WO2008042941A2 - Anticors anti-ephb4 humains et leur utilisation dans le traitement de l'oncologie et de maladies liées a la vasculogenèse - Google Patents

Anticors anti-ephb4 humains et leur utilisation dans le traitement de l'oncologie et de maladies liées a la vasculogenèse Download PDF

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WO2008042941A2
WO2008042941A2 PCT/US2007/080270 US2007080270W WO2008042941A2 WO 2008042941 A2 WO2008042941 A2 WO 2008042941A2 US 2007080270 W US2007080270 W US 2007080270W WO 2008042941 A2 WO2008042941 A2 WO 2008042941A2
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seq
antibody
amino acid
acid sequence
ephb4
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WO2008042941A3 (fr
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Melissa Damschroder
William Dall'acqua
Herren Wu
Emma T. Bowden
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Medimmune, Llc
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    • 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
    • C07K16/2866—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 cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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
    • C07K2317/70—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • the present invention relates to human, humanized, or chimeric anti-EphB4 antibodies that bind to the human EphB4 receptor tyrosine kinase.
  • the present invention is also directed to compositions comprising human, humanized, or chimeric anti-EphB4 antibodies that may act as EphB4 agonists or antagonists and may further mediate one or more of the following: complement-dependent cell-mediated cytotoxicity (CDC), antigen-dependent cell-mediated-cytotoxicity (ADCC), and programmed cell death (apoptosis).
  • CDC complement-dependent cell-mediated cytotoxicity
  • ADCC antigen-dependent cell-mediated-cytotoxicity
  • apoptosis programmed cell death
  • the present invention is further directed to compositions comprising human, humanized, or chimeric anti-EphB4 antibodies of the IgGl and/or IgG3 human isotype, as well as to compositions comprising human, humanized, or chimeric anti-EphB4 antibodies of the IgG2 and/or IgG4 human isotype.
  • the present invention is directed to methods for the treatment of malignancies in human subjects using the therapeutic human, humanized, or chimeric anti-EphB4 antibodies that bind to the human EphB4 receptor tyrosine kinase.
  • the present invention is also directed to methods for the treatment and prevention of a human disorder or disease characterized by an altered bone homeostasis using therapeutic human, humanized, or chimeric anti-EphB4 antibodies that bind to the human EphB4 receptor tyrosine kinase.
  • the present invention is further directed to methods for the treatment and prevention of an angiogenesis-associated disease or disorder in a human using therapeutic human, humanized, or chimeric anti-EphB4 antibodies that bind to the human EphB4 receptor tyrosine kinase.
  • the present invention relates to a method of treating an epithelial migration associated disease or disorder in a human using therapeutic human, humanized, or chimeric anti-EphB4 antibodies that bind to the human EphB4 receptor tyrosine kinase.
  • the present invention relates to human, humanized, or chimeric anti-EphB4 antibodies that bind to the human EphB4 antigen, as well as to compositions comprising those antibodies.
  • the present invention provides chimeric and humanized versions of anti-EphB4 mouse monoclonal antibodies, #47 and #131.
  • anti-EphB4 antibodies may comprise one, two, three, four, five, or all six CDRs of the #47 anti-EphB4 antibody.
  • anti-EphB4 antibodies may comprise one, two, three, four, five, or all six CDRs of the #131 anti-EphB4 antibody.
  • the amino acid sequences of CDRl, CDR2, and CDR3 of the heavy chain variable region of #47 defined according to Kabat are identified as SEQ ID NO: 3 SEQ ID NO:4, and SEQ ID NO:5, respectively.
  • the amino acid sequences of CDRl, CDR2 and CDR3 of the light chain variable region of #47 defined according to Kabat are identified as SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.
  • an anti-EphB4 antibody may comprise one or more framework region of the #47 VH and/or VL region.
  • an anti-EphB4 antibody may comprise one or more framework region of the #131 VH and/or VL region.
  • an anti-EphB4 antibody may comprise one or more CDRs of the anti-EphB4 antibodies #131 or #47 and may further comprise one or more heavy chain framework (FW) regions of a VH region selected from the group consisting of 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO:82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO:106), 3E9 VH (SEQ ID NO:118), 6G2 VH (SEQ ID NO:130), 7A7 VH (SEQ ID NO : 142), 8D 10 VH (SEQ ID NO : 154), 9D9 VH (SEQ ID NO : 166), 9E9 VH (SEQ ID NO : :
  • an anti-EphB4 antibody may comprise one or more CDRs of the anti-EphB4 antibodies #131 or #47 and may further comprise one or more light chain framework (FW) regions of a VL region selected from the group consisting of 22E6 VL
  • an anti-EphB4 antibody may comprise one or more CDRs of the anti-EphB4 antibodies #131 or #47, one or more light chain framework regions of a VL region selected from the group consisting of 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO: 112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO: 148), 8D10 VL (SEQ ID NO : 160), 9D9 VL (SEQ ID NO : 172), 9E9 VL (SEQ ID NO 184), 9E 12
  • a humanized anti-EphB4 antibody may comprise a heavy chain variable region which comprises four framework regions, FWl, FW2, FW3, and FW4, wherein FWl is selected from the group consisting of 22E6 VH FWl (SEQ ID NO:23), 30C2 VH FWl (SEQ ID NO:35), 3G8 VH FWl (SEQ ID NO:47), 4F2 VH FWl (SEQ ID NO:59), 1B7 VH FWl (SEQ ID NO:71), IElO VH FWl (SEQ ID NO:83), 2F4 VH FWl (SEQ ID NO:95), 2G3 VH FWl (SEQ ID NO: 107), 3E9 VH FWl (SEQ ID NO: 119), 6G2 VH FWl (SEQ ID NO: 131), 7A7 VH FWl (SEQ ID NO: 143), 8D10 VH FWl (SEQ ID NO: 22E6 VH FW
  • FW3 is selected from the group consisting of 22E6 VH FW3 (SEQ ID NO:25), 30C2 VH FW3 (SEQ ID NO:37), 3G8
  • a humanized anti-EphB4 monoclonal antibody may comprise a light chain variable region comprising four framework regions, FWl, FW2, FW3, and FW4, wherein FWl is selected from the group consisting of 22E6 VL FWl (SEQ ID NO:29), 30C2 VL FWl (SEQ ID NO:41), 3G8 VL FWl (SEQ ID NO:53), 4F2 VL FWl (SEQ ID NO:65), 1B7 VL FWl (SEQ ID NO:77), IElO VL FWl (SEQ ID NO:89), 2F4 VL FWl (SEQ ID NO: 101), 2G3 VL FWl (SEQ ID NO: 113), 3E9 VL FWl (SEQ ID NO: 125), 6G2 VL FWl (SEQ ID NO: 137), 7A7 VL FWl (SEQ ID NO: 149), 8D10 VL FWl (SEQ ID NO:
  • an anti-EphB4 antibody may comprise a light chain variable region selected from the group consisting of #47 VL (SEQ ID NO:7), #131 VL (SEQ ID NO: 17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO : 100), 2G3 VL (SEQ ID NO : 112), 3E9 VL (SEQ ID NO : 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO: 148), 8D10 VL (SEQ ID NO: 160), 9D9 VL (SEQ ID NO : 172), 9E9 VL (SEQ ID NO : 184), 9E 12 VL
  • the present invention further relates to an anti-EphB4 antibody comprising a heavy chain variable region selected from the group consisting of #47 VH (SEQ ID NO:2), #131 VH (SEQ ID NO: 12), 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO: 82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO:118), 6G2 VH (SEQ ID NO: 130), 7A7 VH (SEQ ID NO: 142), 8D10 VH (SEQ ID NO: 154), 9D9 VH (SEQ ID NO: 166), 9E9 VH (SEQ ID NO : 178), 9E 12 VH (SEQ ID NO
  • an anti-EphB4 antibody may comprise a light chain variable region selected from the group consisting of #47 VL (SEQ ID NO:7), #131 VL (SEQ ID NO: 17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO : 100), 2G3 VL (SEQ ID NO : 112), 3E9 VL (SEQ ID NO : 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO: 148), 8D10 VL (SEQ ID NO: 160), 9D9 VL (SEQ ID NO : 172), 9E9 VL (SEQ ID NO 184), 9E 12 VL
  • a humanized anti-EphB4 antibody comprises a VH sequence designated 22E6 VH (SEQ ID NO:22) and a VL sequence designated 22E6 VL (SEQ ID NO:28).
  • a humanized anti-EphB4 antibody comprises the VH sequence 30C2 VH (SEQ ID NO:34) and the VL sequence 30C2 VL (SEQ ID NO:40).
  • a humanized anti-EphB4 antibody comprises the VH sequence 3G8 VH (SEQ ID NO:46) and the VL sequence 3G8 VL (SEQ ID NO:52). [0021] In a particular embodiment, a humanized anti-EphB4 antibody comprises the VH sequence 4F2 VH (SEQ ID NO:58) and the VL sequence 4F2 VL (SEQ ID NO:64).
  • a humanized anti-EphB4 antibody comprises the VH sequence IElO VH (SEQ ID NO:82) and the VL sequence IElO VL (SEQ ID NO:88).
  • a humanized anti-EphB4 antibody comprises the VH sequence 3E9 VH (SEQ ID NO:118)and the VL sequence 3E9 VL (SEQ ID NO: 124).
  • a humanized anti-EphB4 antibody comprises the VH sequence 10C4 VH (SEQ ID NO:202) and the VL sequence 10C4 VL (SEQ ID NO:208).
  • a humanized anti-EphB4 antibody may bind to human EphB4 with an affinity comparable to that of the mouse monoclonal antibodies #47 and/or #131, or with an affinity comparable to that of the ch#47 antibody comprising #47 VH (SEQ ID NO:
  • a humanized anti-EphB4 antibody may bind to human EphB4 with an affinity comparable to that of the ch#131 antibody comprising #131 VH (SEQ ID NO: 12) and #131 VL (SEQ ID NO: 17).
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding a human, humanized, or chimeric anti-EphB4 antibody of the invention or fragments thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, as defined herein, to polynucleotides that encode a human, humanized, or chimeric antibody of the invention.
  • Another embodiment of the invention is a vector comprising one or more nucleotide sequences encoding a human, humanized, or chimeric anti-EphB4 antibody of the invention or fragments thereof.
  • the present invention further relates to an isolated cell comprising a vector wherein said vector comprises one or more nucleotide sequences encoding a human, humanized, or chimeric anti-EphB4 antibody of the invention or fragments thereof.
  • Chimeric, human, and humanized anti-EphB4 monoclonal antibodies described herein include those of the IgGl, IgG2, IgG3, or IgG4 human isotype.
  • the present invention further relates to pharmaceutical compositions comprising a chimeric, human, and humanized anti-EphB4 antibody.
  • the present invention is directed toward a method of treating a malignancy in a human, comprising administering to a human in need thereof a therapeutically-effective amount of a chimeric, human, or humanized anti-EphB4 monoclonal antibody.
  • the present invention relates to a method of treating an angiogenesis-associated disease or disorder in a human, comprising administering to a human in need thereof a therapeutically-effective amount of a chimeric, human, or humanized anti-EphB4 monoclonal antibody.
  • the present invention relates to a method of treating an epithelial migration associated disease or disorder in a human, comprising administering to a human in need thereof a therapeutically-effective amount of a chimeric, human, or humanized anti-EphB4 monoclonal antibody.
  • the present invention further relates to a method of treating or preventing a human disorder or disease characterized by an altered bone homeostasis, comprising administering to a human in need thereof a therapeutically-effective amount of a chimeric, human, or humanized anti-EphB4 monoclonal antibody.
  • antibody and “antibodies” encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecif ⁇ c antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab ' ) 2 fragments, antibody fragments that exhibit the desired biological activity, disulf ⁇ de-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intrabodies, and epitope- binding fragments of any of the above.
  • multispecific antibodies e.g., bispecif ⁇ c antibodies
  • scFv single-chain Fvs
  • Fab fragments single-chain antibodies
  • F(ab ' ) 2 fragments fragments that exhibit the desired biological activity
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.
  • Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains.
  • Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
  • Such antibodies may be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in segments called Complementarity Determining Regions (CDRs) both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FW).
  • CDRs Complementarity Determining Regions
  • FW framework regions
  • the variable domains of native heavy and light chains each comprise four FW regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FW regions and, with the CDRs from the other chain, contribute to the formation of the antigen- binding site of antibodies (see, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)).
  • the constant domains are generally not involved directly in antigen binding, but may influence antigen binding affinity and may exhibit various effector functions, such as participation of the antibody in ADCC, CDC, and/or apoptosis.
  • the term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are associated with its binding to antigen.
  • the hypervariable regions encompass the amino acid residues of the "complementarity determining regions" or "CDRs” (e.g., residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) of the light chain variable domain and residudes 31-35 (Hl), 50-65 (H2) and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
  • CDRs amino acid residues of the "complementarity determining regions” or "CDRs” (e.g., residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) of the light chain variable domain and residudes 31-35 (Hl), 50-65 (H2) and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma cells that are uncontaminated by other immunoglobulin producing cells.
  • a monoclonal antibody may be produced by cells stably or transiently transfected with the heavy and light chain genes encoding the monoclonal antibody.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring engineering of the antibody by any particular method.
  • the term “monoclonal” is used herein to refer to an antibody that is derived from a clonal population of cells, including any eukaryotic, prokaryotic, or phage clone, and not the method by which the antibody was engineered.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made by any recombinant DNA method ⁇ see, e.g., U.S. Patent No. 4,816,567), including isolation from phage antibody libraries using the techniques described in Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. MoI Biol, 222:581-597 (1991), for example. These methods can be used to produce monoclonal mammalian, chimeric, humanized, human, domain antibodies, diabodies, vaccibodies, linear antibodies, and bispecific antibodies.
  • chimeric antibodies includes antibodies in which at least one portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and at least one other portion of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a nonhuman primate ⁇ e.g., Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences (U.S. Patent No. 5,693,780).
  • "Humanized” forms of nonhuman ⁇ e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • FW region residues of the human immunoglobulin are replaced by corresponding nonhuman residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • a humanized antibody heavy or light chain will comprise substantially all of at least one or more variable domains, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FWs are those of a human immunoglobulin sequence.
  • the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a "human antibody” can be an antibody derived from a human or an antibody obtained from a transgenic organism that has been "engineered” to produce specific human antibodies in response to antigenic challenge and can be produced by any method known in the art. In certain techniques, elements of the human heavy and light chain loci are introduced into strains of the organism derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic organism can synthesize human antibodies specific for human antigens, and the organism can be used to produce human antibody-secreting hybridomas.
  • a human antibody can also be an antibody wherein the heavy and light chains are encoded by a nucleotide sequence derived from one or more sources of human DNA.
  • a fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, or in vitro activated B cells, all of which are known in the art.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • non-specific cytotoxic cells ⁇ e.g., Natural Killer (NK) cells, neutrophils, and macrophages
  • NK Natural Killer
  • neutrophils neutrophils
  • macrophages cytotoxic cells
  • such cells are human cells. While not wishing to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs).
  • NK cells express Fc ⁇ RIII
  • monocytes express Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII and/or Fc ⁇ RIV.
  • FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol, 9:457-92 (1991).
  • an in vitro ADCC assay such as that described in U.S. Patent No. 5,500,362 or 5,821,337 may be performed.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the molecules of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al, Proc. Natl Acad. Sci. (USA), 95:652-656 (1998).
  • "Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to initiate complement activation and lyse a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (CIq) to a molecule (e.g., an antibody) complexed with a cognate antigen.
  • a CDC assay e.g. , as described in Gazzano- Santaro et al, J. Immunol. Methods, 202:163 (1996), may be performed.
  • effector cells are leukocytes which express one or more FcRs and perform effector functions.
  • the cells express at least Fc ⁇ RI, FC ⁇ RII, Fc ⁇ RIII and/or Fc ⁇ RIV and carry out ADCC effector function.
  • human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.
  • Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
  • the FcR is a native sequence human FcR.
  • the FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, and Fc ⁇ RIV subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor”) and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine -based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine -based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • FcR neonatal receptor
  • Fv is the minimum antibody fragment which contains a complete antigen- recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent or covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Affinity of an antibody for an epitope to be used in the treatment(s) described herein is a term well understood in the art and means the extent, or strength, of binding of antibody to epitope. Affinity may be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD or Kd), apparent equilibrium dissociation constant (KD ' or Kd ' ), and IC50 (amount needed to effect 50% inhibition in a competition assay). It is understood that, for purposes of this invention, an affinity is an average affinity for a given population of antibodies which bind to an epitope.
  • KD ' reported herein in terms of mg IgG per mL or mg/mL indicate mg Ig per mL of serum, although plasma can be used.
  • antibody affinity can be measured before and/or during treatment, and the values obtained can be used by a clinician in assessing whether a human patient is an appropriate candidate for treatment.
  • the term "avidity” is a measure of the overall binding strength (i.e., both antibody arms) with which an antibody binds an antigen.
  • Antibody avidity can be determined by measuring the dissociation of the antigen-antibody bond in antigen excess using any means known in the art, such as, but not limited to, by the modification of indirect fluorescent antibody as described by Gray et al, J. Virol. Meth., 44: 11-24. (1993)
  • an "epitope” is a term well understood in the art and means any chemical moiety that exhibits specific binding to an antibody.
  • An "antigen” is a moiety or molecule that contains an epitope, and, as such, also specifically binds to antibody.
  • antibody half-life means a pharmacokinetic property of an antibody that is a measure of the mean survival time of antibody molecules following their administration.
  • Antibody half-life can be expressed as the time required to eliminate 50 percent of a known quantity of immunoglobulin from the patient's body or a specific compartment thereof, for example, as measured in serum or plasma, i.e., circulating half- life, or in other tissues.
  • Half- life may vary from one immunoglobulin or class of immunoglobulin to another. In general, an increase in antibody half- life results in an increase in mean residence time (MRT) in circulation for the antibody administered.
  • MRT mean residence time
  • the term "isotype” refers to the classification of an antibody's heavy or light chain constant region.
  • the constant domains of antibodies are not involved in binding to antigen, but exhibit various effector functions.
  • a given human antibody or immunoglobulin can be assigned to one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM.
  • IgA, IgD, IgE, IgG, and IgM Several of these classes may be further divided into subclasses (isotypes), e.g., IgGl (gamma 1), IgG2 (gamma X), IgG3 (gamma 3), and IgG4 (gamma 4), and IgAl and IgA2.
  • the heavy chain constant regions that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the structures and three-dimensional configurations of different classes of immunoglobulins are well-known.
  • human immunoglobulin classes only human IgGl, IgG2, IgG3, IgG4, and IgM are known to activate complement.
  • Human IgGl and IgG3 are known to mediate ADCC in humans.
  • Human light chain constant regions may be classified into two major classes, kappa and lambda
  • the term “immunogenicity” means that a compound is capable of provoking an immune response (stimulating production of specific antibodies and/or proliferation of specific T cells).
  • the term “antigenicity” means that a compound is recognized by an antibody or may bind to an antibody and induce an immune response.
  • agonist refers to a biologically active ligand or a property of such ligand which binds to its complementary biologically active receptor and activates the latter either to cause a biological response in the receptor or to enhance preexisting biological activity of the receptor.
  • an agonist is an anti-EphB antibody that selectively binds to EphB4 and causes receptor clustering, phosphorylation and/or degradation.
  • antagonist refers to a biologically active ligand or a property of such ligand which binds to its complementary biologically active receptor and inhibits the physiological response of the receptor.
  • an antagonist is an anti-EphB antibody that selectively binds to EphB4 and inhibits receptor clustering, phosphorylation and/or degradation.
  • treat By the terms “treat,” “treating” or “treatment of (or grammatically equivalent terms) it is meant that the severity of the subject's condition is reduced or at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is an inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness.
  • treat By the terms “treat,” “treating” or “treatment of (or grammatically equivalent terms) refer to both prophylactic and therapeutic treatment regimes.
  • a "sufficient amount” or “an amount sufficient to” achieve a particular result refers to an amount of an antibody or composition of the invention that is effective to produce a desired effect, which is optionally a therapeutic effect (i.e., by administration of a therapeutically effective amount).
  • a "sufficient amount” or “an amount sufficient to” can be an amount that is effective to deplete EphB4 expressing cells.
  • a “therapeutically effective” amount as used herein is an amount that provides some improvement or benefit to the subject. Stated in another way, a “therapeutically effective” amount is an amount that provides some alleviation, mitigation, and/or decrease in at least one clinical symptom. Clinical symptoms associated with the disorders that can be treated by the methods of the invention are well-known to those skilled in the art. Further, those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • Figure 1 (A) Amino acid sequence alignment of the #47 VH (SEQ ID NO:2),
  • Figure 2 (A) Amino acid sequence alignment of the #131 VH (SEQ ID NO: 12), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), IElO VH (SEQ ID NO:82), 3E9 VH (SEQ ID NO: 118), 6G2 VH (SEQ ID NO: 130), 9E12 VH (SEQ ID NO: 190), 2G3 VH (SEQ ID NO : 106), 11 H4 VH (SEQ ID NO :214), 7A7 VH (SEQ ID NO : 142), 8D 10 VH (SEQ ID NO: 154), 1B7 VH (SEQ ID NO:70), 2F4 VH (SEQ ID NO:94), 9D9 VH (SEQ ID NO: 166), 9E9 VH (SEQ ID NO: 178), 10C4 VH (SEQ ID NO:202), and 12Cl VH (SEQ ID NO:226) heavy chain variable regions.
  • Binding curves were established using standard ELISA protocols. A chimeric Fab derived from the relevant parental antibody #47 or #131 was included in the assays as positive control. Binding curves displayed in the various panels are as follows: (A) 22E6 and 30C2; (B) IElO, 2F4, 2G3, and 3E9; (C) 6G2, 7A7, 8D10, 1B7, andlOC4; (D) 11H4, 12Cl, 3G8, and 4F2; (E) 9D9, 9E9, 9E12, 2G3, and 3E9. Several of the humanized Fabs display EphB4 binding affinity similar to that of the chimeric Fab generated from the parental murine anti- EphB4 antibody.
