitro and in vivo.
  • the region of Tiel which interacts with E3 can be the target for small molecular weight compounds for Tiel activation or inhibition.
  • E3 functions in one particular Tiel activating assay
  • E3 and other positives in this assay may also have inhibitory effect as to other functions or in other contexts.
  • E3 can inhibit tube formation by HUVEC cells. See below.
  • DMEM fetal calf serum
  • FCS fetal calf serum
  • FCS fetal calf serum
  • the murine BaF3 pre-B lymphocytes were cultured in DMEM supplemented with 10% FCS, glutamine, antibiotics and 2 ng/ml interleukin-3 (Calbiochem).
  • FCS fetal calf serum
  • HDMVECs Human dermal microvascular endothelial cells
  • PromoCell Heidelberg, Germany
  • Immunoprecipitation was carried out from equal amount of cell lysates by incubating with polyclonal anti-human Tiel antibodies (R&D), monoclonal anti-V5 antibodies (Invitrogen) or altogether 23 anti- Tie 1 antibodies (1 ⁇ g/ml) for 1 to 2 h followed by incubation with protein G- Sepharose (Amersham Pharmacia Biotech AB) for 1 h.
  • the imniunoprecipitates were washed twice with PBS-T and twice with PBS, followed by elution with the Laemmli buffer and separation in 8% SDS-PAGE.
  • the blots were probed with the 23 anti-Tiel antibodies (5 ⁇ g/ml) and subsequently anti-human Fc antibodies conjugated with HRP.
  • the cells were then stained with anti-Tiel antibodies (5 ⁇ g/ml) and anti-V5 antibodies for 1 h at room temperature, followed by incubation with FITC- conjugated anti-human antibodies (DAKO, 40 ⁇ g/ml) and TRITC-conjugated anti-mouse antibodies (DAKO, 15 ⁇ g/ml) for 30 min.
  • Hoechst 33258 fluorochrome (Sigma, 0.5 ⁇ g/ml) was used for the staining of the nuclei.
  • BaF3 pre-B cells were stably transfected with a nucleic acid that expresses chimeric receptor containing the extracellular domain of human Tiel fused with the transmembrane and cytoplasmic domains of the mouse erythropoietin receptor.
  • the nucleic acid used was a Tiel-EpoR chimeric cDNA in a pEF-BOS expression vector.
  • the nucleic acid encoding the chimeric receptor was constructed by cloning the PCR amplified extracellular part of human Tiel (bp 37-2316 of X60975) as EcoRI-BglH fragment into mEpoR-pcDNA vector.
  • the cDNA encoding for the chimeric receptor consisting of the extracellular part of Tiel fused with the transmembrane and intracellular domains of EpoR was subcloned into the pEF-BOS expression vector.
  • Vector was linearized and co-transfected into BaF3 cells with pcDNA3.1(+) Zeo vector (Invitrogen). Stable cell pools were generated by selection with 250 ⁇ g/ml Zeocin.
  • the expression of Tiel/EpoR fusion protein in several clones was analyzed by Western blotting with an antibody against EpoR.
  • BaF3 cells expressing the Tiel -EpoR chimera were split in 96- well microtiter plates at 50 000 cells/well in the presence of the indicated concentrations of anti-Tie 1 antibodies.
  • the E3 antibody used in this study was the germ-lined E3 antibody (DX-2220).
  • Zeocin resistant pools not expressing the Tiel -EpoR were used.
  • the viability of the cells was determined by adding MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma), 0.5 mg/ml), followed by further 2 h of culture, addition of an equal volume of cell lysis solution (10% SDS, 10 niM HCl) and incubation overnight at 37°C. Absorbance was measured at 540 nm.
  • the cells were lysed in lysis buffer (RIPA-DOC: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton-X-100, 0.1% SDS, 0.5% DOC, 10 mM EDTA, supplemented with aprotinin, leupeptin, PMSF and sodium vanadate).
  • Clarified lysates from transfected COSl cells or HDMVECs were immunoprecipitated with anti-V5 or anti-Tiel B9, respectively. Proteins were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted using the anti-phosphotyrosine and anti-Tiel (R&D systems) antibodies.
  • Tie-EpoR receptor expressing BaF3 cells can survive and proliferate if the medium contains a Tiel-binding protein, either a natural ligand or an artificial mimetic. Cell survival can be quantitated, e.g., by colorimetric MTT-assay, which measures mitochondrial activity.
  • E3 human umbilical cord endothelial cells
  • HAVEC Human Umbilical vein endothelial cells
  • Ix Trypsin-EDTA
  • AF attachment factor
  • HUVEC monolayers were treated with trypsin/EDTA (500 ⁇ l/dish) at 37°C for 3 min. Trypsin activity was stopped by adding 3 volumes of complete RPMI medium. The cells were carefully scraped, separated by repeated pipetting, and finally washed with PBS.
  • the assay was performed with a streptavidin antibody used as a control (from 1 ng/ml to 10 ⁇ g/ml).
  • the total length of the tube network on the culture surface was quantified at 40x magnification by the METAVUETM Software (Universal Imaging Corporation). Results from triplicate wells were expressed as mean vessel area per field ⁇ SEM (relative units). Each assay was performed at least three times.