  • FIG. 4 Overlay of the DSC thermograms for the chimeric and humanized forms of: (A) Fab #47, and (B) mAb #47.
  • Fab #47 When compared with Fab ch#47, humanization of #47 resulted in a decrease in Tm values as follows: (i) -0.3 0 C to -7.1 0 C for Fab 30C2 when using a reference Tm Fab 30C2 of 79.8 0 C and 73.0 0 C, respectively, and (ii) -3.6 0 C for Fab 22E6.
  • the IgG formats revealed a similar trend: (i) -4.1 0 C to -8 0 C for mAb 30C2 when using a reference Tm 1n Ab 30 C 2 of 75.3 0 C and 71.4 0 C, respectively, and (ii) -3.6 0 C for mAb 22E6.
  • the present invention relates to human, humanized, or chimeric anti-EphB4 antibodies that bind to the human EphB4 protein, as well as to compositions comprising those antibodies.
  • a human, humanized, or chimeric anti-EphB4 antibody may mediate antigen-dependent-cell-mediated- cytotoxicity (ADCC).
  • the present invention is directed toward compositions comprising a human, humanized, or chimeric anti-EphB4 antibody of the IgGl and/or IgG3 human isotype, as well as to a human, humanized, or chimeric anti-EphB4 antibody of the IgG2 and/or IgG4 human isotype, that may mediate human ADCC, CDC, and/or apoptosis.
  • the present invention provides chimeric and humanized versions of the anti- EphB4 mouse monoclonal antibodies #47 and #131.
  • a humanized anti- EphB4 antibody may bind to human EphB4 with an affinity similar to the binding affinity of #47 or #131 murine monoclonal antibody or similar to the binding affinity of a chimeric #47 or #131 antibody.
  • an anti-EphB4 antibody may comprise one, two, three, four, five, or all six of the CDRs of #47 or #131 murine monoclonal antibody.
  • an anti-EphB4 antibody may comprise a heavy chain variable region, VH, comprising at least one CDR sequence selected from the group consisting of #47 VH CDRl (SEQ ID NO:3), #47 VH CDR2 (SEQ ID NO:4), and #47 VH CDR3 (SEQ ID NO:5).
  • an anti-EphB4 antibody may comprise a heavy chain variable region, VH, comprising at least one CDR sequence selected from the group consisting of #13 I VH CDRl (SEQ ID NO: 13), #131 VH CDR2 (SEQ ID NO: 14), and #131 VH CDR3 (SEQ ID NO: 15).
  • an anti-EphB4 antibody may comprise a light chain variable region, VL, comprising at least one CDR sequence selected from the group consisting of #47 VL CDRl (SEQ ID NO:8), #47 VL CDR2 (SEQ ID NO:9), and #47 VL CDR3 (SEQ ID NO:10).
  • an anti-EphB4 antibody may comprise a light chain variable region, VL, comprising at least one CDR sequence selected from the group consisting of #131 VL CDRl (SEQ ID NO:18), #131 VL CDR2 (SEQ ID NO:19), and #131 VL CDR3 (SEQ ID NO:20).
  • the present invention encompasses antibodies that bind to human EphB4, comprising derivatives of the VH domains, VH CDRIs, VH CDR2s, VH CDR3s, VL domains, VL CDRIs, VL CDR2s, or VL CDR3s described herein that may bind to human EphB4.
  • Standard techniques known to those of skill in the art can be used to introduce mutations (e.g., additions, deletions, and/or substitutions) in the nucleotide sequence encoding an antibody, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that are routinely used to generate amino acid substitutions.
  • the VH and/or VL CDRs derivatives may include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, less than 2 amino acid substitutions, or 1 amino acid substitution relative to the original VH and/or VL CDRs of the #47 or #131 anti-EphB4 antibody.
  • the VH and/or VL CDRs derivatives may have conservative amino acid substitutions (e.g.
  • mutants can also be introduced randomly along all or part of the VH and/or VL CDR coding sequences, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined.
  • the present invention further encompasses antibodies that bind to human EphB4, said antibodies or antibody fragments comprising one or more CDRs wherein said CDRs comprise an amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of one or more CDRs of the #47 or #131 anti-EphB4 antibody.
  • the percent identity of two amino acid sequences can be determined by any method known to one skilled in the art, including, but not limited to, BLAST protein searches.
  • an anti-EphB4 antibody may comprise one or more heavy chain framework (FW) regions of aVH region selected from the group consisting of #47 VH (SEQ ID NO:2), #131 VH (SEQ ID NO: 12), 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO:82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO:118), 6G2 VH (SEQ ID NO:130), 7A7 VH (SEQ ID NO:142), 8D10 VH (SEQ ID NO : 154), 9D9 VH (SEQ ID NO : 166), 9E9 VH (SEQ ID NO : 178),
  • FW heavy chain framework
  • an anti-EphB4 antibody may comprise one or more light chain framework (FW) regions of a VL region selected from the group consisting of #47 VL (SEQ ID NO:7), #131 VL (SEQ ID NO: 17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO: 112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO: 148), 8D10 VL (SEQ ID NO : 160), 9D9 VL (SEQ ID NO : 172), 9E9 VL (SEQ ID NO :
  • a humanized anti-EphB4 antibody comprises a heavy chain variable region which may comprise four framework regions, FWl, FW2, FW3, and FW4, wherein FWl is selected from the group consisting of 22E6 VH FWl (SEQ ID NO:23), 30C2 VH FWl (SEQ ID NO:35), 3G8 VH FWl (SEQ ID NO:47), 4F2 VH FWl (SEQ ID NO:59), 1B7 VH FWl (SEQ ID NO:71), IElO VH FWl (SEQ ID NO:83), 2F4 VH FWl (SEQ ID NO:95), 2G3 VH FWl (SEQ ID NO: 107), 3E9 VH FWl (SEQ ID NO: 119), 6G2 VH FWl (SEQ ID NO: 131), 7A7 VH FWl (SEQ ID NO: 143), 8D10 VH FWl (SEQ ID NO:
  • the VH of a humanized anti-EphB4 monoclonal antibody may comprise a framework region that has an amino acid sequence identity within the range of from about 64% to about 100% with the corresponding framework region (i.e., FWl of antibody X as compared to FWl of antibody Y) of a VH region selected from the group consisting of #47 VH (SEQ ID NO :2), # 131 VH (SEQ ID NO : 12), 22E6 VH (SEQ ID NO ).
  • a human or humanized VH framework region of an antibody described herein may have an amino acid sequence identity that is at least 64%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% with a VH region selected from the group consisting of #47 VH (SEQ ID NO:2), #131 VH (SEQ ID NO: 12), 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO:82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO:118), 6G2 VH (SEQ ID NO:130), 7A7 VH (SEQ ID NO:142), 8D10 VH (SEQ ID NO:
  • the human or humanized VH framework regions of an anti-EphB4 antibody described herein may have an amino acid sequence identity with the corresponding framework regions of a VH region selected from the group consisting of #47 VH (SEQ ID NO:2), #131 VH (SEQ ID NO:12), 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO:82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO:118), 6G2 VH (SEQ ID NO:130), 7A7 VH (SEQ ID NO:142), 8D10 VH (SEQ ID NO : 154), 9D9 VH (SEQ ID NO : 166),
  • the mismatch may be a conservative amino acid substitution.
  • a mismatch that is a conservative amino acid substitution is one in which the mismatched amino acid has physical and chemical properties similar to the original amino acid, e.g., the mismatched residue has similar characteristics of polarity (polar or nonpolar), acidity (acidic or basic), side chain structure (e.g., branched or straight, or comprising a phenyl ring, a hydroxyl moiety, or a sulfur moiety) to the original residue.
  • the mismatched residue may be a non-conservative amino acid substitution.
  • a mismatch that is a non-conservative amino acid substitution is one in which the mismatched amino acid does not have physical and chemical properties similar to the original amino acid, e.g., the mismatched residue has a different polarity, acidity, or side chain structure (e.g., branched or straight, or comprising a phenyl ring, a hydroxyl moiety, or a sulfur moiety) as compared to the residue to be replaced.
  • a human or humanized VL framework region of an anti-EphB4 antibody described herein may have an amino acid sequence identity with the corresponding framework region (i.e., FWl of antibody X as compared to FWl of antibody Y) of a VL selected from the group consisting of #47 VL (SEQ ID NO: 7), #131 VL (SEQ ID NO: 17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO : 100), 2G3 VL (SEQ ID NO : 112), 3E9 VL (SEQ ID NO : 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO:
  • a human or humanized VL framework region of an antibody described herein may have an amino acid sequence identity with a framework region of a VL selected from the group consisting of #47 VL (SEQ ID NO:7), #131 VL (SEQ ID NO: 17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO: 112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO: 148), 8D10 VL (SEQ ID NO: 160), 9D9 VL (SEQ ID NO: 172), 9E9 VL (SEQ ID NO:
  • the human or humanized VL framework regions of an antibody described herein may have an amino acid sequence identity with the framework regions of a VL selected from the group consisting of #47 VL (SEQ ID NO: 7), #131 VL (SEQ ID NO: 17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO : 100), 2G3 VL (SEQ ID NO : 112), 3E9 VL (SEQ ID NO : 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO: 148), 8D10 VL (SEQ ID NO: 160), 9D9 VL (SEQ ID NO : 172), 9E9 VL (
  • the mismatch may be a conservative amino acid substitution.
  • a mismatch that is a conservative amino acid substitution is one in which the mismatched amino acid has physical and chemical properties similar to the amino acid replaced, e.g., the mismatched residue has similar characteristics of polarity (polar or nonpolar), acidity (acidic or basic), side chain structure (e.g., branched or straight, or comprising a phenyl ring, a hydroxyl moiety, or a sulfur moiety) to the replaced residue.
  • the mismatch may be a non-conservative amino acid substitution.
  • a mismatch that is a non-conservative amino acid substitution is one in which the mismatched amino acid does not have physical and chemical properties similar to the amino acid replaced, e.g. , the mismatched residue has a different polarity, acidity, or side chain structure (e.g., branched or straight, or comprising a phenyl ring, a hydroxyl moiety, or a sulfur moiety) as compared to the replaced residue.
  • the mismatched residue has a different polarity, acidity, or side chain structure (e.g., branched or straight, or comprising a phenyl ring, a hydroxyl moiety, or a sulfur moiety) as compared to the replaced residue.
  • an anti-EphB4 antibody may comprise a VH region having one or more CDRs of the #131 or #47 anti-EphB4 antibody wherein said VH may further comprise one or more heavy chain framework (FW) regions of aVH region selected from the group consisting of #47 VH (SEQ ID NO:2), #131 VH (SEQ ID NO: 12), 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO:82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO: 118), 6G2 VH (SEQ ID NO: 130), 7A7 VH (SEQ ID NO : 142), 8D 10 VH (SEQ ID NO:2), #1
  • an anti-EphB4 antibody may comprise a VL region having one or more CDRs of the #131 or #47 anti-EphB4 antibody wherein said VL may further comprise one or more light chain framework (FW) regions of a VL region selected from the group consisting of #47 VL (SEQ ID NO:7), #131 VL (SEQ ID NO: 17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO: 112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO : 148), 8D 10 VL (SEQ ID NO:7),
  • an anti-EphB4 antibody may comprise a VL and a VH having one or more CDRs of the #131 or #47 anti-EphB4 antibody, wherein said VL may further comprise one or more light chain framework region of a VL region selected from the group consisting of #47 VL (SEQ ID NO:7), #131 VL (SEQ ID NO: 17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO: 112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO : 148), 8D 10 V
  • an anti-EphB4 antibody may comprise a light chain variable region selected from the group consisting of #47 VL (SEQ ID NO:7), #131 VL (SEQ ID NO: 17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO : 100), 2G3 VL (SEQ ID NO : 112), 3E9 VL (SEQ ID NO : 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO: 148), 8D10 VL (SEQ ID NO: 160), 9D9 VL (SEQ ID NO : 172), 9E9 VL (SEQ ID NO 184), 9E 12 VL
  • the present invention further relates to an anti-EphB4 antibody comprising a heavy chain variable region selected from the group consisting of #47 VH (SEQ ID NO:2), #131 VH (SEQ ID NO: 12), 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO: 82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO:118), 6G2 VH (SEQ ID NO: 130), 7A7 VH (SEQ ID NO: 142), 8D10 VH (SEQ ID NO: 154), 9D9 VH (SEQ ID NO: 166), 9E9 VH (SEQ ID NO : 178), 9E 12 VH (SEQ ID NO
  • a humanized anti-EphB4 antibody may comprise a heavy chain variable region selected from the group consisting of #47 VH (SEQ ID NO:2), #131 VH (SEQ ID NO: 12), 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH
  • a heavy chain comprising a humanized VH may be expressed with a light chain comprising a humanized VL to produce a humanized anti-EphB4 antibody.
  • a humanized anti-EphB4 antibody comprises a VH sequence designated 22E6 VH (SEQ ID NO:22) and a VL sequence designated 22E6 VL (SEQ ID NO:28).
  • a humanized anti-EphB4 antibody comprises the VH sequence 30C2 VH (SEQ ID NO:34) and the VL sequence 30C2 VL (SEQ ID NO:40).
  • a humanized anti-EphB4 antibody comprises the VH sequence 3G8 VH (SEQ ID NO:46) and the VL sequence 3G8 VL (SEQ ID NO:52).
  • a humanized anti-EphB4 antibody comprises the VH sequence 4F2 VH (SEQ ID NO:58) and the VL sequence 4F2 VL (SEQ ID NO:64).
  • a humanized anti-EphB4 antibody comprises the VH sequence IElO VH (SEQ ID NO:82) and the VL sequence IElO VL (SEQ ID NO:88). [0095] In a particular embodiment, a humanized anti-EphB4 antibody comprises the VH sequence 3E9 VH (SEQ ID NO:118)and the VL sequence 3E9 VL (SEQ ID NO: 124).
  • a humanized anti-EphB4 antibody comprises the VH sequence 10C4 VH (SEQ ID NO:202) and the VL sequence 10C4 VL (SEQ ID NO:208).
  • a humanized anti-EphB4 antibody comprises the VH sequence 1B7 VH (SEQ ID NO:70) and the VL sequence 1B7 VL (SEQ ID NO:76).
  • a humanized anti-EphB4 antibody comprises the VH sequence 2F4 VH (SEQ ID NO:94), and the VL sequence 2F4 VL (SEQ ID NO: 100).
  • a humanized anti-EphB4 antibody comprises the VH sequence 2G3 VH (SEQ ID NO:106)and the VL sequence 2G3 VL (SEQ ID NO:112).
  • a humanized anti-EphB4 antibody comprises the VH sequence 6G2 VH (SEQ ID NO:
  • a humanized anti-EphB4 antibody comprises the VH sequence 7A7 VH (SEQ ID NO:142)and the VL sequence 7A7 VL (SEQ ID NO:148).
  • a humanized anti-EphB4 antibody comprises the VH sequence 8D10 VH (SEQ ID NO:154)and the VL sequence 8D10 VL (SEQ ID NO: 160).
  • a humanized anti- EphB4 antibody comprises the VH sequence 9D9 VH (SEQ ID NO: 166) and the VL sequence 9D9 VL (SEQ ID NO: 172).
  • a humanized anti-EphB4 antibody comprises the VH sequence 9E9 VH (SEQ ID NO: 178) and the VL sequence 9E9 VL (SEQ ID NO: 184). In an additional embodiment, a humanized anti-EphB4 antibody comprises the VH sequence 9El 2 VH (SEQ ID NO: 190), and the VL sequence 9El 2 VL (SEQ ID NO: 196). In another embodiment, a humanized anti-EphB4 antibody comprises the VH sequence 11H4 VH (SEQ ID NO:214) and the VK sequence 11H4 VL (SEQ ID NO:220). In yet another embodiment, a humanized anti-EphB4 antibody comprises the VH sequence 12Cl VH (SEQ ID NO:226) and the VK sequence 12Cl VL (SEQ ID NO:232).
  • a humanized VH may comprise a leader sequence of MGDNDIHFAFLSTGVHS (SEQ ID NO: 83).
  • a humanized VK may comprise a leader sequence MDMRVPAQLLGLLLLWLPGAKC (SEQ ID NO :84) selected from the leader peptide of the human VKI-L 12 gene.
  • Anti-EphB4 antibodies described herein may have a high binding affinity for the human EphB4 (hEphB4) antigen.
  • an antibody described herein may have an association rate constant or k on rate (antibody (Ab) + antigen (Ag) kon — > Ab-Ag) of at least 2 X 10 5 M- 1 S “1 , at least 5 X 10 5 M -1 S “1 , at least 10 6 IVT 1 S “1 , at least 5 X 1O 6 IVT 1 S “1 , at least 10 7 M “ 1 S “1 , at least 5 X 10 7 M- 1 S “1 , or at least 10 8 M- 1 S “1 .
  • an anti-EphB4 antibody may have a k off rate ((Ab- Ag) ko// ⁇ antibody (Ab) + antigen (Ag)) of less than 5XlO "1 s “1 , less than 10 "1 s “1 , less than 5xlO "2 s “1 , less than 10 "2 s “1 , less than 5xlO "3 s “1 , less than 10 "3 s “1 , less than 5xlO "4 s “1 , or less than 10 "4 s "1'
  • an antibody of the invention has a k off of less than 5xlO "5 s “1 , less than 10 "5 s “1 , less than 5xlO "6 s “1 , less than 10 "6 s “1 , less than 5xlO "7 s "1 , less than 10 "7 s “1 , less than 5xlO "8 s "
  • an anti-EphB4 antibody may have an affinity constant or K a (k on /k off ) of at least 10 2 M “1 , at least 5 X 10 2 M “1 , at least 10 3 M “1 , at least 5 X 10 3 M “1 , at least 10 4 M “1 , at least 5 X 10 4 M “1 , at least 10 5 M “1 , at least 5 X 10 5 M “1 , at least 10 6 M “1 , at least 5 X 10 6 M “1 , at least 10 7 M “1 , at least 5 X 10 7 M “1 , at least 10 8 M “1 , at least 5 X 10 8 M “1 , at least 10 9 M “1 , at least 5 X 10 9 M “1 , at least 10 10 M “1 , at least 5 X 10 10 M “1 , at least 5 X 10 10 M “1 , at least 10 11 M “1 , at least 5 X 10 11 M “1 , at least 10 12
  • an anti-EphB4 antibody may have a dissociation constant or IQ (k o ff/k on ) of less than 5xlO "2 M, less than 10 "2 M, less than 5xlO "3 M, less than 10 "3 M, less than 5xlO "4 M, less than 10 "4 M, less than 5xlO "5 M, less than 10 "5 M, less than 5xlO "6 M, less than 10 ⁇ 6 M, less than 5x10 7 M, less than 10 ⁇ 7 M, less than 5x10 ⁇ 8 M, less than 10 "8 M, less than 5xlO "9 M, less than 10 "9 M, less than 5x10 ⁇ 10 M, less than 10 "10 M, less than 5x10 11 M, less than 10 "11 M, less than 5x10 ⁇ 12 M, less than 10 ⁇ 12 M, less than 5x10 13 M, less than 10 "13 M, less than 5xlO ⁇ 14 M, less than 10 ⁇
  • An antibody used in accordance with a method described herein may immunospecif ⁇ cally bind to human EphB4 and may have a dissociation constant (K ⁇ ) of less than 3000 pM, less than 2500 pM, less than 2000 pM, less than 1500 pM, less than 1000 pM, less than 750 pM, less than 500 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM as assessed using a method described herein or known to one of skill in the art (e.g., a BIAcore assay, ELISA) (Biacore International AB, Uppsala,
  • K ⁇ dissociation constant
  • an antibody used in accordance with a method described herein may immunospecif ⁇ cally bind to a human EphB4 antigen and may have a dissociation constant (K,) of between 25 to 3400 pM, 25 to 3000 pM, 25 to 2500 pM, 25 to 2000 pM, 25 to 1500 pM, 25 to 1000 pM, 25 to 750 pM, 25 to 500 pM, 25 to 250 pM, 25 to 100 pM, 25 to 75 pM, 25 to 50 pM as assessed using a method described herein or known to one of skill in the art (e.g., a BIAcore assay, ELISA).
  • K dissociation constant
  • an anti-EphB4 antibody used in accordance with a method described herein may immunospecif ⁇ cally bind to hEphB4 and may have a dissociation constant (K ⁇ ) of 500 pM, 100 pM, 75 pM or 50 pM as assessed using a method described herein or known to one of skill in the art (e.g., a BIAcore assay, ELISA).
  • K ⁇ dissociation constant
  • Antibodies like all polypeptides, have an Isoelectric Point (pi), which is generally defined as the pH at which a polypeptide carries no net charge. It is known in the art that protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pi) of the protein. As used herein , the pi value is defined as the pi of the predominant charge form.
  • the pi of a protein may be determined by a variety of methods including but not limited to, isoelectric focusing and various computer algorithms (see, e.g., Bjellqvist et ⁇ l, 1993, Electrophoresis 14:1023).
  • the thermal melting temperatures (Tm) of the Fab domain of an antibody can be a good indicator of the thermal stability of an antibody and may further provide an indication of the shelf-life.
  • a lower Tm indicates more aggregation/less stability, whereas a higher Tm indicates less aggregation/ more stability.
  • antibodies having higher Tm are preferable.
  • Tm of a protein domain e.g., a Fab domain
  • an additional nonexclusive embodiment of the present invention is an anti-EphB4 antibody having certain preferred biochemical characteristics such as a particular isoelectric point (pi) or melting temperature (Tm).