  • E3 is a potent inhibitor of tube formation by HUVECS even at a concentration of 10 ng/ml.
  • the control anti-streptavidin has no effect on the ability of HUVECS to form tubes. This results indicates that E3 can inhibit at least one aspect of angiogenesis.
  • Transfections were carried out using LIPOFECTAMINETM 2000 reagent (Invitrogen, cat. no. 11668019) following the manufacturer's instructions. Five micrograms of plasmid DNA was used per 10-cm dish. Cells were cultured in DMEM (Invitrogen, cat. no. 31966021) supplemented with 10 % "ultra-low IgG" fetal calf serum (Invitrogen, cat. no. 16250078), at 37°C, 5 % CO 2 , in a water saturated atmosphere. Conditioned media were harvested 72 hours and 144 hours after transfection, pooled and sterile filtered.
  • the bead eluates were collected, using the vacuum manifold, into the wells of a 96-well collection plate (UNIPLATE 750, Whatman, cat. no. 7701-5750). Each well of the collection plate contained 60 ⁇ l of 1 M HEPES pH 7.5 buffer to immediately neutralize the eluted fractions. The elution step was performed twice to maximize antibody recovery. The eluted samples were then dialyzed against PBS using dialysis cassettes (Slide- A-Lyser Dialysis Cassettes, MWCO 10,000, Pierce, cat. no. 66380) and protein concentration was determined from the absorbance at 280 nm assuming that a 1 mg/ml solution has an absorbance of 1.35. The quality of the preparations was analyzed by reducing and non-reducing SDS-PAGE.
  • Tiel antibodies were biotinylated using the EZ-link Sulfo-NHS-
  • Sections with the mouse monoclonal anti-tiel antibody (7e8) were treated with biotinylated anti-mouse antibodies (VectaStain) before the addition of streptavidin-HRP.
  • Signal was amplified by using a TSA kit and the visualized by AEC (235 ml NaAc, 15 ml AEC (stock solution: 1600 mg 3-amino-9- ethyl-carbazole and 480 ml N-dimethylformamide), 250 ⁇ l H 2 O 2 ).
  • Tiel expression was upregulated in tumor tissue when compared with matching normal tissue.
  • the anti Tiel antibodies stained other structures in addition to the vessels.
  • some tissue specificity in the expression of certain epitopes was observed.
  • the E3 antibody stained vessels in the lung and kidney but not in the skin while the B2 antibody stained vessels very faintly in other normal tissues than in the breast. Shedding of the ectodomain of Tie 1 into the tumor tissues can explain observed differences.
  • E3 gave the brightest staining in the veins and capillaries of the mammary tissue
  • B2 and 7e8 gave a similar staining while DIl stained those structures rather faintly.
  • the Tiel expression was substantially upregulated, and the E3, B2, and DIl antibodies stained also other structures in addition to vessels.
  • Intracellular Tiel was measured as described above, except for the addition of Saponin to the incubation buffer to a final concentration of 0.1% during incubations.
  • the anti-Tie 1 antibody E3 binds to mouse endothelial cell lines indicating a cross reactivity of E3 with mouse and human Tiel in situ.
  • the binding pattern in mouse cell lines detected by flow cytometry is different from the binding pattern in HUVEC in that in mouse cells there is a greater cell surface staining than that compared to primary human endothelial cell lines.
  • DX-2210 stained positively both mouse endothelial cell lines as well as the HUVEC control cells. There was a shift in the fluorescent signal when the cells were treated with saponin, indicating a significant intracellular pool of sequestered Tiel.
  • platelets were isolated from plasma of healthy donors using the platelet GelSep kit (Biocytex, Marseille, France) kit according to the guidelines of the manufacturer. Platelets were activated by the addition of thrombin to a final concentration of 0.8 U/ml. To distinguish activated from non-activated platelets double staining was performed with Tiel antibodies/control antibodies and antibody CD42 (total platelets) or CD62 (activated platelets).
  • platelets were resuspended in buffer 2 of the GelSep kit, 10% heat inactivated human serum (incubation buffer) and incubated for 1 hour.
  • biotinylated antibodies human anti-Tiel(E3), human anti- streptavidin (A2-SV, an antibody that does not bind Tiel), human anti-FITC and goat anti-Tie (R&D systems) were incubated with 500 000 platelets per test for 1 hour at different dilutions (2 ⁇ g/ml, 10 ⁇ g/ml) for 1 h at room temperature. Platelets were spun down by centrifugation for 10 min at 611 g.
  • the polyclonal goat anti-Tiel antibody indeed binds to platelets under the conditions tested. This binding is lower when platelets are activated. In contrast, the human anti-Tiel antibody E3 shows no significant binding to total platelets, nor to activated platelets (FIG. 1).
  • Anti-Tiel antibodies B2, DIl, E3, the goat polyclonal AF619 (R&D) and negative control antibodies anti-FITC and anti-Streptavidin were used.
  • HUVECS were retrieved from culture dishes by trypsinization and platelets were prepared with the platelet GelSep kit (Biocytex, Marseille, France) kit according to the guidelines of the manufacturer. Per immunoprecipitation experiment 3-5 x 10 6 and 3 x 10 8 CeIIs platelets were used for each antibody tested. Platelets and cells were washed with PBS and spun down at 1400 rpm for 4 minutes and supernatant was removed.