  • an anti-EphB4 antibody has a pi ranging from 5.5 to 9.5.
  • an anti-EphB4 antibody has a pi that ranges from about 5.5 to about 6.0, or about 6.0 to about 6.5, or about 6.5 to about 7.0, or about 7.0 to about 7.5, or about 7.5 to about 8.0, or about 8.0 to about 8.5, or about 8.5 to about 9.0, or about 9.0 to about 9.5.
  • an antibody described herein has a pi that ranges from 5.5-6.0, or 6.0 to 6.5, or 6.5 to 7.0, or 7.0-7.5, or 7.5-8.0, or 8.0-8.5, or 8.5-9.0, or 9.0-9.5.
  • An anti-EphB4 antibody of the present invention may have a pi of at least 5.5, or at least 6.0, or at least 6.3, or at least 6.5, or at least 6.7, or at least 6.9, or at least 7.1, or at least 7.3, or at least 7.5, or at least 7.7, or at least 7.9, or at least 8.1, or at least 8.3, or at least 8.5, or at least 8.7, or at least 8.9, or at least 9.1, or at least 9.3, or at least 9.5.
  • an anti-EphB4 antibody described herein may have a pi of at least about 5.5, or at least about 6.0, or at least about 6.3, or at least about 6.5, or at least about 6.7, or at least about 6.9, or at least about 7.1, or at least about 7.3, or at least about 7.5, or at least about 7.7, or at least about 7.9, or at least about 8.1, or at least about 8.3, or at least about 8.5, or at least about 8.7, or at least about 8.9, or at least about 9.1, or at least about 9.3, or at least about 9.5.
  • solubility by altering the number and location of ionizable residues in the antibody to adjust the pi.
  • the pi of a polypeptide can be manipulated by making the appropriate amino acid substitutions (e.g. , by substituting a charged amino acid such as a lysine, for an uncharged residue such as alanine).
  • amino acid substitutions of an antibody that result in changes of the pi of said antibody may improve solubility and/or the stability of the antibody.
  • amino acid substitutions would be most appropriate for a particular antibody to achieve a desired pi.
  • a substitution is generated in an anti-EphB4 antibody described herein to alter the pi.
  • substitution(s) of the Fc region resulting in altered binding to Fc ⁇ R may also result in a change in the pi.
  • substitution(s) of the Fc region are specifically chosen to effect both the desired alteration in Fc ⁇ R binding and any desired change in pi.
  • an anti-EphB4 antibody of the invention may have a Tm ranging from 65°C to 120 0 C.
  • an anti-EphB4 antibody of the invention may have a Tm ranging from about 75°C to about 120 0 C, or about 75°C to about 85°C, or about 85°C to about 95°C, or about 95°C to about 105 0 C, or about 105 0 C to about 115°C, or about 115°C to about 120 0 C.
  • an anti-EphB4 antibody of the invention may have a Tm ranging from 75°C to 120 0 C, or 75°C to 85°C, or 85°C to 95°C, or 95°C to 105 0 C, or 105 0 C to 115°C, or 115°C to 120 0 C.
  • an anti-EphB4 antibody of the invention may have a Tm of at least about 65°C, or at least about 70 0 C, or at least about 75°C, or at least about 80 0 C, or at least about 85°C, or at least about 90 0 C, or at least about 95°C, or at least about 100 0 C, or at least about 105 0 C, or at least about 110 0 C, or at least about 115°C, or at least about 120 0 C.
  • an anti-EphB4 antibody of the invention may have a Tm of at least 65°C, or at least 70 0 C, or at least 75°C, or at least 80 0 C, or at least 85°C, or at least 90 0 C, or at least 95°C, or at least 100 0 C, or at least 105 0 C, or at least 110 0 C, or at least 115 o C, or at least l20°C.
  • EphB4 is a receptor whose biological activity changes in response to binding a biologically active ligand.
  • an anti-EphB4 antibody may function as an EphB4 agonist.
  • an anti-EphB4 antibody of the invention may cause receptor clustering, phosphorylation and/or degradation upon binding the EphB4 receptor.
  • an anti-EphB4 antibody described herein may function as an EphB4 receptor antagonist.
  • binding of an anti-EphB4 antibody to EphB4 may inhibit the EphB4 receptor clustering, phosphorylation and/or degradation.
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding a human, humanized, or chimeric anti-EphB4 antibody or fragments thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined herein, to polynucleotides that encode a human, humanized, or chimeric antibody that binds to hEphB4.
  • Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2X SSC/0.1% SDS at about 50-65 0 C, highly stringent conditions such as hybridization to filter-bound DNA in 6X SSC at about 45°C followed by one or more washes in 0.1X SSC/0.2% SDS at about 60 0 C, or any other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel, F.M. et al., eds. 1989 Current Protocols in Molecular Biology, vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3).
  • SSC sodium chloride/sodium citrate
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody may also be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody ) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be clonucle
  • the present invention also provides polynucleotide sequences encoding VH and VK framework regions and CDRs of antibodies described herein as well as expression vectors for their efficient expression in mammalian cells.
  • the present invention also relates to a method of treating a malignancy in a human comprising administering to a human in need thereof, a human, humanized or chimeric anti-EphB4 antibody that may mediate human antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), and/or apoptosis in an amount sufficient to deplete the malignant cells.
  • ADCC human antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cell-mediated cytotoxicity
  • apoptosis in an amount sufficient to deplete the malignant cells.
  • the present invention also concerns methods of treating a malignancy in a human comprising administration of a therapeutically effective regimen of a human, humanized, or chimeric anti-EphB4 antibody, which is of the IgGl or IgG3 human isotype.
  • the present invention provides methods of inhibiting angiogenesis and methods of treating angiogenesis-associated diseases.
  • the present invention provides methods of inhibiting or reducing tumor growth and methods of treating an individual suffering from cancer. These methods involve administering to the individual a therapeutically effective amount of one or more polypeptide therapeutic agents as described above. These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans.
  • angiogenesis-associated diseases include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; inflammatory disorders such as immune and non-immune inflammation; chronic articular rheumatism and psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation and wound healing; telangiect
  • angiogenesis-independent cancer refers to a cancer (tumor) where there is no or little neovascularization in the tumor tissue.
  • polypeptide therapeutic agents of the present invention are useful for treating or preventing a cancer (tumor), including, but not limited to, ovarian cancer, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi sarcoma, and leukemia.
  • a cancer tumor including, but not limited to, ovarian cancer, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi sarcoma, and leukemia.
  • the invention can be employed to treat ovarian cancer.
  • the present invention further relates to a method of treating an epithelial migration associated disease or disorder in a human comprising administering to a human in need thereof a therapeutically effective amount of human, humanized, or chimeric anti-EphB4 antibody.
  • the present invention further relates to a method of treating or preventing a human disorder or disease characterized by an altered bone homeostasis, comprising administering to a human in need thereof a therapeutically-effective amount of a chimeric, human, or humanized anti-EphB4 monoclonal antibody.
  • the invention can be employed to treat bone remodeling disease such as osteopetrosis and osteoporosis.
  • Humanized antibodies described herein can be produced using a variety of techniques known in the art, including, but not limited to, CDR-grafting (see e.g. , European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos.
  • FW substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and FW residues to identify FW residues important for antigen binding and sequence comparison to identify unusual FW residues at particular positions. (See, e.g., Queen et al, U.S. Patent No. 5,585,089; and Riechmann et al, 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)
  • a humanized anti-EphB4 antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as "import” residues, which are typically taken from an “import” variable domain.
  • humanized antibodies comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions from human.
  • humanized chimeric antibodies substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FW residues are substituted by residues from analogous sites in rodent antibodies.
  • variable domains both light and heavy
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequences which are most closely related to that of the rodent are then screened for the presences of specific residues that may be critical for antigen binding, appropriate structural formation and/or stability of the intended humanized mAb (Sims et al, J. Immunol, 151 :2296 (1993); Chothia et al, J. MoI Biol, 196:901 (1987), the contents of which are incorporated herein by reference in their entirety).
  • FW sequences matching the desired criteria are then be used as the human donor FW regions for the humanized antibody.
  • Another method uses a particular FW derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same FW may be used for several different humanized anti-EphB4 antibodies (Carter et ah, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol, 151 :2623 (1993), the contents of which are incorporated herein by reference in their entirety).
  • Anti-EphB4 antibodies can be humanized with retention of high affinity for EphB4 and other favorable biological properties.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind EphB4.
  • FW residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, for example affinity for EphB4, is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • a "humanized" antibody may retain a similar antigenic specificity as the original antibody, i.e., in the present invention, the ability to bind human EphB4 antigen.
  • the affinity and/or specificity of binding of the antibody for human EphB4 antigen may be altered using methods of "directed evolution," as described by Wu et al., J. MoI. Biol, 294:151 (1999), the contents of which are incorporated herein by reference herein in their entirety.
  • a monoclonal anti-EphB4 antibody exhibits binding specificity to human EphB4 antigen and may mediate human ADCC, CDC and/or apoptotic mechanisms .
  • Such an antibody can be generated using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • Antibodies are highly specific, being directed against a single antigenic site.
  • An engineered anti-EphB4 antibody can be produced by any means known in the art, including, but not limited to, those techniques described below and improvements to those techniques. Large- scale high-yield production typically involves culturing a host cell that produces the engineered anti-EphB4 antibody and recovering the anti-EphB4 antibody from the host cell culture.
  • Monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al, in Monoclonal Antibodies and T Cell Hybridomas, 563-681 (Elsevier, N.Y., 1981) (said references incorporated herein by reference in their entireties).
  • a mouse or other appropriate host animal such as a hamster or macaque monkey, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • Lymphocytes may also be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
  • a suitable fusing agent such as polyethylene glycol
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • myeloma cells that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63- Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding an anti-EphB4 antibody is readily isolated and sequenced using conventional procedures ⁇ e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of anti-EphB4 antibodies).
  • the hybridoma cells serve as a source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of anti-EphB4 antibodies in the recombinant host cells.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding V H and V L domains are amplified from animal cDNA libraries ⁇ e.g., human or murine cDNA libraries of affected tissues).
  • the DNA encoding the V H and V L domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector.
  • the vector is electroporated in E. coli and the E. coli is infected with helper phage.
  • Phage used in these methods is typically filamentous phage including fd and Ml 3 and the V H and V L domains are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antigen-binding domain that binds to a particular antigen can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al, 1995, J. Immunol. Methods, 182:41-50; Ames et al, 1995, J. Immunol. Methods, 184:177-186;
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below.
  • Techniques to recombinantly produce Fab, Fab' and F(ab') 2 fragments can also be employed using methods known in the art such as those disclosed in PCT Publication No.
  • Antibodies may be isolated from antibody phage libraries generated using the techniques described in McCafferty et al, Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-628 (1991). Marks et al, J. MoI Biol, 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Chain shuffling can be used in the production of high affinity (nM range) human antibodies (Marks et al, Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al , Nuc. Acids. Res., 21 :2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of anti-EphB4 antibodies.
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones.
  • the PCR amplified VH domains can be cloned into vectors expressing a heavy chain constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a light chain constant region, e.g., human kappa or lambda constant regions.
  • the vectors for expressing the VH or VL domains may comprise an EF- l ⁇ promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin.
  • the VH and VL domains may also be cloned into one vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • the polynucleotides encoding the heavy and light chains of an anti-EphB4 antibody of the invention may be subjected to nucleic acid sequence optimization.
  • the final goal of the sequence optimization process is to create a coding region that is transcribed and translated at the highest possible efficiency.
  • Sequence optimization is achieved by a combination of: (i) codon usage optimization, (ii) G/C content adaptation, (iii) elimination of internal splicing sites and premature polyadenylation sites, (iv) disruption of stable RNA secondary structures, (v) elimination of direct repeat sequences, (vi) elimination of sequences that may for stable dsRNA with host cell transcripts, (vii) eliminate sequences targeted by host cell micro RNAs, and (viii) introduction of RNA stabilizing and RNA translocation signals.
  • codon usage optimization e.g., a codon usage optimization
  • G/C content adaptation e.g., G/C content adaptation
  • elimination of internal splicing sites and premature polyadenylation sites e.g., deletion of internal splicing sites and premature polyadenylation sites
  • iv disruption of stable RNA secondary structures
  • elimination of direct repeat sequences e.g., deletion of sequences that may for stable dsRNA with host cell transcripts
  • WO2006015789A2 Bradel-Tretheway et al., J. Virol. Methods 111 :145-56 (2003), Valencik & McDonald, Transgenic Res. 3:269-75 (2001).
  • a sequence may be optimized by engaging the services of a commercial provider ⁇ e.g., GENEART Inc.).
  • anti-EphB4 antibodies herein specifically include chimeric antibodies
  • immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while another portion of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl. Acad. ScL USA, 81 :6851-6855 (1984)).
  • Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a nonhuman primate (e.g. , Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences (U.S. Patent No. 5,693,780).
  • a nonhuman primate e.g. , Old World Monkey, such as baboon, rhesus or cynomolgus monkey
  • human constant region sequences U.S. Patent No. 5,693,780
  • Anti-EphB4 antibodies of compositions and methods described herein can be mutant antibodies.
  • antibody mutant or “altered antibody” refers to an amino acid sequence variant of an anti-EphB4 antibody wherein one or more of the amino acid residues of an anti-EphB4 antibody have been modified.
  • the modifications to the amino acid sequence of an anti-EphB4 antibody include modifications to the sequence that may improve affinity or avidity of the antibody for its antigen, and/or modifications to the Fc portion of the antibody that may improve effector function.
  • the present invention therefore relates to human, humanized, and chimeric anti-EphB4 antibodies disclosed herein as well as altered/mutant derivatives thereof including, but not limited to ones exhibiting altered human EphB4 binding characteristics; e.g. altered association constants koN, dissociation constants koFF, and/or equilibrium constant or binding affinity, K D .
  • the K D of an anti-EphB4 antibody described herein, or an altered/mutant derivative thereof, for human EphB4 may be no more than about 10 "6 M, 10 "7 M, 10 "8 M, or 10 "9 M.
  • modifications may be made to any known anti-EphB4 antibodies or anti- EphB4 antibodies identified as described herein. Such altered antibodies necessarily have less than 100% sequence identity or similarity with a known anti-EphB4 antibody.
  • an altered antibody may have an amino acid sequence that is within the range of from about 25% to about 95% identical or similar to the amino acid sequence of either the heavy or light chain variable domain of an anti-EphB4 antibody as described herein.
  • An altered antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of an anti-EphB4 antibody as described herein.
  • an altered antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of the heavy chain CDRl, CDR2, or CDR3 of an anti-EphB4 antibody as described herein.
  • an altered antibody may maintain human EphB4 binding capability.
  • an anti-EphB4 antibody as described herein may comprise a VH that is at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequence of a VH selected from the group consisting of 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO: 82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO: 118), 6G2 VH (SEQ ID NO:130), 7A7 VH (SEQ ID NO: 142), 8D10 VH (SEQ ID NO:154), 9D
  • an altered antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of FWl, FW2, FW3, or FW4 regions of a VH selected from the group consisting of 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO:82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO:118), 6G2 VH (SEQ ID NO:130), 7A7 VH (SEQ ID NO:142), 8D10 VH (SEQ ID NO: 154), 9D9 VH (SEQ ID NO:
  • an altered antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of the light chain CDRl, CDR2, or CDR3 of an anti-EphB4 antibody as described herein.
  • an anti- EphB4 antibody of the invention may comprise a VL that is at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to an amino acid sequence of a VL region selected from the group consisting of #47 VL (SEQ ID NO:7), #131 VL (SEQ ID NO:17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO: 112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO
  • an altered antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of FWl, FW2, FW3, or FW4 regions of a VL region selected from the group consisting of #47 VL (SEQ ID NO:7), #131 VL (SEQ ID NO: 17), 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO : 100), 2G3 VL (SEQ ID NO : 112), 3E9 VL (SEQ ID NO : 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO: 7S
  • Identity or similarity with respect to a sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) or similar (i.e., amino acid residue from the same group based on common side- chain properties, see below) with anti-EphB4 antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence outside of the variable domain shall be construed as affecting sequence identity or similarity.
  • % identity is a measure of the relationship between two polynucleotides or two polypeptides, as determined by comparing their sequences. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. The alignment of the two sequences is examined and the number of positions giving an exact amino acid or nucleotide correspondence between the two sequences determined, divided by the total length of the alignment and multiplied by 100 to give a % identity figure.
  • This % identity figure may be determined over the whole length of the sequences to be compared, which is particularly suitable for sequences of the same or very similar length and which are highly homologous, or over shorter defined lengths, which is more suitable for sequences of unequal length or which have a lower level of homology.
  • sequences can be aligned with the software clustalw under Unix which generates a file with an ".aln" extension, this file can then be imported into the Bioedit program (Hall, T. A. 1999, BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41 :95-98) which opens the .aln file.
  • Bioedit program Hall, T. A. 1999, BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41 :95-98
  • the Bioedit window one can choose individual sequences (two at a time) and alignment them. This method allows for comparison of the entire sequence.
  • BESTFIT is more suited to comparing two polynucleotide or two polypeptide sequences which are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer.
  • GAP aligns two sequences finding a "maximum similarity" according to the algorithm of Neddleman and Wunsch (J. MoI. Biol, 48:443-354, 1970).
  • GAP is more suited to comparing sequences which are approximately the same length and an alignment is expected over the entire length.
  • the parameters "Gap Weight " and "Length Weight" used in each program are 50 and 3 for polynucleotides and 12 and 4 for polypeptides, respectively.
  • % identities and similarities are determined when the two sequences being compared are optimally aligned.
  • Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res., 25:3389-3402.
  • PSI-Blast can also be used to perform an iterated search which detects distant relationships between molecules (Id ).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See, http://www.ncbi.nlm.nih.gov.
  • FASTA Pearson W.R. and Lipman D.J., Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988, available as part of the Wisconsin Sequence Analysis Package.
  • BLOSUM62 amino acid substitution matrix Henikoff S. and Henikoff J.G., Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992 is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into amino acid sequences before comparison.
  • the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a polynucleotide or a polypeptide sequence of the present invention, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value.
  • one or more amino acid alterations are introduced in one or more of the hypervariable regions of the species- dependent antibody.
  • One or more alterations (e.g., substitutions) of framework region residues may also be introduced in an anti-EphB4 antibody where these result in an improvement in the binding affinity of the antibody mutant for the antigen from the second mammalian species.
  • framework region residues to modify include those which non-covalently bind antigen directly (Amit et al, Science, 233:747-753 (1986)); interact with/effect the conformation of a CDR (Chothia et al, J. MoI.
  • modification of one or more of such framework region residues results in an enhancement of the binding affinity of the antibody for the antigen from the second mammalian species. For example, from about one to about five framework residues may be altered in this embodiment of the invention. Sometimes, this may be sufficient to yield an antibody mutant suitable for use in preclinical trials, even where none of the hypervariable region residues have been altered. Normally, however, an altered antibody will comprise additional hypervariable region alteration(s).
  • the hypervariable region residues which are altered may be changed randomly, especially where the starting binding affinity of an anti-EphB4 antibody for the antigen from the second mammalian species is such that such randomly produced altered antibody can be readily screened.
  • hypervariable region residue(s) are replaced by alanine or polyalanine residue(s) to affect the interaction of the amino acids with the antigen from the second mammalian species.
  • Those hypervariable region residue(s) demonstrating functional sensitivity to the substitutions then are refined by introducing additional or other mutations at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • the Ala-mutants produced this way are screened for their biological activity as described herein.
  • Another procedure for generating such an altered antibody involves affinity maturation using phage display (Hawkins et al., J. MoI. Biol, 254:889-896 (1992) and Lowman et ah, Biochemistry, 30(45): 10832-10837 (1991)). Briefly, several hypervariable region sites ⁇ e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M 13 packaged within each particle. The phage-displayed mutants are then screened for their biological activity ⁇ e.g., binding affinity) as herein disclosed.
  • Mutations in antibody sequences may include substitutions, deletions, including internal deletions, additions, including additions yielding fusion proteins, or conservative substitutions of amino acid residues within and/or adjacent to the amino acid sequence, but that result in a "silent" change, in that the change produces a functionally equivalent anti-EphB4 antibody.
  • Conservative amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • glycine and proline are residues that can influence chain orientation. Non- conservative substitutions will entail exchanging a member of one of these classes for a member of another class.
  • non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the antibody sequence.
  • Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ - Abu, ⁇ -Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids,
  • Any technique for mutagenesis known in the art can be used to modify individual nucleotides in a DNA sequence, for purposes of making amino acid substitution(s) in the antibody sequence, or for creating/deleting restriction sites to facilitate further manipulations.
  • Such techniques include, but are not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA, 82:488 (1985); Hutchinson, C. et al., J. Biol. Chem., 253:6551 (1978)), oligonucleotide-directed mutagenesis (Smith, Ann. Rev.
  • an anti-EphB4 antibody can be modified to produce fusion proteins; i.e., the antibody, or a fragment thereof, fused to a heterologous protein, polypeptide or peptide.
  • the protein fused to the portion of an anti-EphB4 antibody is an enzyme component of Antibody-Directed Enzyme Prodrug Therapy (ADEPT).
  • ADPT Antibody-Directed Enzyme Prodrug Therapy
  • toxins such as ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed anti-viral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxi
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published October 28, 1993.
  • DNA shuffling may be employed to alter the activities of the antiEphB4 antibody or fragments thereof (e.g. , an antibody or a fragment thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al, 1997, Curr.
  • the antibody can further be a binding-domain immunoglobulin fusion protein as described in U.S. Publication 20030118592, U.S. Publication 200330133939, and PCT Publication WO 02/056910, all to Ledbetter et ah, which are incorporated herein by reference in their entireties.