  • lysed cells were lysed in 1 ml lysis buffer containing 5OmM Tris HCL pH 7.5, 150 mM NaCl, 0.5% Deoxycholic acid (DOC) and 0.5% NP-40 for 5 minutes.
  • the lysed cells were spin down for 10 minutes at 14.000 rpm and 5 ⁇ g/ml antibody was added to the supernatant and incubated at 4 0 C on a rotator.
  • 100 ⁇ l/sample protein A beads (Uppsala, Sweden) were washed 3 times with lysis buffer (centrifugation speed: 15 seconds, 2000 rpm) then cell lysates incubated with antibody were added for 30 minutes 4°C.
  • HUVEC HUVEC were trypsinised, washed with PBS and spotted at a density of 60 000 cells on a gelatine coated microscope slide and incubated for 24 hours in a humidified incubator at 37 0 C. Cells were air dried and fixed with 4% paraformaldehyde for 20 minutes at room temperature. The slides were washed with PBS. The slides were incubated with 10% Heat inactivated human serum (incubation buffer).
  • biotinylated antibody E3 and biotinylated negative control antibody A2 were used at a concentration of 10 ⁇ g/ml and incubated for 1 hour at room temperature. Slides were washed twice with PBS. Then, Strepatvidin- R-phycoerythrin (Dako, Glostrup, Denmark) was added and incubated for 1 hour at room temperature. After the last incubation and washing detection of bound antibodies was performed by means of confocal microscopy.
  • E3 binds specifically to HUVEC as detected by confocal microscopy.
  • the staining is pre-dominantly located inside of the cell which suggests a large intracellular pool of Tiel relative to a smaller pool of cell surface localized Tiel .
  • the localization of E3 was consistent with co-localization of Tiel with a cytoskeletal protein.
  • E3 germlined version of E3 in which the LC framework regions were altered to include sequences identical to the DPK4 germline framework regions.
  • the germlined E3 antibody was constructed by engineering a nucleic acid encoding the desired sequence. Changes to nucleic acids encoding the E3 LC variable domain were made by PCR and other standard molecular biological techniques and verified by nucleic acid sequencing.
  • An exemplary germlined light chain variable domain E3 sequence includes:
  • Example 10 Production and Testing of Germlined Anti Tiel E3 - Fab for Binding to Recombinant Tie 1 -Fc in ELISA
  • the soluble expression vector containing the parental E3 Fab and the germlined E3 Fab construct were grown overnight at 30°C in 2xTY broth containing 100 ⁇ g/ml ampicillin and 2% glucose and use 4 ml of this overnight culture to inoculate 400 ml of 2 x TY broth containing 100 ⁇ g/ml ampicillin and 0.1% glucose. Cells were grow at 37 0 C until an OD 600 of 0.8-1.0, ImM IPTG was added and the culture was maintained at 3O 0 C for 4 hours.
  • the cultures were spun down at 4,000 rpm for 15 min at 4 C. The superaatants were discarded and resuspend the pellets resuspended in 4.8 ml of ice cold TES buffer (0.2 M Tris-HCl, 0.5 niM EDTA, 0.5 M sucrose, pH 8.0) containing proteases inhibitors (protease inhibitor cocktail tablets [Roche]: dissolve 1 tablet in 1 ml of water and dilute 50-times in TES buffer). Transfer to 50 ml Falcon tubes and place on ice for 5-10 min. During this incubation, wash the centrifugation bottles with 5.25 ml TES:H 2 O (1:3) containing proteases inhibitors and add this to the cells.
  • TES buffer 0.2 M Tris-HCl, 0.5 niM EDTA, 0.5 M sucrose, pH 8.0
  • proteases inhibitors proteases inhibitors
  • Recombinant purified human Tiel-Fc antigen (Stock 2.45 mg/ml) was biotinylated using the EZ-link Sulfo-NHS-SS-Biotin (Pierce, Cat. 21331). The reaction was performed for 2 hours on ice in 50 mM sodium carbonate buffer, pH 9.6, in the presence of a 5-fold molar excess of biotinylating agent and was stopped by the addition of Tris-HCl, pH 7.5 (50 mM final concentration) followed by a 1-hour incubation on ice. Samples were then dialyzed against PBS. The antigen was then diluted 1/100 fold in HBS and was then captured onto a streptavidin chip.
  • Example 12 Comparison of Affinity of Germlined Anti Tiel E3 -IgG to Parental Anti Tiel E3 for Binding to Recombinant Human Tiel Using BIAcore [0560] In order to evaluate if the binding behavior had been affected in any way by the conversion of the somatic mutations back to germline residues, the germlined antibody was produced and tested as an IgG. The germlined E3-IgG construct used to transiently transfect HEK293T cells and purified.
  • the germlined E3 IgGl stock solution 0.63 mg/ml was diluted 1/50 in a buffer of pH4.5 and the parental E3 IgGl stock solution 0.56 mg/ml (2143-001) was diluted 1/50 in a buffer of pH 4.5.
  • the IgG were directly coated onto a CM5 chip.
  • the surface of the chips was activated with a 7 minute pulse of 0.05M NHS/0.2M EDC and the IgG was flowed over until 780RU germlined E3-IgG and 728 non germlined E3 IgG was coated onto the surface.