  • Anti-EphB4 antibodies of compositions and methods of the invention can be domain antibodies, e.g., antibodies containing the small functional binding units of antibodies, corresponding to the variable regions of the heavy (V R ) or light (V L ) chains of human antibodies.
  • domain antibodies include, but are not limited to, those available from Domantis Limited (Cambridge, UK) and Domantis Inc. (Cambridge, MA, USA) that are specific to therapeutic targets (see, for example, WO04/058821;
  • anti-EphB4 antibodies comprise a EphB4 functional binding unit and a Fc gamma receptor functional binding unit.
  • an anti-EphB4 domain antibody may comprise any one of, or any combination of the CDRs of the heavy or light chains of the #47 or #131 monoclonal antibodies.
  • an anti-EphB4 domain antibody may comprise CDR3 of #47 or #131 VHs together with any combination of the CDRs comprised by the heavy or light chains variable regions of the #47 or #131 monoclonal antibodies.
  • An anti-EphB4 domain antibody may also comprise CDR3 of #47 or #131 VLs together with any combiantion of the CDRs comprised by the heavy or light chains variable regions of the #47 or #131 monoclonal antibodies.
  • an anti-EphB4 domain antibody may comprise CDR3 of #47 or #131 VHs.
  • An anti-EphB4 domain antibody may also comprise CDR3 of #47 or #131 VLs. 5.11. DIABODIES
  • anti-EphB4 antibodies are “diabodies".
  • the term “diabodies” refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H -V L ).
  • V H heavy chain variable domain
  • V L light chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl Acad. Sci. USA, 90:6444-6448 (1993).
  • anti-EphB4 antibodies are Vaccibodies.
  • Vaccibodies are dimeric polypeptides. Each monomer of a vaccibody consists of a scFv with specificity for a surface molecule on APC connected through a hinge region and a C ⁇ 3 domain to a second scFv.
  • vaccibodies containing as one of the scFv's an anti-EphB4 antibody fragment may be used to juxtapose the cells to be destroyed and an effector cell that mediates ADCC. For example, see, Bogen et ah, U.S. Patent Application Publication No. 20040253238.
  • anti-EphB4 antibodies are linear antibodies.
  • Linear antibodies comprise a pair of tandem Fd segments (VH-CH 1 -VH-CH 1 ) which form a pair of antigen-binding regions.
  • Linear antibodies can be bispecif ⁇ c or monospecific. See, Zapata et ah, Protein Eng., 8(10):1057-1062 (1995).
  • an anti-EphB4 antibody is a parent antibody.
  • a "parent antibody” is an antibody comprising an amino acid sequence which may lack, or may be deficient in, one or more amino acid residues in or adjacent to one or more hypervariable regions thereof compared to an altered/mutant antibody as herein disclosed. Thus, the parent antibody may have a shorter hypervariable region than the corresponding hypervariable region of an antibody mutant as herein disclosed.
  • the parent polypeptide may comprise a native antibody sequence (i.e., a naturally occurring, including a naturally occurring allelic variant) or an antibody sequence with pre-existing amino acid sequence modifications (such as other insertions, deletions and/or substitutions) of a naturally occurring sequence.
  • the parent antibody may be a humanized antibody or a human antibody.
  • Antibody fragments comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof.
  • antibody fragments include Fab, Fab ' , F(ab ' ) 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecif ⁇ c antibodies formed from antibody fragments.
  • these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24:107-117 (1992) and Brennan et al, Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells.
  • the antibody fragments can be isolated from the antibody phage libraries discussed above.
  • Fab ' -SH fragments can also be directly recovered from E. coli and chemically coupled to form F(ab ' ) 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • F(ab ' ) 2 fragments can be isolated directly from recombinant host cell culture.
  • the antibody of choice is a single-chain Fv fragment (scFv). See, for example, WO 93/16185.
  • the antibody is not a Fab fragment.
  • Bispecif ⁇ c antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a single antigen. Other such antibodies may bind a first cell marker and further bind a second cell surface marker. An anti-EphB4 binding arm may also be combined with an arm which binds to a triggering molecule on a leukocyte such as a T cell receptor molecule (e.g., CD2 or CD3 ), or Fc receptors for IgG (Fc ⁇ R), so as to focus cellular defense mechanisms to the target cell. Bispecif ⁇ c antibodies may also be used to localize cytotoxic agents.
  • a T cell receptor molecule e.g., CD2 or CD3
  • Fc receptors for IgG Fc ⁇ R
  • bispecif ⁇ c antibodies possess an EphB4 binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon- ⁇ , vinca alkaloid, ricin A chain, methola-exate or radioactive isotope hapten).
  • cytotoxic agent e.g., saporin, anti-interferon- ⁇ , vinca alkaloid, ricin A chain, methola-exate or radioactive isotope hapten.
  • Bispecif ⁇ c antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab ' ): bispecific antibodies).
  • an anti-EphB4 antibody of compositions and methods of the invention is bispecif ⁇ c
  • the anti-EphB4 antibody may be human or humanized and may have specificity for human EphB4 and an epitope on a T cell or may be capable of binding to a human effector cell such as, for example, a monocyte/macrophage and/or a natural killer cell to effect cell death.
  • the present invention provides formulation of proteins comprising a variant Fc region. That is, a non naturally occurring Fc region, for example an Fc region comprising one or more non naturally occurring amino acid residues. Also encompassed by the variant Fc regions of present invention are Fc regions which comprise amino acid deletions, additions and/or modifications.
  • Fc region as used herein includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • IgA and IgM Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (C ⁇ 2 and C ⁇ 3) and the hinge between Cgammal (C ⁇ l) and Cgamma2 (C ⁇ 2).
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA).
  • the "EU index as set forth in Kabat” refers to the residue numbering of the human IgGl EU antibody as described in Kabat et al. supra.
  • Fc may refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein.
  • An Fc variant protein may be an antibody, Fc fusion, or any protein or protein domain that comprises an Fc region including, but not limited to, proteins comprising variant Fc regions, which are non naturally occurring variants of an Fc.
  • Polymorphisms have been observed at a number of Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and thus slight differences between the presented sequence and sequences in the prior art may exist.
  • the present invention encompasses Fc variant proteins which have altered binding properties for an Fc ligand (e.g., an Fc receptor, CIq) relative to a comparable molecule (e.g., a protein having the same amino acid sequence except having a wild type Fc region).
  • Fc ligand e.g., an Fc receptor, CIq
  • a comparable molecule e.g., a protein having the same amino acid sequence except having a wild type Fc region.
  • binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (K D ), dissociation and association rates (k off and k on respectively), binding affinity and/or avidity.
  • K D equilibrium dissociation constant
  • k off and k on respectively dissociation and association rates
  • affinity and/or avidity e.g., avidity
  • a binding molecule e.g., a Fc variant protein such as an antibody
  • K D equilibrium dissociation constant
  • k off and k on respectively binding
  • the affinities and binding properties of an Fc domain for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art for determining Fc-Fc ⁇ R interactions, i.e., specific binding of an Fc region to an Fc ⁇ R including but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE® analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration).
  • in vitro assay methods biochemical or immunological based assays
  • ELISA enzyme-linked immunoabsorbent assay
  • RIA radioimmunoassay
  • kinetics e.g., BIACORE® analysis
  • indirect binding assays e
  • These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • the Fc variant protein has enhanced binding to one or more Fc ligand relative to a comparable molecule.
  • the Fc variant protein has an affinity for an Fc ligand that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold greater than that of a comparable molecule.
  • the Fc variant protein has enhanced binding to an Fc receptor.
  • the Fc variant protein has enhanced binding to the Fc receptor Fc ⁇ RIIIA. In still another specific embodiment, the Fc variant protein has enhanced binding to the Fc receptor FcRn. In yet another specific embodiment, the Fc variant protein has enhanced binding to CIq relative to a comparable molecule. [0188]
  • the serum half-life of proteins comprising Fc regions may be increased by increasing the binding affinity of the Fc region for FcRn. In one embodiment, the Fc variant protein has enhanced serum half life relative to comparable molecule.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., Natural Killer (NK) cells, neutrophils, and macrophages
  • NK Natural Killer
  • macrophages a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins.
  • NK Natural Killer
  • IgG antibodies directed to the surface of target cells "arm" the cytotoxic cells and are absolutely required for such killing. Lysis of the target cell is extracellular, requires direct cell-to-cell contact, and does not involve complement.
  • ADCC activity the cell-mediated cytotoxicity resulting from the activity of an Fc fusion protein.
  • ADCC activity the cell-mediated cytotoxicity resulting from the activity of an Fc fusion protein.
  • the ability of any particular Fc variant protein to mediate lysis of the target cell by ADCC can be assayed.
  • an Fc variant protein of interest is added to target cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis is generally detected by the release of label (e.g.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the Fc variant protein of interest may also be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., 1998, Proc. Natl. Acad. ScL USA 95:652-656.
  • an Fc variant protein has enhanced ADCC activity relative to a comparable molecule.
  • an Fc variant protein has
  • an Fc variant protein has enhanced binding to the Fc receptor Fc ⁇ RIIIA and has enhanced ADCC activity relative to a comparable molecule.
  • the Fc variant protein has both enhanced ADCC activity and enhanced serum half life relative to a comparable molecule.
  • complement dependent cytotoxicity and “CDC” refer to the lysing of a target cell in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (CIq) to a molecule, an antibody for example, complexed with a cognate antigen.
  • CIq first component of the complement system
  • a CDC assay e.g. as described in Gazzano-Santoro et al., 1996, J. Immunol. Methods, 202:163, may be performed.
  • an Fc variant protein has enhanced CDC activity relative to a comparable molecule.
  • an Fc variant protein has CDC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold or at least 50 fold or at least 100 fold greater than that of a comparable molecule.
  • the Fc variant protein has both enhanced CDC activity and enhanced serum half life relative to a comparable molecule.
  • the present invention provides formulations, wherein the Fc region comprises a non naturally occurring amino acid residue at one or more positions selected from the group consisting of 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443 as numbered by the EU index as set forth in Kabat.
  • the Fc region may comprise a non naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Patents 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040217, WO 05/092925 and WO 06/020114).
  • the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non naturally occurring amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241 R.
  • the Fc region comprises at least one non naturally occurring amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 2
  • the Fc region may comprise additional and/or alternative non naturally occurring amino acid residues known to one skilled in the art (see, e.g., U.S. Patents 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).
  • the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least a non naturally occurring amino acid at one or more positions selected from the group consisting of 239, 330 and 332, as numbered by the EU index as set forth in Kabat.
  • the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat.
  • the Fc region may further comprise addtional non naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat.
  • the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat and at least one non naturally occurring amino acid at one or more positions are selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.
  • the Fc variants of the present invention may be combined with other known Fc variants such as those disclosed in Ghetie et al., 1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol 147:2657- 2662; Lund et al, 1992, MoI Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537- 1543; Hutchins et al., 1995, Proc Natl Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett.
  • Fc regions which comprise deletions, additions and/or modifications. Still other modifications/substitutions/additions/deletions of the Fc domain will be readily apparent to one skilled in the art.
  • amino acid substitutions and/or deletions can be generated by mutagenesis methods, including, but not limited to, site- directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985) ), PCR mutagenesis (Higuchi, in “PCR Protocols: A Guide to Methods and Applications", Academic Press, San Diego, pp. 177-183 (1990)), and cassette mutagenesis (Wells et al., Gene 34:315-323 (1985)).
  • site-directed mutagenesis is performed by the overlap-extension PCR method (Higuchi, in "PCR Technology: Principles and Applications for DNA Amplification", Stockton Press, New York, pp. 61-70 (1989)).
  • the technique of overlap-extension PCR can also be used to introduce any desired mutation(s) into a target sequence (the starting DNA).
  • the first round of PCR in the overlap- extension method involves amplifying the target sequence with an outside primer (primer 1) and an internal mutagenesis primer (primer 3), and separately with a second outside primer (primer 4) and an internal primer (primer 2), yielding two PCR segments (segments A and B).
  • the internal mutagenesis primer (primer 3) is designed to contain mismatches to the target sequence specifying the desired mutation(s).
  • the products of the first round of PCR (segments A and B) are amplified by PCR using the two outside primers (primers 1 and 4).
  • the resulting full-length PCR segment (segment C) is digested with restriction enzymes and the resulting restriction fragment is cloned into an appropriate vector.
  • the starting DNA e.g., encoding an Fc fusion protein, an antibody or simply an Fc region
  • the primers are designed to reflect the desired amino acid substitution.
  • an Fc variant protein comprises one or more engineered glycoforms, i.e., a carbohydrate composition that is covalently attached to the molecule comprising an Fc region.
  • Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function.
  • Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example DI N-acetylglucosaminyltransferase III (GnTIl 1), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed.
  • one or more enzymes for example DI N-acetylglucosaminyltransferase III (GnTIl 1)
  • GnTIl 1 DI N-acetylglucosaminyltransferase III
  • the glycosylation of antibodies utilized in accordance with the invention is modified.
  • an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • One or more amino acid substitutions can also be made that result in elimination of a glycosylation site present in the Fc region (e.g., Asparagine 297 of IgG).
  • aglycosylated antibodies may be produced in bacterial cells which lack the necessary glycosylation machinery.
  • An antibody can also be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GIcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. See, for example, Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat.
  • an anti-EphB4 antibody of the invention may be desirable to modify an anti-EphB4 antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating malignancies, for example.
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and/or antibody-dependent cellular cytotoxicity (ADCC). See, Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, B., J. Immunol., 148:2918-2922 (1992).
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al , Cancer Research, 53:2560-2565 (1993).
  • An antibody can also be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See, Stevenson et al, Anti-Cancer Drug Design, 3:219-230 (1989).
  • the anti-EphB4 antibody can be produced on a commercial scale using methods that are well-known in the art for large scale manufacturing of antibodies. For example, this can be accomplished using recombinant expressing systems such as, but not limited to, those described below. 5.21. RECOMBINANT EXPRESSION SYSTEMS
  • Recombinant expression of an antibody or variant thereof generally requires construction of an expression vector containing a polynucleotide that encodes the antibody.
  • a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well-known in the art. See, e.g., U.S. Patent No. 6,331,415, which is incorporated herein by reference in its entirety.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
  • the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a portion thereof, or a heavy or light chain CDR, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication Nos.
  • variable domain of the antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
  • anti-EphB4 antibodies can be made using targeted homologous recombination to produce all or portions of the anti-EphB4 antibodies (see, U.S. Patent Nos. 6,063,630, 6,187,305, and 6,692,737). In certain embodiments, anti-EphB4 antibodies can be made using random recombination techniques to produce all or portions of the anti-EphB4 antibodies (see, U.S. Patent Nos. 6,361,972, 6,524,818, 6,541,221, and 6,623,958).
  • Anti-EphB4 antibodies can also be produced in cells expressing an antibody from a genomic sequence of the cell comprising a modified immunoglobulin locus using Cre- mediated site-specific homologous recombination (see, U.S. Patent No. 6,091,001).
  • the host cell line may be derived from human or nonhuman species including but not limited to mouse, and Chinese hamster. Where human or humanized antibody production is desired, the host cell line should be a human cell line. These methods may advantageously be used to engineer stable cell lines which permanently express the antibody molecule. [0207] Once the expression vector is transferred to a host cell by conventional techniques, the transfected cells are then cultured by conventional techniques to produce an antibody.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention or fragments thereof, or a heavy or light chain thereof, or portion thereof, or a single-chain antibody of the invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • a variety of host-expression vector systems may be utilized to express an anti- EphB4 antibody or portions thereof that can be used in the engineering and generation of anti-EphB4 antibodies (see, e.g., U.S. Patent No. 5,807,715).
  • mammalian cells such as Chinese hamster ovary cells (CHO)
  • CHO Chinese hamster ovary cells
  • a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene, 45:101 (1986); and Cockett et al., Bio/Technology, 8:2 (1990)).
  • a host cell strain may be chosen which modulates the expression of inserted antibody sequences, or modifies and processes the antibody gene product in the specific fashion desired. Such modifications (e.g. , glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the antibody or portion thereof expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeIa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), CRL7O3O and HsS78Bst cells.
  • human cell lines developed by immortalizing human lymphocytes can be used to recombinantly produce monoclonal human anti-EphB4 antibodies.
  • the human cell line PER.C6. (Crucell, Netherlands) can be used to recombinantly produce monoclonal human anti-EphB4 antibodies.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such an antibody is to be produced, for the generation of pharmaceutical compositions comprising an anti-EphB4 antibody, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al, EMBO, 12:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, 1989, J. Biol Chem., 24:5503-5509 (1989)); and the like.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione- S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to glutathione-agarose affinity matrix followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to introduce athrombin and/or factor Xa protease cleavage sites into the expressed polypeptide so that the cloned target gene product can be released from the GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into non- essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter).
  • a number of virus based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome ⁇ e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts ⁇ e.g., see, Logan & Shenk, Proc.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon should generally be in frame with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. ⁇ see, e.g., Bittner et ah, Methods in Enzymol, 153:51-544(1987)).
  • Stable expression can be used for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express the antibody molecule may be generated.
  • Host cells can be transformed with an appropriately engineered vector comprising expression control elements ⁇ e.g., promoter, enhancer, transcription terminators, polyadenylation sites, etc.), and a selectable marker gene.
  • expression control elements e.g., promoter, enhancer, transcription terminators, polyadenylation sites, etc.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells that stably integrated the plasmid into their chromosomes to grow and form foci which in turn can be cloned and expanded into cell lines. Plasmids that encode an anti-EphB4 antibody can be used to introduce the gene/cDNA into any cell line suitable for production in culture.
  • a number of selection systems may be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al, Cell, 11 :223 (1977)), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. ScL USA, 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al, Cell, 22:8-17 (1980)) genes can be employed in tk " , hgprt " or aprT cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al, Proc. Natl. Acad. Sci. USA, 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3 : 87-95 (1991);
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see, Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. Academic Press, New York (1987)).
  • vector amplification for a review, see, Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. Academic Press, New York (1987)).
  • a marker in the vector system expressing antibody is amplif ⁇ able
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et ah, MoI. Cell. Biol, 3:257 (1983)).
  • Antibody expression levels may be amplified through the use recombinant methods and tools known to those skilled in the art of recombinant protein production, including technologies that remodel surrounding chromatin and enhance transgene expression in the form of an active artificial transcriptional domain.
  • the host cell may be co-transfected with two expression vectors, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical or different selectable markers.
  • a single vector which encodes, and is capable of expressing, both heavy and light chain polypeptides may also be used.
  • the light chain should be placed 5' to the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:562- 65 (1986); and Kohler, 1980, Proc. Natl. Acad. Sci. USA, 77:2197 (1980)).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigens Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigens Protein A or Protein G, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification. 5.21.1. ANTIBODY PURIFICATION AND ISOLATION
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et ah,
  • Bio/Technology, 10:163-167 (1992) describe a procedure for isolating antibodies which are secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody mutant is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a commercially available protein concentration filter for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and/or affinity chromatography either alone or in combination with other purification steps.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody mutant.
  • Protein A can be used to purify antibodies that are based on human ⁇ l, ⁇ 2, or ⁇ 4 heavy chains (Lindmark et ah, J. Immunol. Methods, 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et ah, EMBO J, 5:15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the Bakerbond ABX resin J. T. Baker, Phillipsburg, NJ
  • Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, andperformed at low salt concentrations (e.g., from about 0-0.25 M salt).
  • An anti-EphB4 antibody used in compositions and methods of the invention may be a human antibody or a humanized antibody that may mediate apoptosis and/or human ADCC, or can be selected from known anti-EphB4 antibodies that may mediate apoptosis and/or human ADCC.
  • anti-EphB4 antibodies can be chimeric antibodies.
  • an anti-EphB4 antibody can be a monoclonal human, humanized, or chimeric anti-EphB4 antibody.
  • An anti-EphB4 antibody used in compositions and methods of the invention may be a human antibody or a humanized antibody of the IgGl or IgG3 human isotype or any IgGl or IgG3 allele found in the human population.
  • an anti-EphB4 antibody used in compositions and methods of the invention can be a human antibody or a humanized antibody of the IgG2 or IgG4 human isotype or any IgG2 or IgG4 allele found in the human population.
  • murine antibodies #47 and #131 as described herein or other commercially available anti-EphB4 antibodies can be chimerized, humanized, or made into human antibodies.
  • known anti-EphB4 antibodies that can be used include, but are not limited to, IDl (Ig2a/kappa) (Abnova, Taiwan) and 38-1E (IgG2a/ kappa) (Inada T. et al, Blood, 89(8):2757-65 (1997)).
  • the antibody is an isotype switched variant of a known antibody (e.g., to an IgGl or IgG3 human isotype) such as those described above.
  • Binding assays can be used to identify antibodies that bind the human EphB4 antigen. Binding assays may be performed either as direct binding assays or as competition- binding assays. Binding can be detected using for example standard ELISA, flow cytometry or immunocytochemistry assays. In a direct binding assay, a candidate antibody is tested for binding to human EphB4 antigen. In certain embodiments, the screening assays comprise, in a second step, determining the ability to cause cell death or apoptosis of cells expressing human EphB4.
  • Competition-binding assays assess the ability of a candidate antibody to compete with a known anti-EphB4 antibody or other compound that binds human EphB4.
  • the human EphB4 antigen is contacted with a candidate antibody under conditions that allow binding of the candidate antibody to the human EphB4 antigen.
  • the binding may take place in solution or on a solid surface.
  • the candidate antibody may have been previously labeled for detection. Any detectable compound can be used for labeling, such as ,but not limited to, a luminescent, fluorescent, or radioactive isotope or group containing same, or a nonisotopic label, such as an enzyme or dye.
  • a candidate antibody is evaluated for its ability to inhibit or displace the binding of a known anti-EphB4 antibody (or other compound) to the human EphB4 antigen.
  • a labeled known binder of EphB4 may be mixed with the candidate antibody, and placed under conditions in which the interaction between them would normally occur, with and without the addition of the candidate antibody.
  • the amount of labeled known binder of EphB4 that binds the human EphB4 may be compared to the amount bound in the presence or absence of the candidate antibody.
  • the binding assay is carried out with one or more components immobilized on a solid surface to facilitate antibody antigen complex formation and detection.
  • the solid support could be, but is not restricted to, polyvinylidene fluoride polycarbonate, polystyrene, polypropylene, polyethylene, glass, nitrocellulose, dextran, nylon, polyacrylamide and agarose.
  • the support configuration can include beads, membranes, microparticles, the interior surface of a reaction vessel such as a microtiter plate, test tube or other reaction vessel.
  • the immobilization of human EphB4, or other component can be achieved through covalent or non-covalent attachments.
  • the attachment may be indirect, i.e., through an attached antibody.
  • the human EphB4 antigen and negative controls are tagged with an epitope, such as glutathione S-transferase (GST) so that the attachment to the solid surface can be mediated by a commercially available antibody such as anti-GST (Santa Cruz Biotechnology).
  • GST glutathione S-transferase
  • such an affinity binding assay may be performed using the human EphB4 antigen which is immobilized to a solid support.
  • the non-mobilized component of the binding reaction in this case the candidate anti-EphB4 antibody, is labeled to enable detection.
  • labeling methods are available and may be used, such as luminescent, chromophore, fluorescent, or radioactive isotope or group containing same, and nonisotopic labels, such as enzymes or dyes.
  • the candidate anti-EphB4 antibody is labeled with a fluorophore such as fluorescein isothiocyanate (FITC, available from Sigma Chemicals, St. Louis).
  • FITC fluorescein isothiocyanate
  • Such an affinity binding assay may be performed using the human EphB4 antigen immobilized on a solid surface.
  • Anti-EphB4 antibodies are then incubated with the antigen and the specific binding of antibodies is detected by methods known in the art including, but not limited to, BiaCore Analyses, ELISA, FMET and RIA methods.
  • the label remaining on the solid surface may be detected by any detection method known in the art. For example, if the candidate anti-EphB4 antibody is labeled with a fluorophore, a fluorimeter may be used to detect complexes.
  • the human EphB4 antigen can be added to binding assays in the form of intact cells that express human EphB4 antigen, or isolated membranes containing human EphB4 antigen.
  • direct binding to human EphB4 antigen may be assayed in intact cells in culture or in animal models in the presence and absence of the candidate anti-EphB4 antibody.
  • a labeled candidate anti-EphB4 antibody may be mixed with cells that express human EphB4 antigen, or with crude extracts obtained from such cells, and the candidate anti-EphB4 antibody may be added. Isolated membranes may be used to identify candidate anti-EphB4 antibodies that interact with human EphB4.
  • cells may be genetically engineered to express human EphB4 antigen.
  • Membranes can be harvested by standard techniques and used in an in vitro binding assay.
  • Labeled candidate anti-EphB4 antibody ⁇ e.g. , fluorescent labeled antibody
  • Soluble human EphB4 antigen may also be recombinantly expressed and utilized in non-cell based assays to identify antibodies that bind to human EphB4 antigen.
  • the recombinantly expressed human EphB4 polypeptides can be used in the non-cell based screening assays. Peptides corresponding to one or more of the binding portions of human EphB4 antigen, or fusion proteins containing one or more of the binding portions of human EphB4 antigen can also be used in non-cell based assay systems to identify antibodies that bind to portions of human EphB4 antigen.
  • the recombinantly expressed human EphB4 is attached to a solid substrate such as a test tube, microtiter well or a column, by means well-known to those in the art (see, Ausubel et al., supra). The test antibodies are then assayed for their ability to bind to human EphB4 antigen.
  • the binding reaction may also be carried out in solution.
  • the labeled component is allowed to interact with its binding partner(s) in solution. If the size differences between the labeled component and its binding partner(s) permit such a separation, the separation can be achieved by passing the products of the binding reaction through an ultraf ⁇ lter whose pores allow passage of unbound labeled component but not of its binding partner(s) or of labeled component bound to its partner(s). Separation can also be achieved using any reagent capable of capturing a binding partner of the labeled component from solution, such as an antibody against the binding partner and so on.
  • a phage library can be screened by passing phage from a continuous phage display library through a column containing purified human EphB4 antigen, or derivative, analog, fragment, or domain, thereof, linked to a solid phase, such as plastic beads.
  • a solid phase such as plastic beads.
  • Phage isolated from the column can be cloned and affinities can be measured directly. Knowing which antibodies and their amino acid sequences confer the strongest binding to human EphB4 antigen, computer models can be used to identify the molecular contacts between EphB4 antigen and the candidate antibody.
  • the solid support is membrane containing human EphB4 antigen attached to a microtiter dish.
  • Candidate antibodies for example, can bind cells that express library antibodies cultivated under conditions that allow expression of the library members in the microtiter dish. Library members that bind to the human EphB4 are harvested. Such methods, are generally described by way of example in Parmley and Smith, 1988, Gene, 73:305-318; Fowlkes et al, 1992, BioTechniques, 13:422-427; PCT
  • Antibodies identified as binding to human EphB4 antigen can be of any of the types or modifications of antibodies described above. 5.22.2. SCREENING OF ANTIBODIES FOR HUMAN ADCC EFFECTOR FUNCTION
  • Antibodies of the human IgG class which have functional characteristics such a long half-life in serum and the ability to mediate various effector functions are used in certain embodiments of the invention ⁇ Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 1 (1995)).
  • the human IgG class antibody is further classified into the following 4 subclasses: IgGl, IgG2, IgG3 and IgG4.
  • C ⁇ 2 domain the second domain of C region of the antibody
  • C ⁇ 2 domain several amino acid residues in the hinge region and the second domain of C region (hereinafter referred to as "C ⁇ 2 domain") of the antibody are important ⁇ Eur. J. Immunol, 23, 1098 (1993), Immunology, 86, 319 (1995), Chemical Immunology, 65, 88 (1997)) and that a sugar chain in the C ⁇ 2 domain ⁇ Chemical Immunology, 65, 88 (1997)) is also important.
  • Anti-EphB4 antibodies can be modified with respect to effector function, e.g., so as to enhance ADCC and/or complement dependent cytotoxicity (CDC) of the antibody.
  • CDC complement dependent cytotoxicity
  • This may be achieved by introducing one or more amino acid substitutions in the Fc region of an antibody. Cysteine residue(s) may also be introduced in the Fc region, allowing for interchain disulfide bond formation in this region. In this way a homodimeric antibody can be generated that may have improved internalization capability and or increased complement- mediated cell killing and ADCC (Caron et al, J. Exp. Med. , 176 : 1191 - 1195 ( 1992) and
  • Heterobifunctional cross-linkers can also be used to generate homodimeric antibodies with enhanced anti-tumor activity (Wolff et al , Cancer Research, 53:2560-2565 (1993)).
  • Antibodies can also be engineered to have two or more Fc regions resulting in enhanced complement lysis and ADCC capabilities (Stevenson et al, Anti-Cancer Drug Design, (3)219-230 (1989)).
  • Fc ⁇ RI CD64
  • Fc ⁇ RII CD32
  • Fc ⁇ RIII CD 16
  • Fc ⁇ RIV Fc ⁇ RIV
  • Fc ⁇ RII and Fc ⁇ RIII are further classified into Fc ⁇ RIIa and Fc ⁇ RIIb, and Fc ⁇ RIIIa and Fc ⁇ RIIIb, respectively.
  • Fc ⁇ R is a membrane protein belonging to the immunoglobulin superfamily
  • Fc ⁇ RII, Fc ⁇ RIII, and Fc ⁇ RIV have an ⁇ chain having an extracellular region containing two immunoglobulin-like domains
  • Fc ⁇ RI has an ⁇ chain having an extracellular region containing three immunoglobulin-like domains, as a constituting component
  • the ⁇ chain is involved in the IgG binding activity.
  • Fc ⁇ RI and Fc ⁇ RIII have a ⁇ chain or ⁇ chain as a constituting component which has a signal transduction function in association with the ⁇ chain ⁇ Annu. Rev. Immunol, 18, 709 (2000), Annu. Rev. Immunol, 19, 275
  • an in vitro ADCC assay can be used, such as that described in U.S. Patent No. 5,500,362 or 5,821,337.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • the ability of any particular antibody to mediate lysis of the target cell by complement activation and/or ADCC can be assayed.
  • the cells of interest are grown and labeled in vitro; the antibody is added to the cell culture in combination with immune cells which may be activated by the antigen antibody complexes; i.e., effector cells involved in the ADCC response.
  • the antibody can also be tested for complement activation. In either case, cytolysis of the target cells is detected by the release of label from the lysed cells.
  • antibodies can be screened using the patient's own serum as a source of complement and/or immune cells. The antibodies that are capable of mediating human ADCC in the in vitro test can then be used therapeutically in that particular patient.
  • ADCC activity of the molecule of interest may also be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et ah, Proc. Natl. Acad. Sci. (USA) 95:652- 656 (1998).
  • Antibodies of the present invention may be capable or may have been modified to have the ability of inducing ADCC and/or CDC.
  • Assays to determine ADCC function can be practiced using human effector cells to assess human ADCC function. Such assays may also include those intended to screen for antibodies that induce, mediate, enhance, block cell death by necrotic and/or apoptotic mechanims.
  • Such methods including assays utilizing viable dyes, methods of detecting and analyzing caspases, and assays measuring DNA breaks can be used to assess the apoptotic activity of cells cultured in vitro with an anti- EphB4 antibody of interest.
  • Annexin V or TdT-mediated dUTP nick-end labeling (TUNEL) assays can be carried out as described in Decker et al. , Blood (USA) 103:2718-2725 (2004) to detect apoptotic activity.
  • the TUNEL assay involves culturing the cell of interest with fluorescein-labeled dUTP for incorporation into DNA strand breaks. The cells are then processed for analysis by flow cytometry.
  • the Annexin V assay detects the appearance of phosphatidylserine (PS) on the outside of the plasma membrane of apoptotic cells using a fluorescein-conjugated Annexin V that specifically recognizes the exposed PS molecules.
  • a viable dye such as propidium iodide can be used to exclude late apoptotic cells.
  • the cells are stained with the labeled Annexin V and are analyzed by flow cytometry.
  • therapeutic agents or toxins can be conjugated to chimerized, human, or humanized anti-EphB4 antibodies for use in compositions and methods of the invention.
  • these conjugates can be generated as fusion proteins.
  • therapeutic agents and toxins include, but are not limited to, members of the enediyne family of molecules, such as calicheamicin and esperamicin.
  • Chemical toxins can also be taken from the group consisting of duocarmycin ⁇ see, e.g., U.S. Patent No. 5,703,080 and U.S. Patent No.
  • methotrexate methotrexate
  • doxorubicin methotrexate
  • melphalan chlorambucil
  • ARA-C vindesine
  • mitomycin C cis-platinum
  • etoposide bleomycin and 5-fluorouracil.
  • chemotherapeutic agents also include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (Ara-C), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see, U.S. Patent No. 4,675,187), Melphalan, and other related nitrogen mustards.
  • anti-EphB4 antibodies are conjugated to a cytostatic, cytotoxic or immunosuppressive agent wherein the cytotoxic agent is selected from the group consisting of an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid.
  • a cytostatic, cytotoxic or immunosuppressive agent wherein the cytotoxic agent is selected from the group consisting of an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid.
  • the cytotoxic agent is paclitaxel, docetaxel, CC- 1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin- 10, echinomycin, combretastatin, calicheamicin, maytansine, DM-I, auristatin E, AEB, AEVB, AEFP, MMAE (see,US Patent Publication No. US 2005-0238649 Al) , or netropsin.
  • the cytotoxic agent of an anti-EphB4 antibody- cytotoxic agent conjugate of the invention is an anti-tubulin agent.
  • the cytotoxic agent is selected from the group consisting of a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, and a dolastatin.
  • the cytotoxic agent is vincristine, vinblastine, vindesine, vinorelbine, VP- 16, camptothecin, paclitaxel, docetaxel, epithilone A, epithilone B, nocodazole, coichicine, colcimid, estramustine, cemadotin, discodermolide, maytansine, DM-I, AEFP, auristatin E, AEB, AEVB, AEFP, MMAE or eleutherobin.
  • an anti-EphB4 antibody is conjugated to the cytotoxic agent via a linker, wherein the linker is peptide linker.
  • an anti-EphB4 antibody is conjugated to the cytotoxic agent via a linker, wherein the linker is a val-cit linker, a phe-lys linker, a hydrazone linker, or a disulfide linker.
  • Any methods known to one of skill in the art may be utilized for the generation of conjugated human, humanized and chimeric anti-EphB4 antibodies, for example the methods disclosed in International Publication Nos. WO 07/103288, WO 07/008848, WO 07/008603, WO 06/132670, WO 05/082023, and WO 05/077090.
  • the anti-EphB4 antibody of an anti-EphB4 antibody- cytotoxic agent conjugate is conjugated to the cytotoxic agent via a linker, wherein the linker is hydrolyzable at a pH of less than 5.5. In a specific embodiment the linker is hydrolyzable at a pH of less than 5.0.
  • the anti-EphB4 antibody of an anti-EphB4 antibody- cytotoxic agent conjugate is conjugated to the cytotoxic agent via a linker, wherein the linker is cleavable by a protease.
  • the protease is a lysosomal protease.
  • the protease is, inter alia, a membrane-associated protease, an intracellular protease, or an endosomal protease.
  • plant toxins such as ricin, abrin, modeccin, botulina, and diphtheria toxins.
  • combinations of the various toxins could also be coupled to one antibody molecule thereby accommodating variable cytotoxicity.
  • Illustrative of toxins which are suitably employed in combination therapies of the invention are ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed anti-viral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example, Pastan et ah, Cell, 47:641 (1986), and Goldenberg et al, Cancer Journal for Clinicians, 44
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published October 28, 1993.
  • Suitable toxins and chemotherapeutic agents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995), and in Goodman And Gilman's The Pharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985). Other suitable toxins and/or chemotherapeutic agents are known to those of skill in the art.
  • the present invention further encompasses antibodies (including antibody fragments or variants thereof) comprising or conjugated to a radiocative agent suitable for diagnostic purposes.
  • suitable radioactive materials include, but are not limited to, iodine (1211, 1231, 1251, 1311), carbon (14C), sulfur (35S), tritium (3H), indium (11 Hn, 112In, 113mln, 115mln), technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (135Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 9OY, 47Sc, 186Re, 188Re, 142Pr, 105Rh, and 97Ru.
  • an anti-EphB4 antibody of the invention may be coupled or conjugated to a radioactive metal ion utilized for therapeutic purposes.
  • radioactive ions examples include, but are not limited to, alpha-emitters such as 213Bi, or other radioisotopes such as 103Pd, 135Xe, 1311, 68Ge, 57Co, 65Zn, 85Sr, 32P, 35S, 9OY, 153Sm, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113 Sn, 9OY, 117Tin, 186Re, 188Re and 166Ho.
  • an antibody or fragment thereof is attached to macrocyclic chelators that chelate radiometal ions, including but not limited to, 177Lu, 9OY, 166Ho, and 153Sm, to polypeptides.
  • the macrocyclic chelator is 1,4,7,10-tetraazacyclod- odecane-N,N',N",N'"-tetraacetic acid (DOTA).
  • DOTA is attached to the an antibody of the invention or fragment thereof via a linker molecule.
  • linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res 4(10):2483-90, 1998; Peterson et al., Bioconjug Chem 10(4):553-7, 1999; and Zimmerman et al., Nucl Med Biol 26(8):943-50, 1999 which are hereby incorporated by reference in their entirety.
  • An anti-EphB4 antibody of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g. , a peptidyl chemotherapeutic agent, see, WO81/01145) to an active anti-cancer drug.
  • a prodrug e.g. , a peptidyl chemotherapeutic agent, see, WO81/01145
  • the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
  • Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as ⁇ -galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs
  • Antibodies with enzymatic activity can be used as well to convert the prodrugs into free active drugs (see, e.g., Massey, Nature 328:457-458 (1987)).
  • Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme as desired to portions of a human affected by a malignancy.
  • Antibodies of this invention may be covalently bound to the enzymes by techniques well-known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Fusion proteins comprising at least the antigen-binding region of an anti-EphB4 antibody linked to at least a functionally active portion of an enzyme may also be constructed using recombinant DNA techniques well-known in the art (see, e.g., Neuberger et al, Nature, 312:604-608 (1984)).
  • Covalent modifications of an anti-EphB4 antibody are included within the scope of this invention. They may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of an anti-EphB4 antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
  • Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
  • ⁇ -haloacetates and corresponding amines
  • iodo-reagents may also be used.
  • Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ -(5- imidozoyl)prop ionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para- bromophenacyl bromide also is useful; the reaction can be performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing ⁇ -amino-containing residues and/or ⁇ -amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues generally requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the ⁇ -amino groups of lysine as well as the arginine epsilon-amino group.
  • tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125 I or 131 I to prepare labeled proteins for use in radioimmunoassay.
  • R and R' are different alkyl groups, such as l-cyclohexyl-3-(2-morpholinyl— 4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide.
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated form of these residues falls within the scope of this invention.
  • Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation.
  • the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.
  • cancer or one or more symptoms thereof may be prevented, treated, managed or ameliorated by the administration of an anti-EphB4 mAb in combination with the administration of one or more therapies such as, but not limited to, chemotherapies, radiation therapies, hormonal therapies, and/or biological therapies/immunotherapies .
  • therapies such as, but not limited to, chemotherapies, radiation therapies, hormonal therapies, and/or biological therapies/immunotherapies .
  • methods of the invention encompass the administration of one or more angiogenesis antagonists such as but not limited to:
  • Angiostatin (plasminogen fragment); antiangiogenic antithrombin III; Angiozyme; ABT-627; Bay 12-9566; Benefm; Bevacizumab; BMS-275291; cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagen XVIII fragment); Fibronectin fragment; Gro-beta; Halofuginone; Heparinases; Heparin hexasaccharide fragment; HMV833; Human chorionic gonadotropin (hCG); IM-862;
  • Interferon alpha/beta/gamma Interferon inducible protein (IP-IO); Interleukin-12; Kringle 5 (plasminogen fragment); Marimastat; Metalloproteinase inhibitors (TIMPs); 2- Methoxyestradiol; MMI 270 (CGS 27023 A); MoAb IMC-ICl 1; Neovastat; NM-3; Panzem; PI-88; Placental ribonuclease inhibitor; Plasminogen activator inhibitor; Platelet factor-4 (PF4); Prinomastat; Prolactin 16kD fragment; Proliferin-related protein (PRP); PTK 787/ZK 222594; Retinoids; Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SUl 1248; Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1 (TSP-I); TNP-
  • methods of the invention encompass the administration of one or more immunomodulatory agents, such as but not limited to, chemotherapeutic agents and non-chemotherapeutic immunomodulatory agents.
  • chemotherapeutic agents include methotrexate, cyclosporin A, leflunomide, cisplatin, ifosfamide, taxanes such as taxol and paclitaxol, topoisomerase I inhibitors (e.g., CPT-I l, topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, temodal, cytochalasin B, gramicidin D, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
  • non-chemotherapeutic immunomodulatory agents include, but are not limited to, anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH IH (Ilex)), anti-CD2 antibodies (e.g., IL
  • anti-CD 11 a antibodies e.g. , Xanelim (Genentech)
  • anti-B7 antibodies e.g. , IDEC-114)
  • anti-cytokine receptor antibodies e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)
  • anti-IL-4 receptor antibodies e.g., anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, and anti-IL-12 receptor antibodies
  • anti-cytokine antibodies e.g., anti-IFN antibodies, anti-TNF- ⁇ antibodies, anti-IL-l ⁇ antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8 (Abgenix)), anti-IL-12 antibodies and anti-IL-23 antibodies
  • CTLA4-immunoglobulin LFA-3TIP (Biogen, International Publication No.
  • soluble cytokine receptors e.g., the extracellular domain of a TNF- ⁇ receptor or a fragment thereof, the extracellular domain of an IL- l ⁇ receptor or a fragment thereof, and the extracellular domain of an IL-6 receptor or a fragment thereof
  • cytokines or fragments thereof e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-11, IL-12, IL-15, IL-23, TNF- ⁇ , TNF- ⁇ , interferon (IFN)- ⁇ , IFN- ⁇ , IFN- ⁇ , and GM-CSF
  • anti- cytokine antibodies e.g., anti-IL-2 antibodies, anti-IL-4 antibodies, anti-IL-6 antibodies, anti-IL-10 antibodies, anti-IL-12 antibodies, anti-IL-15 antibodies, anti-TNF
  • an immunomodulatory agent is an immunomodulatory agent other than a chemotherapeutic agent.
  • an immunomodulatory agent is an immunomodulatory agent other than a cytokine or hemapoietic such as IL-I, IL-2, IL-4, IL- 12, IL- 15, TNF, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , M-CSF, G- CSF, IL-3 or erythropoietin.
  • an immunomodulatory agent is an agent other than a chemotherapeutic agent and a cytokine or hemapoietic factor.
  • methods of the invention encompass the administration of one or more anti-inflammatory agents, such as but not limited to, non- steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, beta- agonists, anticholingeric agents, and methyl xanthines.
  • NSAIDs non- steroidal anti-inflammatory drugs
  • beta- agonists beta- agonists
  • anticholingeric agents methyl xanthines
  • NSAIDs include, but are not limited to, aspirin, ibuprofen, celecoxib (CELEBREXTM), diclofenac (VOLTARENTM), etodolac (LODINETM), fenoprofen (NALFONTM), indomethacin (INDOCINTM), ketoralac (TORADOLTM), oxaprozin (DAYPROTM), nabumentone (RELAFENTM), sulindac (CLINORILTM), tolmentin (TOLECTINTM), rofecoxib (VIOXXTM), naproxen (ALEVETM, NAPROSYNTM), ketoprofen (ACTRONTM) and nabumetone (RELAFENTM).