  • AU flow cells were subsequently deactivated with a 7 minute pulse of IM ethanolamine hydrochloride pH 8.5. All analysis was performed in HBS buffer.
  • Mouse Tie 1-Fc antigen (0.5 mg/ml stock) was biotinylated using established procedures and after dilution 1/100 fold in HBS this was then used for capturing to a streptavidin chip. This was coated to a resonance value of 740RU. All analysis was performed in HBS buffer.
  • the parental Fab E3 0.587 mg/ml (11740 nM) was diluted 1/587 in HBS + BSA to obtain a stock of 20 nM and the germlined Fab E3 0.025 mg/ml (500 nM) was diluted 1/25 in HBS + BSA to obtain a stock of 20 nM.
  • Example 14 Comparison of Affinity of Germlined Anti Tiel E3 -IgG to Parental Anti Tiel E3 for Binding to Recombinant Mouse Tiel Using BIAcore
  • the germlined antibody was produced and tested as an IgG.
  • the germlined E3 was reformatted to an IgG as described. This was then used to transiently transfect HEK293T cells using established procedures.
  • the IgG was purified from the culture supernatant using protein A column chromatography using established procedures and the subsequent IgG was then tested for binding activity using surface plasmon resonance (BIAcore).
  • the germlined E3 IgGl stock solution 0,63 mg/ml (2146-002) was diluted 1/50 in a buffer of pH 4.5 and the parental E3 IgGl stock solution 0,56 mg/ml (2143-001) was diluted 1/50 in a buffer of pH 4.5.
  • the IgG were directly coated via onto a CM5 chip.
  • the surface of the chips was activated with a 7 minute pulse of 0.05M NHS/0.2M EDC and the IgG was flowed over until 780RU germlined E3-IgG and 728 non germlined E3 IgG was coated onto the surface.
  • AU flow cells were subsequently deactivated with a 7 minute pulse of IM ethanolamine hydrochloride pH8,5.
  • AU analysis was performed in HBS buffer. Purified recombinant mouse Tiel Fc was diluted 1/6,5 in HBS to obtain a 40OnM stock solution. Serial dilutions were made to obtain 20OnM, 10OnM, 5OnM and 25 nM Tiel Fc stocks. For analysis of the association phase samples were injected at 30 ⁇ l/min for 8,3 minutes using kinject program. This was followed by a 40 minutes dissociation phase. Any antigen remaining associated to the surface was stripped from the IgG surface at a flow of 50 ⁇ l/min with two injections of 1OmM glycine pHl,5 for 30seconds. AU samples were run and analyzed in duplicate
  • Germlined E3 (DX-2220) and its parental antibody (DX-2200) were evaluated in the tube formation assay in a collagen type-I matrix.
  • Human Umbilical vein endothelial cells (HUVEC) (freshly isolated) were obtained by treating human umbilical cord veins with Trypsin-EDTA (Ix) (Gibco/Invitrogen) for 20-25 minutes at 37°C. The cells were then cultured in a T-25 flask coated with attachment factor (AF), (Cascade Biologies) in RPMI 1640 medium supplemented with 10 % FCS, 0.4 % BBE, 1% 1-glutamin, 1% penicillin/streptomycin.
  • AF attachment factor
  • HUVECs (passage 2) were seeded in their culture medium (40 x 10 3 /50 ⁇ l/well of a 96-well plate) on a collagen gel (50 ⁇ l of collagen 1 1.5mg/ml) prepared by mixing 7.5 volumes of 2mg/ml collagen (Collagen R; Serva, Heidelberg, Germany), 1 volume of 1OX MEM, 1.5 volume of NaHCO3 (15.6 mg/ml) and ⁇ 1 volume of NaOH to adjust the pH to 7.4. After Ih 30 min., the culture medium was then discarded and the cells were covered with a new layer of collagen (1.5 mg/ml, new preparation, 50 ⁇ l/well).
  • E3 and germlined E3 were evaluated for their ability to inhibit tube formation in vitro using mouse endothelial cell line (LEII).
  • LEII lung mouse endothelial cell line was cultured in a T-25 flask in MEM medium with GLUTAMAXTM (Life Technologies Ltd., Paisley, Scotland) supplemented with 10 % FCS, and 1% penicillin/streptomycin. During culturing, the cells were kept in a proliferative state by culturing them in a split ratio 1:5 at an approximate density of the monolayer of about 80%. LEII monolayers were treated with trypsin/EDTA (500 ⁇ l/dish) at 37°C for 3 min. Trypsin activity was stopped by adding 3 volumes of complete MEM medium.
  • LEII cells were seeded in their culture medium (20-40 X 103/50 ⁇ l/well of a 96-well plate) on a basement membrane (BIOCOATTM Angiogenesis System; Becton Dickinson). After polymerization of the MATRIGELTM (30 min at 37 0 C, 5% CO 2 environment) the endothelial cell suspension resuspended in complete culture medium in the presence of the desired molecules (4.105 cells/ml; 50 ⁇ l/well) was added to each well. The angiogenesis assay plate was then incubated for 16 to 18 hours at 37°C, 5% CO 2 atmosphere.