  • NSAIDs function by inhibiting a cyclooxygenase enzyme (e.g., COX-I and/or COX-2).
  • a cyclooxygenase enzyme e.g., COX-I and/or COX-2
  • steroidal anti-inflammatory drugs include, but are not limited to, glucocorticoids, dexamethasone (DECADRONTM), cortisone, hydrocortisone, prednisone (DELTASONETM), prednisolone, triamcinolone, azulfidine, and eicosanoids such as prostaglandins, thromboxanes, and leukotrienes.
  • methods of the invention encompass the administration of one or more antiviral agents (e.g., amantadine, ribavirin, rimantadine, acyclovir, famciclovir, foscarnet, ganciclovir, trifluridine, vidarabine, didanosine, stavudine, zalcitabine, zidovudine, interferon), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), anti-emetics (e.g., alprazolam, dexamethoasone, domperidone, dronabinol, droperidol, granisetron, haloperidol, haloperidol, iorazepam, methylprednisolone, metoclopramide, nabilone, ondansetron, prochlor
  • antiviral agents e
  • anti-cancer agents that can be used in various embodiments of the invention, including pharmaceutical compositions and dosage forms and kits, include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine
  • ALL-TK antagonists altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol
  • Simvastatin, and Atorvastatin loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonaf ⁇ de; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk;
  • plasminogen activator inhibitor platinum complex; platinum compounds; platinum-triamine complex; porf ⁇ mer sodium; porf ⁇ romycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase
  • Additional anti-cancer drugs are 5-fluorouracil and leucovorin. These two agents may be useful when used in methods employing thalidomide and a topoisomerase inhibitor.
  • an anti-cancer agent is not a chemotherapeutic agent.
  • the present invention also comprises the administration of an anti-EphB4 mAb in combination with the administration of one or more therapies such as, but not limited to, anti-cancer agents such as those disclosed in Table 1, for the treatment of breast, ovary, melanoma, prostate, colon and lung cancers as described above.
  • therapies such as, but not limited to, anti-cancer agents such as those disclosed in Table 1, for the treatment of breast, ovary, melanoma, prostate, colon and lung cancers as described above.
  • the dosages and/or the frequency of administration listed in Table 1 may be decreased. Table 1.
  • Anti-cancer agents such as those disclosed in Table 1, for the treatment of breast, ovary, melanoma, prostate, colon and lung cancers as described above.
  • Lupron® subcutaneous mark
  • ketoconazole Cream 2% cream applied once or
  • Agent prednisone Oral Initial dosage may vary from
  • estramustine Oral 14 mg/ kg of body weight Daily given in 3 or 4 phosphate (capsule) (i.e. one 140 mg capsule for divided doses sodium each 10 kg or 22 Ib of body
  • VP- 16 (100 mg) dacarbazine Intravenous 2-4.5 mg/kg Once a day for 10 days.
  • DTIC-Dome® May be repeated at 4 week intervals polifeprosan 20 wafer placed 8 wafers, each containing 7.7 with carmustine in resection mg of carmustine, for a total implant (BCNU) cavity of 61.6 mg, if size and shape
  • the invention also encompasses administration of an anti-EphB4 mAb in combination with radiation therapy comprising the use of x-rays, gamma rays and other sources of radiation to destroy the cancer cells.
  • the radiation treatment is administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source.
  • the radiation treatment is administered as internal therapy or brachytherapy wherein a radiaoactive source is placed inside the body close to cancer cells or a tumor mass.
  • Cancer therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the Physician 's Desk Reference (56 th ed., 2002).
  • the present invention relates to pharmaceutical compositions comprising human, humanized, or chimeric anti-EphB4 antibodies of the IgGl or IgG3 human isotype.
  • the present invention also relates to pharmaceutical compositions comprising human or humanized anti-EphB4 antibodies of the IgG2 or IgG4 human isotype.
  • pharmaceutical compositions of the invention comprise anti-EphB4 antibodies that may mediate ADCC, complement-dependent cellular cytoxicity, or apoptosis.
  • the present invention also relates to pharmaceutical compositions comprising monoclonal human, humanized, or chimerized anti-EphB4 antibodies that can be produced by means known in the art.
  • angiogenesis-associated diseases including, but not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; inflammatory disorders such as immune and nonimmune inflammation; chronic articular rheumatism and psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation and wound healing; telan
  • the invention can be employed to treat or prevent cancer (tumor), including, but not limited to, ovarian carcinoma, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi sarcoma, and leukemia.
  • cancer tumor
  • HNSCC squamous cell carcinoma of the head and neck
  • leukemia squamous cell carcinoma of the head and neck
  • the invention can be employed to treat bone remodeling disease such as osteopetrosis and osteoporosis.
  • compositions of the invention contain as the active ingredient human, humanized, or chimeric anti-EphB4 antibodies.
  • the formulations contain naked antibody, immunoconjugate, or fusion protein in an amount effective for producing the desired response in a unit of weight or volume suitable for administration to a human patient, and are preferably sterile.
  • the response can be measured by determining the physiological effects of the anti-EphB4 antibody composition using assays known to one of ordinary skill in the art. 5.25.1. PHARMACEUTICAL FORMULATIONS
  • An anti-EphB4 antibody composition may be formulated with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients.
  • Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • Such pharmaceutically acceptable preparations may also routinely contain compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, boric, formic, malonic, succinic, and the like.
  • pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the antibodies of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • anti-EphB4 antibody compositions can be prepared for storage by mixing the antibody or immunoconjugate having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington 's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1999)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyr r olidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • Anti-EphB4 antibody compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • Anti-EphB4 antibody compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, anti-EphB4 antibody compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of anti-EphB4 antibody, which is preferably isotonic with the blood of the recipient.
  • This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • carrier formulation suitable for various routes of administration can be the same or similar to that described for RITUXANTM. See, Physicians ' Desk Reference (Medical Economics Company, Inc., Montvale, NJ, 2005), pp. 958-960 and 1354-1357, which is incorporated herein by reference in its entirety.
  • anti-EphB4 antibody compositions are formulated for intravenous administration with sodium chloride, sodium citrate dihydrate, polysorbate 80, and sterile water where the pH of the composition is adjusted to approximately 6.5.
  • a vascular barrier comprising endothelial cells of the vasculature and the subendothelial matrix. Still, the vascular barrier is a more notable problem for the uptake of therapeutic antibodies by solid tumors.
  • Intralymphatic routes of administration such as subcutaneous or intramuscular injection, or by catheterization of lymphatic vessels, may also provide a useful means of delivering anti-EphB4 antibodies.
  • anti-EphB4 antibodies of compositions and methods of the invention are self-administered subcutaneously.
  • the composition is formulated as a lyophilized drug or in a liquid buffer (e.g., PBS and/or citrate) at about 50 mg/mL.
  • a liquid buffer e.g., PBS and/or citrate
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active compound preferably those with complementary activities that do not adversely affect each other.
  • it may be desirable to further provide an immunosuppressive agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the formulations to be used for in vivo administration are typically sterile.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing an anti-EphB4 antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No.
  • copolymers of L-glutamic acid and ⁇ -ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-gly colic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 0 C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devized for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulf ⁇ de interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. In certain embodiments, the pharmaceutically acceptable carriers used in compositions of the invention do not affect human ADCC or CDC.
  • Anti-EphB4 antibody compositions disclosed herein may also be formulated as immunoliposomes.
  • a "liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as anti-EphB4 antibodies disclosed herein) to a human.
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • Liposomes containing antibodies of the invention are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. ScL USA, 82:3688 (1985); Hwang et al, Proc. Natl. Acad. Sci.
  • Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257:286-288 (1982) via a disulfide interchange reaction. A therapeutic agent can also be contained within the liposome. See, Gabizon et al., J. National Cancer Inst., (19)1484 (1989).
  • compositions include, but are not limited to:
  • a sterile, preservative-free liquid concentrate for intravenous (i.v.) administration of anti-EphB4 antibody supplied at a concentration of 10 mg/ml in either 100 mg (10 niL) or 500 mg (50 rnL) single-use vials.
  • the product can be formulated for i.v. administration using sodium chloride, sodium citrate dihydrate, polysorbate and sterile water for injection.
  • the product can be formulated in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5.
  • a sterile, lyophilized powder in single-use glass vials for subcutaneous (s.c.) injection can be formulated with sucrose, L-histidine hydrochloride monohydrate, L-histidine and polysorbate 20.
  • each single-use vial can contain 150 mg anti-EphB4 antibody, 123.2 mg sucrose, 6.8 mg L-histidine hydrochloride monohydrate, 4.3 mg L-histidine, and 3 mg polysorbate 20.
  • Reconstitution of the single-use vial with 1.3 ml sterile water for injection yields approximately 1.5 ml solution to deliver 125 mg per 1.25 ml (100 mg/ml) of antibody.
  • a sterile, preservative-free lyophilized powder for intravenous (i.v.) administration can be formulated with ⁇ -trehalose dihydrate, L-histidine HCl, histidine and polysorbate 20 USP.
  • each vial can contain 440 mg anti-EphB4 antibody, 400 mg ⁇ , ⁇ -trehalose dihydrate, 9.9 mg L-histidine HCl, 6.4 mg L-histidine, and 1.8 mg polysorbate 20, USP.
  • each single-use vial can contain 100 mg antibody, 500 mg sucrose, 0.5 mg polysorbate 80, 2.2 mg monobasic sodium phosphate monohydrate, and 6.1 mg dibasic sodium phosphate dihydrate. No preservatives are present. Following reconstitution with 10 ml sterile water for injection, USP, the resulting pH is approximately 7.2.
  • the product can be formulated with sodium chloride, monobasic sodium phosphate dihydrate, dibasic sodium phosphate dihydrate, sodium citrate, citric acid monohydrate, mannitol, polysorbate 80 and water for injection, USP.
  • Sodium hydroxide may be added to adjust pH to about 5.2.
  • each syringe can be formulated to deliver 0.8 ml (40 mg) of drug product.
  • Each 0.8 ml contains 40 mg anti-EphB4 antibody, 4.93 mg sodium chloride, 0.69 mg monobasic sodium phosphate dihydrate, 1.22 mg dibasic sodium phosphate dihydrate, 0.24 mg sodium citrate, 1.04 citric acid monohydrate, 9.6 mg mannitol, 0.8 mg polysorbate 80 and water for injection, USP.
  • SWFI sterile water for injection
  • USP sterile water for injection
  • the product can be formulated with sucrose, histidine hydrochloride monohydrate, L-histidine, and polysorbate.
  • a 75 mg vial can contain 129.6 mg or 112.5 mg of an anti-EphB4 antibody, 93.1 mg sucrose, 1.8 mg L-histidine hydrochloride monohydrate, 1.2 mg L-histidine, and 0.3 mg polysorbate 20, and is designed to deliver 75 mg of the antibody in 0.6 ml after reconstitution with 0.9 ml SWFI, USP.
  • a 150 mg vial can contain 202.5 mg or 175 mg anti-EphB4 antibody, 145.5 mg sucrose, 2.8 mg L-histidine hydrochloride monohydrate, 1.8 mg L-histidine, and 0.5 mg polysorbate 20, and is designed to deliver 150 mg of the antibody in 1.2 ml after reconstitution with 1.4 ml SWFI, USP.
  • a sterile, hyophilized product for reconstitution with sterile water for injection can be formulated as single -use vials for intramuscular (IM) injection using mannitol, histidine and glycine.
  • IM intramuscular
  • each single-use vial can contain 100 mg anti-EphB4 antibody, 67.5 mg of mannitol, 8.7 mg histidine and 0.3 mg glycine, and is designed to deliver 100 mg antibody in 1.0 ml when reconstituted with 1.0 ml sterile water for injection.
  • each single-use vial can contain 50 mg anti-EphB4 antibody, 40.5 mg mannitol, 5.2 mg histidine and 0.2 mg glycine, and is designed to deliver 50 mg of antibody when reconstituted with 0.6 ml sterile water for injection.
  • a sterile, preservative-free solution for intramuscular (IM) injection supplied at a concentration of 100 mg/ml.
  • the product can be formulated in single-use vials with histidine, glycine, and sterile water for injection.
  • each single-use vial can be formulated with 100 mg antibody, 4.7 mg histidine, and 0.1 mg glycine in a volume of 1.2 ml designed to deliver 100 mg of antibody in 1 ml.
  • each single-use vial can be formulated with 50 mg antibody, 2.7 mg histidine and 0.08 mg glycine in a volume of 0.7 ml or 0.5 ml designed to deliver 50 mg of antibody in 0.5 ml.
  • apharmaceutical composition of the invention is stable at 4 0 C.
  • a pharmaceutical composition of the invention is stable at room temperature. 5.25.2. ANTIBODY HALF-LIFE
  • the half-life of an anti-EphB4 antibody of compositions and methods of the invention is at least about 4 to 7 days.
  • the mean half-life of an anti-EphB4 antibody of compositions and methods of the invention is at least about 2 to 5 days, 3 to 6 days, 4 to 7 days, 5 to 8 days, 6 to 9 days, 7 to 10 days, 8 to 11 days, 8 to 12, 9 to 13, 10 to 14, 11 to 15, 12 to 16, 13 to 17, 14 to 18, 15 to 19, or 16 to 20 days.
  • the mean half-life of an anti-EphB4 antibody of compositions and methods of the invention is at least about 17 to 21 days, 18 to 22 days, 19 to 23 days, 20 to 24 days, 21 to 25, days, 22 to 26 days, 23 to 27 days, 24 to 28 days, 25 to 29 days, or 26 to 30 days.
  • the half-life of an anti-EphB4 antibody of compositions and methods of the invention can be up to about 50 days.
  • the half-lives of antibodies of compositions and methods of the invention can be prolonged by methods known in the art. Such prolongation can in turn reduce the amount and/or frequency of dosing of the antibody compositions .
  • Antibodies with improved in vivo half-lives and methods for preparing them are disclosed in U.S. Patent No. 6,277,375; and International Publication Nos. WO 98/23289 and WO 97/3461.
  • the serum circulation of anti-EphB4 antibodies in vivo may also be prolonged by attaching inert polymer molecules such as high molecular weight polyethyleneglycol (PEG) to the antibodies with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of the antibodies or via epsilon-amino groups present on lysyl residues.
  • PEG polyethyleneglycol
  • Linear or branched polymer derivatization that results in minimal loss of biological activity will be used.
  • the degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies.
  • Unreacted PEG can be separated from antibody-PEG conjugates by size-exclusion or by ion-exchange chromatography.
  • PEG-derivatized antibodies can be tested for binding activity as well as for in vivo efficacy using methods known to those of skill in the art, for example, by immunoassays described herein.
  • compositions and methods of the invention can be conjugated to albumin in order to make the antibody more stable in vivo or have a longer half-life in vivo.
  • the techniques are well known in the art, see, e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 413, 622, all of which are incorporated herein by reference. 5.25.3. ADMINISTRATION AND DOSING
  • compositions of the invention can be by any route, including but not limited to intravenous, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via an aerosol), buccal (e.g., sub-lingual), topical (i.e., both skin and mucosal surfaces, including airway surfaces), intrathecal, intraarticular, intraplural, intracerebral, intra-arterial, intraperitoneal, oral, intralymphatic, intranasal, rectal or vaginal administration, by perfusion through a regional catheter, or by direct intralesional injection.
  • intravenous intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via an aerosol), buccal (e.g., sub-lingual), topical (i.e., both skin and mucosal surfaces, including airway surfaces), intrathecal, intraarticular, intraplural, intracerebral, intra-arterial, intraperitoneal, oral, intralymphatic,
  • compositions of the invention are administered by intravenous push or intravenous infusion given over defined period (e.g., 0.5 to 2 hours).
  • Compositions of the invention can be delivered by peristaltic means or in the form of a depot, although the most suitable route in any given case will depend, as is well known in the art, on such factors as the species, age, gender and overall condition of the subject, the nature and severity of the condition being treated and/or on the nature of the particular composition (i.e., dosage, formulation) that is being administered.
  • the route of administration is via bolus or continuous infusion over a period of time, once or twice a week.
  • the route of administration is by subcutaneous injection, optionally once or twice weekly.
  • compositions, and/or methods of the invention are administered on an outpatient basis.
  • the dose of a composition comprising anti-EphB4 antibody is measured in units of mg/kg of patient body weight. In other embodiments, the dose of a composition comprising anti-EphB4 antibody is measured in units of mg/kg of patient lean body weight (i.e., body weight minus body fat content). In yet other embodiments, the dose of a composition comprising anti-EphB4 antibody is measured in units of mg/m 2 of patient body surface area. In yet other embodiments, the dose of a composition comprising anti-EphB4 antibody is measured in units of mg per dose administered to a patient. Any measurement of dose can be used in conjunction with compositions and methods of the invention and dosage units can be converted by means standard in the art.
  • compositions of the invention may be extrapolated from dose-response curves derived in vitro test systems or from animal model ⁇ e.g., the cotton rat or monkey) test systems. Models and methods for evaluation of the effects of antibodies are known in the art (Wooldridge et al, Blood, 89(8): 2994-2998 (1997)), incorporated by reference herein in its entirety).
  • therapeutic regimens standard in the art for antibody therapy can be used with compositions and methods of the invention.
  • Examples of dosing regimens that can be used in methods of the invention include, but are not limited to, daily, three times weekly (intermittent), weekly, or every 14 days. In certain embodiments, dosing regimens include, but are not limited to, monthly dosing or dosing every 6-8 weeks.
  • dosages are generally higher and/or frequency of administration greater for initial treatment as compared with maintenance regimens.
  • anti-EphB4 antibodies bind to tumor cells and may result in efficient ⁇ i.e., at low dosage) depletion of the tumor cells . Higher degrees of binding may be achieved where the density of human EphB4 on the surface of a patient's tumor cells is high.
  • dosages of the antibody are at least about 0.0005, 0.001, 0.05, 0.075, 0.1, 0.25, 0.375, 0.5, 1, 2.5, 5, 10, 20, 37.5, or 50 mg/m 2 and/or less than about 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 60, 50, 37.5, 20, 15, 10, 5, 2.5, 1, 0.5, 0.375, 0.1, 0.075 or 0.01 mg/m 2 .
  • the dosage is between about 0.0005 to about 200 mg/m 2 , between about 0.001 and 150 mg/m 2 , between about 0.075 and 125 mg/m 2 , between about 0.375 and 100 mg/m 2 , between about 2.5 and 75 mg/m 2 , between about 10 and 75 mg/m 2 , and between about 20 and 50 mg/m 2 .
  • the dosage of anti-EphB4 antibody used is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5 mg/kg of body weight of a patient.
  • the dose of naked anti-EphB4 antibody used is at least about 1 to 10, 5 to 15, 10 to 20, or 15 to 25 mg/kg of body weight of a patient.
  • the dose of anti-EphB4 antibody used is at least about 1 to 20, 3 to 15, or 5 to 10 mg/kg of body weight of a patient. In other embodiments, the dose of anti-EphB4 antibody used is at least about 5, 6, 7, 8, 9, or 10 mg/kg of body weight of a patient.
  • a single dosage unit of the antibody (optionally in a pharmaceutically acceptable carrier as part of a pharmaceutical composition) can be at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,
  • dose is up to 1 g per single dosage unit.
  • All of the above doses are exemplary and can be used in conjunction with compositions and methods of the invention, however where an anti-EphB4 antibody is used in conjunction with a toxin or radiotherapeutic agent the lower doses described above may be preferred. In certain embodiments, where the patient has low levels of EphB4 density, the lower doses described above may be preferred.
  • the dose or amount of the chimeric antibody is greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 mg/kg of patient body weight.
  • the dose or amount of the chimeric antibody is less than about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg of patient body weight.
  • antibodies and/or compositions of this invention can be administered at a dose lower than about 375 mg/m 2 ; at a dose lower than about 37.5 mg/m 2 ; at a dose lower than about 0.375 mg/m 2 ; and/or at a dose between about 0.075 mg/m 2 and about 125 mg/m 2 .
  • dosage regimens comprise low doses, administered at repeated intervals.
  • compositions of the invention can be administered at a dose lower than about 375 mg/m 2 at intervals of approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 days.
  • a dosage of about 125 mg/m 2 or less of an antibody or antigen-binding fragment results in tumor cell depletion for a period of at least about 7, 14, 21, 30, 45, 60, 90, 120, 150, or 200 days.
  • a dosage of about 37.5 mg/m 2 or less depletes tumor cells for a period of at least about 7, 14, 21, 30, 45, 60, 90, 120, 150, or 200 days.
  • a dosage of about 0.375 mg/m 2 or less results in depletion of tumor cells for at least about 7, 14, 21, 30, 45 or 60 days.
  • a dosage of about 0.075 mg/m 2 or less results in depletion of tumor cells for a period of at least about 7, 14, 21, 30, 45, 60, 90, 120, 150, or 200 days.
  • a dosage of about 0.01 mg/m 2 , 0.005 mg/m 2 or even 0.001 mg/m 2 or less results in depletion of tumor cells for at least about 3, 5, 7, 10, 14, 21, 30, 45, 60, 90, 120, 150, or 200 days.
  • the dosage can be administered by any suitable route, but is optionally administered by a subcutaneous route.
  • the dose can be escalated or reduced to maintain a constant dose in the blood or in a particular tissue, such as, but not limited to, ovary, bone marrow.
  • the dose is escalated or reduced by about 2%, 5%, 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% in order to maintain a desired level of an antibody of compositions and methods of the invention.
  • the dosage can be adjusted and/or the infusion rate can be reduced based on patient's immunogenic response to compositions and methods of the invention.
  • a loading dose of an anti-EphB4 antibody and/or composition of the invention can be administered first followed by a maintenance dose until the malignancy being treated progresses.
  • a patient may be pretreated with compositions and methods of the invention to detect, minimize immunogenic response, or minimize adverse effects of compositions and methods of the invention.
  • the tolerance, toxicity and/or efficacy of the compositions and/or treatment regimens of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population), the ED50 (the dose therapeutically effective in 50% of the population), and IC50 (the dose effective to achieve a 50% inhibition).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapies that exhibit large therapeutic indices may be preferred.
  • While therapies that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to EphB4-expressing cells in order to minimize potential damage to EphB4 negative cells and, thereby, reduce side effects.
  • Data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages of the compositions and/or treatment regimens for use in humans.
  • the dosage of such agents may lie within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • a therapeutically effective dose can be estimated by appropriate animal models.
  • the dose can be scaled for human use according to art-accepted formulas, for example, as provided by Freireich et ah, Quantitative comparison of toxicity of anticancer agents in mouse, rat, monkey, dog, and human, Cancer Chemotherapy Reports, NCI 1966 40:219-244. Data obtained from cell culture assays can be useful for predicting potential toxicity. Animal studies can be used to formulate a specific dose to achieve a circulating plasma concentration range that includes the IC50 ⁇ i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma drug levels may be measured, for example, by high performance liquid chromatography, ELISA, or by cell based assays.
  • assays for EphB4 density can be employed to further characterize the patient's diagnosis.
  • Methods of determining the density of antibody binding to cells are known to those skilled in the art ⁇ See, e.g., Sato et al., J. Immunology 165:6635-6643 (2000)).
  • Other standard methods include Scatchard analysis.
  • the antibody or fragment can be isolated, radiolabeled, and the specific activity of the radiolabeled antibody determined.
  • the antibody is then contacted with a target cell expressing EphB4.
  • the radioactivity associated with the cell can be measured and, based on the specific activity, the amount of antibody or antibody fragment bound to the cell determined.
  • Fluorescence activated flow cytometry can also be employed.
  • the antibody or antibody fragment is bound to a target cell expressing EphB4.
  • a second reagent that binds to the antibody is then added, for example, a flourochrome labeled antiimmunoglobulin antibody. Flourochrome staining can then be measured and used to determine the density of antibody or antibody fragment binding to the cell.
  • the antibody or antibody fragment can be directly labeled with a detectable label, such as a fluorophore, and bound to a target cell. The ratio of label to protein is determined and compared with standard beads with known amounts of label bound thereto. Comparison of the amount of label bound to the cell with the known standards can be used to calculate the amount of antibody bound to the cell.
  • the present invention provides a method for detecting in vitro or in vivo the presence and/or density of EphB4 in a sample or individual. This can also be useful for monitoring disease and effect of treatment and for determining and adjusting the dose of the antibody to be administered.
  • the in vivo method can be performed using imaging techniques such as PET (positron emission tomography) or SPECT (single photon emission computed tomography).
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • the in vivo method can be performed by contacting a sample to be tested, optionally along with a control sample, with a human anti-EphB4 antibody under conditions that allow for formation of a complex between an antibody of the invention and the human EphB4 antigen. Complex formation is then detected ⁇ e.g. , using fluorescent activated flow cytometry or Western blotting).
  • a complex is detected in both samples and any statistically significant difference in the formation of complexes between the samples is indicative of the presence of human EphB4 in the test sample.
  • the density of EphB4 may influence the determination and/or adjustment of the dosage and/or treatment regimen used with an anti-EphB4 antibody of compositions and methods of the invention. For example, where density of EphB4 is high, it may be possible to use anti-EphB4 antibodies that less efficiently deplete tumor cells. In certain embodiments, where the patient treated using compositions and methods of the invention has a low EphB4 density, a higher dosage of an anti-EphB4 antibody of compositions and methods of the invention may be used.
  • EphB4 density of a sample can be compared to an average EphB4 density of a similar sample for humans or for a particular patient population, or EphB4 density can be compared to EphB4 levels in the patietn prior to therapy or prior to onset of a disease or disorder.
  • Anti-EphB4 antibody compositions used in the therapeutic regimen/protocols can be naked antibodies, immunoconjugates and/or fusion proteins.
  • Compositions of the invention can be used as a single agent therapy or in combination with other therapeutic agents or regimens.
  • Anti-EphB4 antibodies or immunoconjugates can be administered prior to, concurrently with, or following the administration of one or more therapeutic agents.
  • Therapeutic agents that can be used in combination therapeutic regimens with compositions of the invention include any substance that inhibits or prevents the function of cells and/or causes destruction of cells. Examples include, but are not limited to, radioactive isotopes, chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • the therapeutic regimens described herein, or any desired treatment regimen can be tested for efficacy using a transgenic animal model which expresses human EphB4 antigen in place of native EphB4 antigen.
  • an anti-EphB4 antibody treatment regimen can be tested in an animal model to determine efficacy before administration to a human.
  • Anti-EphB4 antibodies, compositions and methods may be practiced to treat angiogenesis-associated diseases including, but not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; inflammatory disorders such as immune and nonimmune inflammation; chronic articular rheumatism and psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound
  • angiogenesis-independent cancer refers to a cancer (tumor) where there is no or little neovascularization in the tumor tissue.
  • the therapeutic antibodies of the present invention are useful for treating or preventing a cancer (tumor), including, but not limited to, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi sarcoma, and leukemia.
  • a cancer tumor including, but not limited to, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi sarcoma, and leukemia.
  • the invention can be employed to treat ovarian cancer.
  • the invention can be employed to treat bone remodeling disease such as osteopetrosis and osteoporosis.
  • anti-EphB4 immunotherapy encompasses the administration of any of the anti-EphB4 antibodies of the invention in accordance with any therapeutic regimen described herein.
  • Anti-EphB4 antibodies can be administered as naked antibodies, or immunoconjugates or fusion proteins.
  • Anti-EphB4 immunotherapy encompasses the administration of an anti-EphB4 antibody as a single agent therapeutic for the treatment of a malignancy.
  • Anti-EphB4 immunotherapy encompasses methods of treating an early stage disease resulting from a malignancy.
  • Anti-EphB4 immunotherapy encompasses methods of treating a malignancy wherein an anti-EphB4 antibody mediates ADCC.
  • Anti-EphB4 immunotherapy encompasses methods of treating a malignancy wherein an anti-EphB4 antibody mediates apoptosis.
  • Anti-EphB4 immunotherapy encompasses methods of treating a malignancy wherein an anti-EphB4 antibody is administered before the patient has received any treatment for the malignancy, whether that therapy is chemotherapy, radio chemical based therapy or surgical therapy.
  • a human subject having a malignancy can be treated by administering a human or humanized antibody that may be able to mediate human ADCC.
  • any anti-EphB4 antibody that may mediate ADCC can be used in the human subjects (including murine and chimeric antibodies); however, human and humanized antibodies may be preferred.
  • a human subject having a malignancy can be treated by administering a human or humanized antibody that may be able to mediate apoptosis.
  • a human or humanized antibody that may be able to mediate apoptosis.
  • any anti-EphB4 antibody that may mediate apoptosis can be used in the human subjects (including murine and chimeric antibodies); however, human and humanized antibodies may be preferred.
  • Antibodies of IgGl or IgG3 human isotypes are in some cases preferred for therapy.
  • the IgG2 or IgG4 human isotypes can be used as well, provided they have the relevant effector function.
  • Such effector function can be assessed by measuring the ability of the antibody in question to mediate target cell killing in vitro or in vivo.
  • tumor burden can be measured and used in connection with compositions and methods of the invention. Methods for measuring tumor burden are well known in the art and include, but are not limited to the following embodiments.
  • PET scans can be used to measure metabolic activity and identify areas of higher activity which are indicative of tumors.
  • CT scans and MRI can also be used to examine soft tissue for the presence and size of tumors.
  • bone scan can be used to measure tumor volume and location.
  • tumor burden can be measured by examining the blood flow into and out of a tumor using doppler technology ⁇ e.g., ultrasound). In such embodiments, changes in blood flow over time or deviations from normal blood flow in the appropriate tissue of a patient can be used to calculate an estimate to tumor burden.
  • doppler technology ⁇ e.g., ultrasound
  • tumor cells are depleted and/or tumor burden is decreased while ADCC function is maintained.
  • an anti-EphB4 antibody is administered as a single agent therapy
  • the invention contemplates use of different treatment regimens.
  • an anti-EphB4 antibody used in compositions and methods of the invention is a naked antibody.
  • the dose of naked anti-EphB4 antibody used is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5 mg/kg of body weight of a patient.
  • the dose of naked anti-EphB4 antibody used is at least about 1 to 10, 5 to 15, 10 to 20, or 15 to 25 mg/kg of body weight of a patient. In certain embodiments, the dose of naked anti-EphB4 antibody used is at least about 1 to 20, 3 to 15, or 5 to 10 mg/kg of body weight of a patient. In other embodiments, the dose of naked anti-EphB4 antibody used is at least about 5, 6, 7, 8, 9, or 10 mg/kg of body weight of a patient. [0331] In certain embodiments, the dose comprises about 375 mg/m 2 of anti-EphB4 antibody administered weekly for 4 to 8 consecutive weeks. In certain embodiments, the dose is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg of body weight of the patient administered weekly for 4 to 8 consecutive weeks.
  • doses are single dose injections.
  • the doses are administered over a period of time.
  • the doses are administered multiple times over a period of time. The period of time may be measured in days, weeks, or months.
  • Multiple doses of an anti-EphB4 antibody can be administered at intervals suitable to achieve a therapeutic benefit while balancing toxic side effects. For example, where multiple doses are used, it may be preferred to time the intervals to allow for recovery of the patient's monocyte count prior to the repeat treatment with antibody. This dosing regimen may optimize the efficiency of treatment, since the monocyte population reflects ADCC function in the patient.
  • compositions of the invention are administered to a human patient as long as the patient is responsive to therapy. In other embodiments, compositions of the invention are administered to a human patient as long as the patient's disease does not progress. In related embodiments, compositions of the invention are administered to a human patient until a patient's disease does not progress or has not progressed for a period of time, then the patient is not administered compositions of the invention unless the disease reoccurs or begins to progress again. For example, a patient can be treated with any of the above doses for about 4 to 8 weeks, during which time the patient is monitored for disease progression.
  • the patient will not be administered compositions of the invention until that patient relapses, i.e., the disease being treated reoccurs or progresses. Upon this reoccurrence or progression, the patient can be treated again with the same dosing regimen initially used or using other doses described above.
  • compositions of the invention can be administered as a loading dose followed by multiple lower doses (maintenance doses) over a period of time.
  • the doses may be timed and the amount adjusted to maintain effective B cell depletion.
  • the loading dose is about 10, 11, 12, 13, 14, 15, 16, 17, or 18 mg/kg of patient body weight and the maintenance dose is at least about 5 to 10 mg/kg of patient body weight.
  • the maintenance dose is administered at intervals of every 7, 10, 14 or 21 days. The maintenance doses can be continued indefinitely, until toxicity is present, until platelet count decreases, until there is no disease progression, until the patient exhibits immunogenicity, or until disease progresses to a terminal state.
  • compositions of the invention are administered to a human patient until the disease progresses to a terminal stage.
  • doses of anti-EphB4 antibody administered may be spaced to allow for recovery of monocyte count.
  • a composition of the invention may be administered at intervals of every 8, 9, 10, 11, 12, 13,
  • an anti-EphB4 antibody is conjugated to or administered in conjunction with a toxin
  • the dose of anti-EphB4 antibody can be adjusted based on the toxin dose and that the toxin dose will depend on the specific type of toxin being used. Typically, where a toxin is used, the dose of anti-EphB4 antibody will be less than the dose used with a naked anti-EphB4 antibody.
  • the appropriate dose can be determined for a particular toxin using techniques well known in the art. For example, a dose range study can be conducted to determine the maximum tolerated dose of anti-EphB4 antibody when administered with or conjugated to a toxin.
  • the dose of the anti-EphB4 antibody will vary depending on the radiotherapeutic used.
  • a two step process is used. First, the human patient is administered a composition comprising a naked anti-EphB4 antibody and about 6, 7, 8, 9, or 10 days later a small amount of the radiotherapeutic is administered. Second, once the tolerance, distribution, and clearance of the low dose therapy has been determined, the patient is administered a dose of the naked anti-EphB4 antibody followed by a therapeutic amount of the radiotherapeutic is administered.
  • Anti-EphB4 immunotherapy can be used in conjunction with other therapies including but not limited to, chemotherapy, radioimmunotherapy (RIT), chemotherapy and external beam radiation (combined modality therapy, CMT), or combined modality radioimmunotherapy (CMRIT) alone or in combination, etc.
  • RIT radioimmunotherapy
  • CMT combined modality therapy
  • CMRIT combined modality radioimmunotherapy
  • an anti-EphB4 antibody therapy of the present invention can be administered in conjunction with CHOP
  • an anti-EphB4 immunotherapy is in conjunction with a cytotoxic radionuclide or radiotherapeutic isotope.
  • a cytotoxic radionuclide or radiotherapeutic isotope for example, an alpha-emitting isotope such as 225 Ac, 224 Ac, 211 At, 212 Bi, 213 Bi, 212 Pb, 224 Ra, or 223 Ra.
  • the cytotoxic radionuclide may also be a beta-emitting isotope such as 186 Re, 188 Re, 90 Y, 131 I, 67 Cu, 177 Lu, 153 Sm, 166 Ho, or 64 Cu.
  • the cytotoxic radionuclide may emit Auger and low energy electrons and include the isotopes 125 I, 123 I or 77 Br. In other embodiments the isotope may be 198 Au, 32 P, and the like. In certain embodiments, the amount of the radionuclide administered to the subject is between about 0.001 mCi/kg and about 10 mCi/kg.
  • the amount of the radionuclide administered to the subject is between about 0.1 mCi/kg and about 1.0 mCi/kg. In other embodiments, the amount of the radionuclide administered to the subject is between about 0.005 mCi/kg and 0.1 mCi/kg.
  • an anti-EphB4 immunotherapy is in conjunction with a chemical toxin or chemotherapeutic agent.
  • the chemical toxin or chemotherapeutic agent may be selected from the group consisting of an enediyne such as calicheamicin and esperamicin; duocarmycin, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin and 5-fluorouracil.
  • Suitable chemical toxins or chemotherapeutic agents that can be used in combination therapies with an anti-EphB4 immunotherapy include members of the enediyne family of molecules, such as calicheamicin and esperamicin. Chemical toxins can also be taken from the group consisting of duocarmycin (see, e.g., U.S. Pat. No. 5,703,080 and U.S. Pat. No. 4,923,990), methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin and 5-fluorouracil.
  • duocarmycin see, e.g., U.S. Pat. No. 5,703,080 and U.S. Pat. No. 4,923,990
  • methotrexate doxorubicin
  • melphalan chlorambucil
  • ARA-C ARA-C
  • chemotherapeutic agents also include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see, U.S. Pat. No. 4,675,187), Melphalan and other related nitrogen mustards.
  • CVB (1.5 g/m 2 cyclophosphamide, 200-400 mg/m 2 etoposide, and 150-200 mg/m 2 carmustine) can be used in combination therapies of the invention.
  • CVB is a regimen used to treat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51 :18 (1993).
  • Other suitable combination chemotherapeutic regimens are well-known to those of skill in the art. See, for example, Freedman et al. , “Non-Hodgkin 's Lymphomas," in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al.
  • first generation chemotherapeutic regimens for treatment of intermediate-grade non-Hodgkin's lymphoma include C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone).
  • a useful second generation chemotherapeutic regimen is m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone and leucovorin), while a suitable third generation regimen is MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and leucovorin).
  • Additional useful drugs include phenyl butyrate and brostatin-1.
  • both chemotherapeutic drugs and cytokines are co-administered with an antibody, immunoconjugate or fusion protein according to the present invention.
  • the cytokines, chemotherapeutic drugs and antibody, immunoconjugate or fusion protein can be administered in any order, or together.
  • Illustrative of toxins which are suitably employed in combination therapies of the invention are ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published October 28, 1993.
  • Suitable toxins and chemotherapeutic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND GILMAN' S THE PHARMACOLOGICAL BASIS OF
  • An anti-EphB4 immunotherapy of the present invention may also be in conjunction with a prodrug-activating enzyme which converts a prodrug (e.g. , a peptidyl chemotherapeutic agent, see, WO81/01145) to an active anti-cancer drug.
  • a prodrug e.g. , a peptidyl chemotherapeutic agent, see, WO81/01145
  • the enzyme component of such combinations includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Harbor (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al. (ed.), pp. 247-267, Humana Press (1985).
  • Prodrugs that can be used in combination with anti-EphB4 antibodies include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
  • compositions and methods of the invention may enable the postponement of toxic therapy and may help avoid unnecessary side effects and the risks of complications associated with chemotherapy and delay development of resistance to chemotherapy.
  • toxic therapies and/or resistance to toxic therapies is delayed in patients administered compositions and methods of the invention delay for up to about 6 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • a compound that enhances monocyte or macrophage function can be used in conjunction with an anti-EphB4 immunotherapy.
  • Such compounds include, without limitation, cytokines such as interleukins (e.g., IL-12), and interferons (e.g., alpha or gamma interferon).
  • the compound that enhances monocyte or macrophage function or enhancement can be formulated in the same pharmaceutical composition as the antibody, immunoconjugate or antigen-binding fragment.
  • the antibody/fragment and the compound can be administered concurrently (within a period of hours of each other), can be administered during the same course of therapy, or can be administered sequentially (i.e., the patient first receives a course of the antibody/fragment treatment and then a course of the compound that enhances macrophage/monocyte function or vice versa).
  • the compound that enhances monocyte or macrophage function is administered to the human subject prior to, concurrently with, or following treatment with other therapeutic regimens and/or compositions of the invention.
  • the human subject has a blood leukocyte, monocyte, neutrophil, lymphocyte, and/or basophil count that is within the normal range for humans.
  • Normal ranges for human blood leukocytes (total) is about 3.5- about 10.5 (10 9 /L).
  • Normal ranges for human blood neutrophils is about 1.7- about 7.0 (10 9 /L)
  • monocytes is about 0.3- about 0.9 (10 9 /L)
  • lymphocytes is about 0.9- about 2.9 (10 9 /L)
  • basophils is about 0- about 0.3 (10 9 /L)
  • eosinophils is about 0.05- about 0.5 (10 9 /L).
  • the human subject has a blood leukocyte count that is less than the normal range for humans, for example at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 (10 9 /L) leukocytes.
  • An anti-EphB4 immunotherapy may also be in conjunction with an immunoregulatory agent.
  • an immunoregulatory agent in this approach, a chimeric, human or humanized anti-EphB4 antibody can be used.
  • immunoregulatory agent refers to substances that act to suppress, mask, or enhance the immune system of the host. This would include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5 -substituted pyrimidines (see, U.S. Pat. No.
  • azathioprine or cyclophosphamide, if there is an adverse reaction to azathioprine
  • bromocryptine bromocryptine
  • glutaraldehyde which masks the MHC antigens, as described in U.S. Pat. No.
  • anti-idiotypic antibodies for MHC antigens and MHC fragments include cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone, methylprednisolone, and dexamethasone; cytokine or cytokine receptor antagonists including anti-interferon- ⁇ , - ⁇ , or - ⁇ antibodies; anti-tumor necrosis factor- ⁇ antibodies; anti-tumor necrosis factor- ⁇ antibodies; anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, for example anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO
  • cytokines include, but are not limited to lymphokines, monokines, and traditional polypeptide hormones.
  • cytokines include growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoiotin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor; transforming growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇ ; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
  • the methods further include administering to the subject one or more immunomodulatory agents, for example a cytokine.
  • Suitable cytokines may be selected from the group consisting of interleukin-1 (IL-I), IL-2, IL-3, IL-12, IL-15, IL-18, G-CSF, GM-CSF, thrombopoietin, and ⁇ interferon.
  • immunoregulatory agents are administered at the same time or at separate times from anti-EphB4 antibodies.
  • the preferred immunoregulatory agent will depend on many factors, including the type of disorder being treated, as well as the patient's history, but the agent frequently may be selected from cyclosporin A, a glucocorticosteroid (for example prednisone or methylprednisolone), OKT-3 monoclonal antibody, azathioprine, bromocryptine, heterologous anti-lymphocyte globulin, or a mixture thereof.
  • Agents that act on the tumor neovasculature can also be used in conjunction with anti-EphB4 immunotherapy and include tubulin-binding agents such as combrestatin A4 (Griggs et ah, Lancet Oncol. 2:82, (2001)) and angiostatin and endostatin (reviewed in Rosen, Oncologist 5:20 (2000), incorporated by reference herein).
  • Immunomodulators suitable for use in combination with anti-EphB4 antibodies include, but are not limited to, of ⁇ - interferon, ⁇ - interferon, and tumor necrosis factor alpha (TNF ⁇ ).
  • the therapeutic agents used in combination therapies using compositions and methods of the invention are peptides.
  • the therapeutic agents used in combination therapies using compositions and methods of the invention are antibodies or fragments thereof.
  • pharmaceutical compounds that may be used for combinatory anti-angiogenesis therapy include: (1) inhibitors of release of "angiogenic molecules," such as bFGF (basic fibroblast growth factor); (2) neutralizers of angiogenic molecules, such as an anti-.beta.bFGF antibodies; and (3) inhibitors of endothelial cell response to angiogenic stimuli, including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-penicillamine and gold thiomalate, vitamin D. sub.3 analogs, alpha-interferon, and the like.