  • the total length of the tube network was then quantified at 40 X magnification by the METAVUETM Software (Universal Imaging Corporation). Results from triplicate wells were expressed as mean vessel area per field ⁇ SEM (relative units). Each assay was performed at least two times. Germlined E3 is a potent inhibitor of tube formation in mouse endothelial cells.
  • the slides were cooled down in fresh citrate buffer for 20 min and rinsed with distilled water.
  • the slides were hydrogen peroxide treated, (0.3 % H 2 O 2 in PBS), and preincubated with PBS, 5 % FCS, 5% heat inactivated human serum (HS) for 1 hour. Between antibody incubations slides were washed 3 times 5 minutes in PBS.
  • Biotinylated antibodies E3 and A2-SV were diluted to a concentration of 10 ⁇ g/ml in PBS 5 10 % HS and incubated for 1 hour at RT. Slides were then incubated with an avidin-HRP (Dako) for 30 minutes at room temperature.
  • Staining was detected by AEC (Vector Laboratories, Burlingame) and H 2 O 2 .
  • the peroxidase reaction was stopped with water and slides were counter-stained with haematoxylin.
  • the tissues were evaluated for their binding reactivity.
  • the staining pattern was consistent with staining of mouse endothelial cell Tiel and also with Tiel expressed by the E3 binds to Tiel expressed by SW480 tumor cells in a mouse xenograft.
  • the germlined variant of the E3 IgG antibody was evaluated in an in vivo assay for angiogenesis induced by bFGF in MATRIGELTM plugs.
  • Growth factor reduced MATRIGELTM (BD Biosciences, catalog # 354230) was supplemented with 80 ng/ml of bFGF (R&D Systems, catalog #234-FSE).
  • the A2-SV or an IgG4 E3 antibody (10 ⁇ g/ml) or PBS was injected subcutaneously into the abdominal area of NMRI nu/nu mice (150 ⁇ l of Matri gel/plug).
  • mice were injected with MATRIGELTM supplemented with bFGF and soluble VEGFR-I (10 ⁇ g/ml) as a positive control for an angiogenic inhibitor.
  • MATRIGELTM supplemented with bFGF and soluble VEGFR-I (10 ⁇ g/ml) as a positive control for an angiogenic inhibitor.
  • mice were anesthetized and perfused through heart with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS).
  • PFA paraformaldehyde
  • PBS phosphate buffered saline
  • MATRIGELTM plugs and a piece of liver were removed and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin (H&E).
  • mice were treated as described above, and then anesthetized eight days post-implantation and injected with fluorescein-conjugated tomato (lycopersicon esculentum) lectin (100 ⁇ g in 200 ⁇ l of PBS; Vector, catalog #FL-1171) into the tail vein. After five min circulation the animals were perfused through the heart with 10 ml of PBS followed by 10 ml of 4% PFA in PBS. MATRIGELTM plugs and pieces of kidney and liver were removed and frozen in OCT (Tissue-Tek). Nuclei were visualized on sections by using VECTASHIELD® mounting medium containing DAPI (Vector) and analyzed under fluorescence microscopy.
  • fluorescein-conjugated tomato (lycopersicon esculentum) lectin 100 ⁇ g in 200 ⁇ l of PBS; Vector, catalog #FL-1171
  • MATRIGELTM plugs and pieces of kidney and liver were removed and frozen in OCT (Tissue-Tek). Nuclei were
  • E3 can inhibit bFGF-induced angiogenesis in vivo.
  • mice were anesthetized and injected with fluorescein-conjugated tomato (Lycopercicon esculentum) lectin into the tail vein.
  • the MATRIGELTM plugs and liver and kidneys were removed and frozen in OCT media.
  • sections were cut and either stained for endothelial cell content using an anti-CD-31 antibody or analyzed under fluorescence microscopy to assess the amount of functional blood vessels (tomato lectin staining) (data not shown).
  • the amount of blood vessels per unit area was quantitated.
  • a candidate protein for example, E3 or E3b antibody
  • binds a complex member such as Tiel, Tie2, or an angiopoietin is tested for its ability to antagonize formation of a heteromeric complex that includes Tiel, Tie2, and Ang, by inhibiting its formation or disrupting the heteromeric complex once it forms.
  • cells expressing Tiel and Tie2 are treated with Ang for a period of time sufficient to allow binding of Ang to Tiel and/or Tie2.
  • the cells are contacted with the candidate protein for a period of time sufficient to allow disruption of the complex.
  • the cells are treated with a membrane non-permeable cross-linker, such as DTSSP, to chemically cross-link the proteins.
  • Cell lysates are prepared and subjected to immunoprecipitation with an antibody specific to a complex member.
  • the immunoprecipitated proteins are separated by SDS-PAGE electrophoresis and immunoblotted with antibodies specific to the complex members.
  • a positive control immunoprecipitation-immunoblot is also performed in which cells expressing Tiel and Tie2 are treated with Ang but not with the candidate protein or are treated with a nonspecific protein. If treatment with the candidate protein decreases the amount of a complex member- that is not bound by the immunoprecipitating antibody- associated with the immunoprecipitated member as compared to the positive control, the candidate protein is an antagonist of complex formation.
  • Candidate proteins that antagonize complex formation, by inhibiting complex formation or by disrupting complexes, are then tested for their effects on angiogenesis in an assay described herein.
  • Tie2 amino acid sequence is as follows:
  • CEKACELHTF GRTCKERCSG QEGCKSYVFC LPDPYGCSCA TGWKGLQCNE 300
  • a strategy was designed to repair this mutation.
  • the introduction of the germlined residue was facilitated by the presence of internal restriction sites in the framework flanking regions of the CDRs.
  • the design of the HC-CDRl -CDR2 sublibrary, present in FAB 310 library was made in such a way that the shuffling of every CDR is allowed by the presence of unique restriction sites in the framework flanking regions. Since the valine residue to be corrected is located in FR3 region, 3' near the Xbal site, a primer was designed containing both the Xbal sequence and the corrected methionine germline residue.
  • E3b a soluble Fab expression vector containing the germlined E3 antibody
  • DX-2220 a soluble Fab expression vector containing the germlined E3 antibody
  • the PCR fragment from Example 22 was digested overnight with 50 U/ ⁇ gXbal restriction enzyme, followed by a 5 hours digestion with 25 U/ ⁇ g BsiER.
  • the cleaved PCR product was then purified on a 1% TAE-agarose preparative gel. Ligation into the similarly-digested phagemid expression vector (pMIDl) containing the Tiel E3 germlined light chain sequence was performed for three hours at room temperature.
  • pMIDl similarly-digested phagemid expression vector
  • Fab E3b Five nanograms of the newly-ligated material were electroporated into TGl bacterial cells. Verification of the correction of the mutation was performed by sequence determination of the heavy chain of 20 randomly picked isolates. The resulting coding construct contained sequences that encode a germlined HC and a germlined LC sequence in a Fab format (termed Fab E3b).
  • the E3b Fab antibody was reformatted to a human IgGl . This construct was then used to transiently transfect HEK293T cells. Plasmid preparations for transient cell transfections were obtained using the Qiagen filter Plasmid Maxi kit (Qiagen, cat. no. 12263). HEK293T cells (GenHunter Corp., cat. no. Q401) were seeded 24 hours before transfection; 220 x 10 6 cells were plated per CELLSTACK® culture vessel (CeIlST ACK®- 10 Chamber, Corning, cat. no. 3271). Transfections were carried out using the GeneJuice® reagent (VWR, cat. no.
  • novg70967-3) following the manufacturer's instructions.
  • 650 micrograms of plasmid DNA was used per CELLSTACK®.
  • Cells were cultured in DMEM (Invitrogen, cat. no. 31966021) supplemented with 10 % "ultra-low IgG" fetal calf serum (Invitrogen, cat. no. 16250078), at 37 0 C, 5 % CO 2 , in a water saturated atmosphere.
  • Conditioned media were harvested 72, 144 and 216 hours after transfection, pooled and sterile filtered.
  • the E3b antibody was further purified by cation exchange.
  • the antibody was dialyzed against 50 mM sodium citrate, pH 5.0, and loaded on a HiLoad 26/10 SP Sepharose HP column (GE Healthcare, cat. no. 17-1138-01) equilibrated in the same buffer.
  • the antibody was eluted with 50 mM sodium citrate, pH 5.0, containing 1 M NaCl (linear gradient on 10 column volumes). Fractions containing the antibody were pooled and dialyzed against PBS.
  • Antibody concentration was calculated from the absorbance at 280 nm, assuming that a protein concentration of 1 mg/ml has an absorbance of 1.36.
  • E3 IgGl stock solution (0.41 mg/ml) were diluted 50-fold in 10 mM sodium acetate, pH 4.5.
  • the IgGs were directly coated on a CM5 chip.
  • the surface of the chip was activated with a 7-minute pulse of 0.05 M NHS / 0.2 M EDC and the IgG was run over the chip until 823 RU of germlined E3b and 788 RU of parental E3 were coated on the surface.
  • AU flow cells were subsequently deactivated with a 7-minute pulse of 1 M ethanolamine hydrochloride, pH 8.5. AU further experiments were performed in HBS buffer.
  • Recombinant human Tiel/Fc was coated on a CM5 chip.
  • the surface of the chip was first activated with a 7-minute pulse of 0.05 M NHS / 0.2 M EDC, then Tiel/Fc (2 ⁇ g/ml in 10 mM sodium acetate, pH 4.0) was run over the chip surface until 750 RUs were coated on the surface. All flow cells were subsequently deactivated with a 7-minute pulse of 1 M ethanolamine hydrochloride, pH 8.5. All further experiments were performed in HBS buffer.
  • HUVECs (passage 3) were seeded in their culture medium (40 x 10 3 /50 ⁇ l/well of a 96-well plate) on a collagen gel (50 ⁇ l of collagen I 1.5mg/ml) prepared by mixing 7.5 volumes of 2mg/ml collagen (Collagen R; Serva, Heidelberg, Germany), 1 volume of 1OX MEM, 1.5 volume OfNaHCO 3 (15.6 mg/ml) and ⁇ 1 volume of NaOH to adjust the pH to 7.4. After Ih 30 min, the culture medium was then discarded and the cells were covered with a new layer of collagen (1.5mg/ml, new preparation, 50 ⁇ l/well).
  • Example 28 Exemplary Tiel Binding Sequences

Abstract

Tie1 et Tie2 sont des protéines tyrosine kinases réceptrices comportant un domaine transmembranaire. Tie1 et Tie2 sont présents sur des cellules endothéliales. La présente invention a trait à des agents, tels que des anticorps, de liaison à Tie1, Tie2, et à l'angiopoiétine, comprenant ceux qui sont inhibiteurs de l'activité de cellules endothéliales et de l'angiogenèse. Les agents peuvent être utilisés pour le traitement de troubles associés à l'angiogenèse.
PCT/US2005/028413 2004-08-12 2005-08-09 Proteines de liaison au complexe tie WO2006020706A2 (fr)

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CA002576886A CA2576886A1 (fr) 2004-08-12 2005-08-09 Proteines de liaison au complexe tie
EP05784935A EP1789451A4 (fr) 2004-08-12 2005-08-09 Protéines de liaison au complexe tie
AU2005272848A AU2005272848A1 (en) 2004-08-12 2005-08-09 Tie complex binding proteins
JP2007525760A JP2008532476A (ja) 2004-08-12 2005-08-09 複合結合タンパク質

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PCT/US2004/026116 WO2005019267A2 (fr) 2003-08-12 2004-08-12 Ligands se fixant au tie 1
US10/916,840 US7348001B2 (en) 2003-08-12 2004-08-12 Tie1-binding ligands
USPCT/US2004/026116 2004-08-12
US10/916,840 2004-08-12
US11/049,536 2005-02-02
US11/049,536 US7871610B2 (en) 2003-08-12 2005-02-02 Antibodies to Tie1 ectodomain

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US10280219B2 (en) 2003-09-10 2019-05-07 Amgen Fremont Inc. Antibodies to M-CSF
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EP2371961A1 (fr) * 2005-03-25 2011-10-05 National Research Council of Canada Procédé d'isolation de polypeptides solubles
US8293233B2 (en) 2005-03-25 2012-10-23 National Research Council Of Canada Method for isolation of soluble polypeptides
US11091536B2 (en) 2005-03-25 2021-08-17 National Research Council Of Canada Method for isolation of soluble polypeptides
US10150807B2 (en) 2005-03-25 2018-12-11 National Research Council Of Canada Method for isolation of soluble polypeptides
EP1915394B1 (fr) 2005-05-20 2021-01-27 Lonza Biologics plc. Expression de niveau eleve d'un anticorps recombinant d'une cellule hote de mammifere
WO2007079218A2 (fr) 2005-12-30 2007-07-12 Dyax Corp. Proteines de liaison a la metalloproteinase
US8333968B2 (en) 2006-03-10 2012-12-18 Zymogenetics, Inc. Methods of inhibiting inflammation with antagonists to IL-17A, IL-17F, and IL-23P19
US10562967B2 (en) 2006-03-10 2020-02-18 Zymogenetics, Inc. Treating inflammation with IL-17/IL-23 bispecific antibodies
US9994634B2 (en) 2006-03-10 2018-06-12 Zymogenetics, Inc. Use of an IL-17/IL-23 bispecific antibody for treating inflammation
US9464134B2 (en) 2006-03-10 2016-10-11 Zymogenetics, Inc. Polynucleotides encoding antagonists of IL-17A, IL-17F, and IL-23p19
US7910703B2 (en) 2006-03-10 2011-03-22 Zymogenetics, Inc. Antagonists to IL-17A, IL-17F, and IL-23P19 and methods of use
US8496936B2 (en) 2006-03-10 2013-07-30 Zymogenetics, Inc. Antagonists of IL-17A, IL-17F, and IL-23P19
US8992922B2 (en) 2006-03-10 2015-03-31 Zymogenetics, Inc. Antagonists of IL-17A, IL-17F, and IL-23P19
US8147829B2 (en) 2006-03-28 2012-04-03 Biogen Idec Ma Inc. Anti-IGR-1R antibodies and uses thereof
EP1999149A4 (fr) * 2006-03-28 2010-01-20 Biogen Idec Inc Anticorps anti-igf-1r et utilisations de ceux-ci
JP2009532027A (ja) * 2006-03-28 2009-09-10 バイオジェン・アイデック・エムエイ・インコーポレイテッド 抗ｉｇｆ−１ｒ抗体およびその使用
EP1999149A2 (fr) * 2006-03-28 2008-12-10 Biogen Idec MA Inc. Anticorps anti-igf-1r et utilisations de ceux-ci
EP2073826A4 (fr) * 2006-10-17 2010-12-15 Dyax Corp Thérapie de combinaison séquentielle
EP2073826A2 (fr) * 2006-10-17 2009-07-01 Dyax Corporation Thérapie de combinaison séquentielle
KR101598229B1 (ko) 2007-02-16 2016-02-26 메리맥 파마슈티컬즈, 인크. Ｅｒｂｂ3에 대한 항체 및 이의 용도
US9487588B2 (en) 2007-02-16 2016-11-08 Merrimack Pharmaceuticals, Inc. Antibodies against the ectodomain of ERBB3 and uses thereof
WO2008100624A3 (fr) * 2007-02-16 2009-01-15 Merrimack Pharmaceuticals Inc Anticorps contre erbb3 et leur utilisation
CN101674846B (zh) * 2007-02-16 2014-07-02 梅里麦克制药股份有限公司 ErbB3抗体及其用途
KR20100014426A (ko) * 2007-02-16 2010-02-10 메리맥 파마슈티컬즈, 인크. Ｅｒｂｂ3에 대한 항체 및 이의 용도
EA020465B1 (ru) * 2007-02-16 2014-11-28 Мерримак Фармасьютикалз, Инк. ИЗОЛИРОВАННЫЕ МОНОКЛОНАЛЬНЫЕ АНТИТЕЛА, КОТОРЫЕ СВЯЗЫВАЮТСЯ С ErbB3, НАБОРЫ И КОМПОЗИЦИИ, ИХ СОДЕРЖАЩИЕ, И ИХ ПРИМЕНЕНИЕ
US8961966B2 (en) 2007-02-16 2015-02-24 Merrimack Pharmaceuticals, Inc. Antibodies against ERBB3 and uses thereof
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US7846440B2 (en) 2007-02-16 2010-12-07 Merrimack Pharmaceuticals, Inc. Antibodies against ErbB3 and uses thereof
US8691225B2 (en) 2007-02-16 2014-04-08 Merrimack Pharmaceuticals, Inc. Antibodies against the ectodomain of ErbB3 and uses thereof
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JP2010538625A (ja) * 2007-09-14 2010-12-16 グルベル，ジェンス 核酸担持ウイルス様粒子を用いた遺伝子発現の下方制御
US10030051B2 (en) 2008-01-03 2018-07-24 The Scripps Research Institute Antibody targeting through a modular recognition domain
EP2671891A2 (fr) 2008-06-27 2013-12-11 Amgen Inc. Inhibition d'ang-2 pour traiter la sclérose en plaques
US8623592B2 (en) 2008-08-15 2014-01-07 Merrimack Pharmaceuticals, Inc. Methods and systems for predicting response of cells to a therapeutic agent
WO2010072740A3 (fr) * 2008-12-23 2010-10-21 Astrazeneca Ab Agents de liaison ciblés dirigés contre α5β1 et leurs applications
US8895001B2 (en) 2010-03-11 2014-11-25 Merrimack Pharmaceuticals, Inc. Use of ErbB3 inhibitors in the treatment of triple negative and basal-like breast cancers
US9518130B2 (en) 2010-03-11 2016-12-13 Merrimack Pharmaceuticals, Inc. Use of ERBB3 inhibitors in the treatment of triple negative and basal-like breast cancers
US10526381B2 (en) 2011-05-24 2020-01-07 Zygenia, Inc. Multivalent and monovalent multispecific complexes and their uses
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US10316105B2 (en) 2011-08-19 2019-06-11 Regeneron Pharmaceuticals, Inc. Anti-TIE2 antibodies and uses thereof
US10023641B2 (en) 2011-08-19 2018-07-17 Regeneron Pharmaceuticals, Inc. Anti-TIE2 antibodies and uses thereof
WO2013028442A1 (fr) * 2011-08-19 2013-02-28 Regeneron Pharmaceuticals, Inc Anticorps anti-tie2 et utilisations associées
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US9017670B2 (en) 2011-08-19 2015-04-28 Regeneron Pharmaceuticals, Inc. Anti-Tie2 antibodies and uses thereof
EP3415533A1 (fr) * 2011-08-19 2018-12-19 Regeneron Pharmaceuticals, Inc. Anticorps anti-tie2 et utilisations associées
CN103874709B (zh) * 2011-08-19 2016-12-21 瑞泽恩制药公司 抗tie2抗体及其用途
US10752702B2 (en) 2011-08-19 2020-08-25 Regeneron Pharmaceuticals, Inc. Anti-TIE2 antibodies and uses thereof
US9546218B2 (en) 2011-08-19 2017-01-17 Regeneron Pharmaceuticals, Inc. Anti-Tie2 antibodies and uses thereof
CN106963946A (zh) * 2011-08-19 2017-07-21 瑞泽恩制药公司 抗tie2抗体及其用途
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US10442864B2 (en) 2011-08-19 2019-10-15 Regeneron Pharmaceuticals, Inc. Anti-Tie2 antibodies and uses thereof
EP3424530A1 (fr) 2013-03-15 2019-01-09 Zyngenia, Inc. Complexes multispécifiques monovalents et multivalents et leurs utilisations
US10150800B2 (en) 2013-03-15 2018-12-11 Zyngenia, Inc. EGFR-binding modular recognition domains
US9688761B2 (en) 2013-12-27 2017-06-27 Merrimack Pharmaceuticals, Inc. Biomarker profiles for predicting outcomes of cancer therapy with ERBB3 inhibitors and/or chemotherapies
US10273304B2 (en) 2013-12-27 2019-04-30 Merrimack Pharmaceuticals, Inc. Biomarker profiles for predicting outcomes of cancer therapy with ERBB3 inhibitors and/or chemotherapies
US10577412B2 (en) * 2015-04-12 2020-03-03 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Anti-plasmodium parasite antibodies
EP3283518B1 (fr) * 2015-04-12 2022-05-04 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Anticorps anti-plasmodium pour parasite
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EP1789451A4 (fr) 2009-12-02
AU2005272848A2 (en) 2006-02-23
WO2006020706A3 (fr) 2006-10-12
EP1789451A2 (fr) 2007-05-30

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