  • angiogenic molecules such as bFGF (basic fibroblast growth factor)
  • neutralizers of angiogenic molecules such as an anti-.beta.bFGF antibodies
  • inhibitors of endothelial cell response to angiogenic stimuli including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-
  • angiogenesis there are a wide variety of compounds that can be used to inhibit angiogenesis, for example, peptides or agents that block the VEGF -mediated angiogenesis pathway, endostatin protein or derivatives, lysine binding fragments of angiostatin, melanin or melanin-promoting compounds, plasminogen fragments (e.g., Kringles 1-3 of plasminogen), tropoin subunits, antagonists of vitronectin .alpha..sub.v.beta..sub.3, peptides derived from Saposin B, antibiotics or analogs (e.g., tetracycline, or neomycin), dienogest-containing compositions, compounds comprising a MetAP-2 inhibitory core coupled to a peptide, the compound EM- 138, chalcone and its analogs, and naaladase inhibitors.
  • plasminogen fragments e.g., Kringles 1-3 of plasminogen
  • an anti-EphB4 immunotherapy is in conjunction with one or more calicheamicin molecules.
  • the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations.
  • Structural analogues of calicheamicin which may be used include, but are not limited to, ⁇ l 1 , ⁇ 2 1 , ⁇ 3 ! , N-acetyl- ⁇ l 1 , PSAG and 011 Hinman et al, Cancer Research 53:3336-3342 (1993) and Lode et al, Cancer Research 58: 2925-2928 (1998)).
  • a fusion protein comprising an anti-EphB4 antibody and a cytotoxic agent may also be made, e.g., by recombinant techniques or peptide synthesis.
  • an anti-EphB4 antibody may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pretargeting wherein the antagonist-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” ⁇ e.g., avidin) which is conjugated to a therapeutic agent ⁇ e.g., a radionucleotide).
  • a treatment regimen includes compounds that mitigate the cytotoxic effects of an anti-EphB4 antibody composition.
  • Such compounds include analgesics (e.g., acetaminophen), bisphosphonates, antihistamines (e.g., chlorpheniramine maleate), and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins).
  • analgesics e.g., acetaminophen
  • bisphosphonates e.g., antihistamines (e.g., chlorpheniramine maleate)
  • steroids e.g., dexamethasone, retinoids, deltoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins.
  • the therapeutic agent used in combination with an anti-EphB4 immunotherapy is a small molecule (i.e., inorganic or organic compounds having a molecular weight of less than about 2500 daltons).
  • libraries of small molecules may be commercially obtained from Specs and BioSpecs B. V. (Rijswijk, The Netherlands), Chembridge Corporation (San Diego, CA), Comgenex USA Inc. (Princeton, NJ), and Maybridge Chemicals Ltd. (Cornwall PL34 OHW, United Kingdom).
  • an anti-EphB4 immunotherapy can be administered in combination with an anti-bacterial agent.
  • anti-bacterial agents include proteins, polypeptides, peptides, fusion proteins, antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit and/or reduce a bacterial infection, inhibit and/or reduce the replication of bacteria, or inhibit and/or reduce the spread of bacteria to other cells or subjects.
  • anti-bacterial agents include, but are not limited to, antibiotics such as penicillin, cephalosporin, imipenem, axtreonam, vancomycin, cycloserine, bacitracin, chloramphenicol, erythromycin, clindamycin, tetracycline, streptomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, spectinomycin, trimethoprim, norfloxacin, rifampin, polymyxin, amphotericin B, nystatin, ketocanazole, isoniazid, metronidazole, and pentamidine.
  • antibiotics such as penicillin, cephalosporin, imipenem, axtreonam, vancomycin, cycloserine, bacitracin, chloramphenicol, erythromycin, clindamycin, tetracycline, streptomycin, tobramycin, gentamicin,
  • an anti-EphB4 immunotherapy can be administered in combination with an anti-fungal agent.
  • anti-fungal agents include, but are not limited to, azole drugs (e.g. , miconazole, ketoconazole (NIZORAL ® ), caspofungin acetate (CANCIDAS ® ), imidazole, triazoles (e.g., fluconazole (DIFLUCAN ® )), and itraconazole (SPORANOX ® )), polyene (e.g., nystatin, amphotericin B (FUNGIZONE ® ), amphotericin B lipid complex (“ABLC”) (ABELCET ® ), amphotericin B colloidal dispersion (“ABCD”) (AMPHOTEC ® ), liposomal amphotericin B (AMBISONE ® )), potassium iodide (KI), pyrimidine (e.g., flucytosine), a compound that cata
  • an anti-EphB4 immunotherapy can be administered in combination with one or more of the agents described above to mitigate the toxic side effects that may accompany administration of compositions of the invention.
  • an anti-EphB4 immunotherapy can be administered in combination with one or more agents that are well known in the art for use in mitigating the side effects of antibody administration, chemotherapy, toxins, or drugs.
  • compositions of the invention may be administered in combination with or in treatment regimens with high-dose chemotherapy (melphalan, melphalan/prednisone (MP), vincristine/doxorubicin/dexamethasone (VAD), liposomal doxorubicin/vincristine, dexamethasone (DVd), cyclophosphamide, etoposide/dexamethasone/cytarabine, cisplatin (EDAP)), stem cell transplants (e.g., autologous stem cell transplantation or allogeneic stem cell transplantation, and/or mini-allogeneic (non-myeloablative) stem cell transplantation), radiation therapy, steroids (e.g., corticosteroids, dexamethasone, thalidomide/dexamethasone, prednisone, melphal
  • the additional antibody or antibodies and/or agents can be administered in any sequence relative to the administration of the antibody of this invention.
  • the additional antibody or antibodies can be administered before, concurrently with, and/or subsequent to administration of an anti-EphB4 antibody or immunoconjugate to the human subject.
  • the additional antibody or antibodies can be present in the same pharmaceutical composition as an antibody of the invention, and/or present in a different pharmaceutical composition.
  • an antibody of this invention and the dose of the additional antibody or antibodies can be the same or different, in accordance with any of the teachings of dosage amounts and modes of administration as provided in this application and as are well known in the art. 5.33. USE OF ANTI-EphB4 ANTIBODIES FOR DIAGNOSTIC PURPOSES
  • the present invention also encompasses anti-EphB4 antibodies, and compositions thereof, that immunospecif ⁇ cally bind to the human EphB4 antigen, which anti-EphB4 antibodies are conjugated to a diagnostic or detectable agent.
  • the antibodies are human or humanized anti-EphB4 antibodies.
  • Such anti-EphB4 antibodies can be useful for monitoring or prognosing the development or progression of a disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.
  • Such diagnosis and detection can be accomplished by coupling an anti-EphB4 antibody that immunospecif ⁇ cally binds to the human EphB4 antigen to a detectable substance including, but not limited to, various enzymes, such as but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to, streptavidinlbiotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but
  • any detectable label that can be readily measured can be conjugated to an anti-EphB4 antibody and used in diagnosing a disease or disorder.
  • the detectable substance may be coupled or conjugated either directly to an antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, e.g., U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as a diagnostics according to the present invention.
  • the invention provides for diagnostic kits comprising an anti-EphB4 antibody conjugated to a diagnostic or detectable agent. 5.34. KITS
  • the invention provides a pharmaceutical pack or kit comprising one or more containers filled with a composition of the invention for the prevention, treatment, management or amelioration of a human malignancy, or one or more symptoms thereof, potentiated by or potentiating a malignancy.
  • kits that can be used in the above-described methods.
  • a kit comprises a composition of the invention, in one or more containers.
  • a kit comprises a composition of the invention, in one or more containers, and one or more other prophylactic or therapeutic agents useful for the prevention, management or treatment of a human malignancy, or one or more symptoms thereof, potentiated by or potentiating a malignancy in one or more other containers.
  • the kit may further comprise instructions for preventing, treating, managing or ameliorating a malignancy, as well as side effects and dosage information for method of administration.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • a chimeric, humanized, or human monoclonal antibody or fragment thereof that binds a human EphB4 antigen wherein the antibody comprises a VL domain comprising at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-10 and 18-20, and further comprising one or more framework region of a light chain variable region selected from the group consisting of 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO: 112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO : 148), 8D 10 VL (
  • the CDR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18-20.
  • the VL domain comprises an amino acid sequence selected from the group consisting of 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO: 112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO: 148), 8D10 VL (SEQ ID NO: 160), 9D9 VL (SEQ ID NO: 172), 9E9 VL (SEQ ID NO: 184), 9El
  • the antibody further comprises a VH domain comprising at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3-5 and 13-15, and further comprising one or more framework region of a heavy chain variable region selected from the group consisting of 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO:82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO:118), 6G2 VH (SEQ ID NO: 130), 7A7 VH (SEQ ID NO: 142), 8D10 VH (SEQ ID NO: 154), 9D9 VH (SEQ ID NO : 166), 9E9 VH (
  • a chimeric, humanized, or human monoclonal antibody or fragment thereof that binds a human EphB4 antigen wherein the antibody comprises a VH domain comprising at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3-5 and 13-15 and further comprising one or more framework region of a heavy chain variable region selected from the group consisting of 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO:82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO: 118), 6G2 VH (SEQ ID NO: 130), 7A7 VH (SEQ ID NO: 142), 8D10 VH (SEQ ID NO:
  • the antibody further comprises a VL domain comprising at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-10 and 18-20, and further comprising one or more framework region of a light chain variable region selected from the group consisting of 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO:112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO:148), 8D10 VL (SEQ ID NO: 160), 9D9 VL (SEQ ID NO: 172), 9E9 VL (S
  • VH domain comprises an amino acid sequence selected from the group consisting of 22E6 VH (SEQ ID NO:22), 30C2 VH (SEQ ID NO:34), 3G8 VH (SEQ ID NO:46), 4F2 VH (SEQ ID NO:58), 1B7 VH (SEQ ID NO:70), IElO VH (SEQ ID NO:82), 2F4 VH (SEQ ID NO:94), 2G3 VH (SEQ ID NO: 106), 3E9 VH (SEQ ID NO: 118), 6G2 VH (SEQ ID NO: 130), 7A7 VH (SEQ ID NO: 142), 8D10 VH (SEQ ID NO: 154), 9D9 VH (SEQ ID NO: 166), 9E9 VH (SEQ ID NO: 178), 9El 2 VH (SEQ ID NO: 190), 10C4 VH (SEQ ID NO:202), 11H4 VH (SEQ ID NO:
  • the antibody further comprises a VL domain comprising at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-10 and 18-20, and further comprising at least one framework region of a light chain variable region selected from the group consisting of 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO:112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO:148), 8D10 VL (SEQ ID NO: 160), 9D9 VL (SEQ ID NO : 172), 9E9 VL (
  • VL domain comprising an amino acid sequence selected from the group consisting of 22E6 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO:40), 3G8 VL (SEQ ID NO:52), 4F2 VL (SEQ ID NO:64), 1B7 VL (SEQ ID NO:76), IElO VL (SEQ ID NO:88), 2F4 VL (SEQ ID NO: 100), 2G3 VL (SEQ ID NO: 112), 3E9 VL (SEQ ID NO: 124), 6G2 VL (SEQ ID NO: 136), 7A7 VL (SEQ ID NO: 148), 8D10 VL (SEQ ID NO: 160), 9D9 VL (SEQ ID NO: 172), 9E9 VL (SEQ ID NO: 184), 9El 2 VL (SEQ ID NO: 196), 10C4 VL (SEQ ID NO:208), 11H4 VL (SEQ ID NO:28), 30C2 VL (SEQ ID NO
  • the antibody is selected from the group consisting of: (a) an antibody comprising a VH comprising the amino acid sequence of 22E6 VH (SEQ ID NO:22) and VL comprising the amino acid sequence of 22E6 VL (SEQ ID NO:28); and (b) an antibody comprising a VH comprising the amino acid sequence of 30C2 VH (SEQ ID NO:34) and VL comprising the amino acid sequence of 30C2 VL (SEQ ID NO:40).
  • an antibody comprising a VH comprising the amino acid sequence of 1B7 VH (SEQ ID NO: 70) and VL comprising the amino acid sequence of 1B7 VL (SEQ ID NO:76);
  • an antibody comprising a VH comprising the amino acid sequence of 2G3 VH (SEQ ID NO: 106) and VL comprising the amino acid sequence of 2G3 VL (SEQ ID NO:112);
  • a vector comprising the nucleic acid of embodiment 13. [[00338811]] 1155.. An isolated cell comprising the vector of embodiment 14.
  • a method of producing an antibody comprising culturing the isolated cell of embodiment 16 under conditions sufficient for the production of the antibody and recovering the antibody from the culture.
  • a pharmaceutical composition comprising the antibody as in any of embodiments 1-12 in a pharmaceutically-acceptable carrier.
  • angiogenesis-associated diseases and disorders including, but not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; inflammatory disorders such as immune and non- immune inflammation; chronic articular rheumatism and psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation and wound healing; telangiect
  • a method of treating or preventing a malignancy including, but not limited to, ovarian cancer, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi sarcoma, and leukemia comprising administering to a human in need thereof a therapeutically-effective amount of the antibody of embodiments 2, 6 or 12.
  • a malignancy including, but not limited to, ovarian cancer, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi sarcoma, and leukemia
  • a method of treating or preventing a bone remodeling disease such as osteopetrosis and osteoporosis comprising administering to a human in need thereof a therapeutically-effective amount of the antibody of embodiments 2, 6 or 12.
  • RNA is isolated and purified from a #47 or # 131 anti-EphB4 antibody secreting cell line using a Straight A's mRNA Purification kit (Novagen, Madison, WI) according to the manufacturer's instructions.
  • cDNA is synthesized with a First Strand cDNA synthesis kit (Novagen) as recommended by the manufacturer.
  • Amplification of both V H and V L genes is carried out using the IgGVH and Ig ⁇ VL oligonucleotides from the Mouse Ig- Primer Set (Novagen) as suggested by the manufacturer.
  • the chimeric Fab of murine anti-EphB4 antibodies #47 and #131 are cloned into an M13-based phage expression vector.
  • the cloning is carried out essentially as described in US20050048617, US Application 11/377,148 (filed on 3/17/2006, Docket No. AE650CP1), and PCT/US2006/009745 (filed on 3/17/2006, Docket No. AE650PCT2); all of which are incorporated hereby by reference in their entirety).
  • #47 and #131 heavy and light chain genes are synthesized by PCR from the corresponding pSTBlue-1 clones (see Section 6.1.1) using the appropriate combinations of primers listed in Table 2. Forward (“for" in Table 2) primers are biotinylated to allow minus strand purification.
  • An M13-based phage expression vector is used to express a chimeric Fab fragment containing the first constant domain of a human ⁇ l heavy chain and the constant domain of a human kappa (K) light chain under the control of the lacZ promoter.
  • Cloning is carried out by hybridization mutagenesis, Kunkel et al, Methods Enzymol. 154:367-382 (1987), as described Wu et al, Methods MoL Biol. 207: 213-233 (2003).
  • minus single-stranded DNA corresponding to the VH and VL of #47 or #131 is purified from the final PCR products by ethanol precipitation after dissociation of the double-stranded PCR product using sodium hydroxide and elimination of the biotinylated plus strand by streptavidin-coated magnetic beads as described (H. Wu, et al, Methods MoI. Biol. 207: 213- 233(2003); H. Wu, Methods MoI. Biol. 207: 197-212 (2003)).
  • Equimolar amounts of #47 or #13 IVH and VL minus strands were mixed to construct chimeric Fab #47 or #131, respectively.
  • the different mixes are individually annealed to a positive strand preparation of the M13-based vector and followed by second strand synthesis as described by Kunkel et al (Methods Enzymol. 154, 367-382 (1987)). DNA is electroporated into XLl-Blue for plaque formation on XLl-Blue bacterial lawn or production of Fab fragments as described Wu et al, Methods MoI. Biol. 207: 213-233 (2003).
  • a polynucleotide encoding a human EphB4-Fc fusion protein (SEQ ID NO: 237) consisting of human EphB4 fused with the Fc portion of a human IgGl, ⁇ see US20050084873) can be generated via overlap PCR following standard protocols.
  • Commercially available human cDNA may be used as template for the PCR reactions ⁇ e.g., FirstChoice® PCR-Ready and RACE-Ready cDNA from Ambion).
  • Human EphB4-Fc fusion protein can be expressed in human embryonic kidney (HEK) 293 cells and purified by protein G affinity chromatography using standard protocols.
  • Human EphB4-Fc biotinylation is carried out using an EZ-Link Sulfo-NHS-LC-Biotinylation Kit according to the manufacturer's instructions (Pierce, Rockford, IL).
  • #47 anti-EphB4 antibody is humanized using "framework shuffling" of human heavy and light (K) germline genes.
  • the procedure is carried out essentially as previously described (US20050048617, US Application 11/377,148 (filed on 3/17/2006, Docket No. AE650CP1), PCT/US2006/009745 (filed on 3/17/2006, Docket No. AE650PCT2), all of which are incorporated hereby by reference in their entirety).
  • the corresponding V H and V L framework-shuffled libraries are all generated by total in vitro synthesis (Dall'Acqua, W. F. et al, Methods 36:43-60 (2005); Wu, H., Methods MoI. Biol. 207:197- 212. (2003)) using PCR by overlap extension (Ho, S. N. et al., Gene 77:51-59 (1989)).
  • Oligonucleotide primers having the nucleotide sequence of SEQ ID NO:240-333 were used for the in vitro synthesis of V H framework-shuffled libraries comprising the #47 VH CDRs.
  • Oligonucleotide primers having the nucleotide sequence of SEQ ID NO:428-507 were used for the in vitro synthesis of V L framework- shuffled libraries comprising the #47 VL CDRs.
  • #47 anti-EphB4 antibody is humanized using two distinct approaches (A and
  • Approach A involves a one-step strategy in which both its framework-shuffled heavy and light chains are simultaneously selected for binding to human EphB4-Fc. Screening of -1200 clones from this #47 antibody derived library resulted in the identification of one particular clone, named 30C2 thereafter, which consistently exhibited binding affinity within 2-fold that of exhibited by its chimeric counterpart and above that of an irrelevant antibody (by - 30- fold) using a single point ELISA.
  • Approach B involves a two-step humanization process in which the light and heavy chains of anti-EphB4 antibody #47 are successively humanized.
  • a #47-derived framework shuffled light chain library is first paired with the chimeric Fd portion of clone 47. Screening of- 600 clones from this Fab library for binding to human EphB4-Fc lead to the selection of 18 humanized light chains. These are then combined with a #47-derived framework shuffled heavy chain library.
  • Clones 30C2 and 22E6 are further characterized by dideoxynucleotide sequencing using a ABI3000 genomic analyzer. The translated amino acid sequence of clones clones 30C2 and 22E6 is shown in Figure 1.
  • #131 anti-EphB4 antibody is humanized using "framework shuffling" of human heavy and light (K) germline genes. The procedure is carried out essentially as previously described (US20050048617, US Application 11/377,148 (filed on 3/17/2006, Docket No. AE650CP1), PCT/US2006/009745 (filed on 3/17/2006, Docket No. AE650PCT2), all of which are incorporated hereby by reference in their entirety). In particular, the corresponding v H and v L framework-shuffled libraries are all generated by total in vitro synthesis (Dall'Acqua, W. F.
  • Oligonucleotide primers having the nucleotide sequence of SEQ ID NO:334-427 were used for the in vitro synthesis of V H framework-shuffled libraries comprising the #131 VH CDRs.
  • Oligonucleotide primers having the nucleotide sequence of SEQ ID NO:508-587 were used for the in vitro synthesis of V L framework-shuffled libraries comprising the # 131 VL CDRs .
  • #131 anti-EphB4 antibody is humanized using two distinct approaches (A and B).
  • Approach A involves a one-step strategy in which both its framework-shuffled heavy and light chains are simultaneously selected for binding to human EphB4-Fc.
  • Screening of clones from this #131 -derived library resulted in the identification of two particular clones, named 3G8 and 4F2 thereafter, which consistently exhibited a signal similar to that exhibited by its chimeric counterpart and above that of an irrelevant antibody using a single point ELISA.
  • the clones are further characterized by dideoxynucleotide sequencing using an ABI3000 genomic analyzer.
  • the translated amino acid sequence of clones 3G8 and 4F2 is reported in Figure 2.
  • Approach B involves a two-step humanization process in which the light and heavy chains of monoclonal antibody #131 are successively humanized.
  • a #131 -derived framework shuffled light chain library is first paired with the chimeric Fd portion of clone #131. Screening of clones from this Fab library for binding to human EphB4-Fc lead to the selection of 9 humanized light chains. These are then combined with a #131 -derived framework shuffled heavy chain library.
  • a secondary screen using Fab fragments expressed in periplasmic extracts prepared from 15 ml- bacterial culture is carried out. More precisely, two ELISAs are used: (i) a functional ELISA in which individual wells of a 96-well Maxisorp Immunoplate are coated with 500 ng of human EphB4-Fc and blocked with 3%BSA/PBS for 2h at 37 0 C. 2-fold serially diluted samples are then added and incubated for Ih at room temperature. Incubation with a goat anti-human kappa horseradish peroxydase (HRP) conjugate then follows.
  • HRP horseradish peroxydase
  • HRP activity is detected with TMB substrate and the reaction quenched with 0.2 M H 2 SO 4 . Plates are read at 450 nm; (ii) an anti-human Fab quantification ELISA that is carried out essentially as described. (Wu et al, Methods MoI. Biol. 207: 213-233 (2003)). Briefly, individual wells of a 96-well Immulon Immunoplate are coated with 100 ng of a goat anti-human Fab antibody and then incubated with 2-fold serially diluted samples (starting at a 1/25 dilution) or standard (human IgG Fab, 500-3.91 ng/ml).
  • HRP activity is detected with TMB substrate and the reaction quenched with 0.2 M H 2 SO 4 . Plates are read at 450 nm.
  • EphB4 binding curves for humanized versions of #47 and #131 anti-EphB4 antibody are shown in Figure 3.
  • Chimeric Fab of #47 or #131 was included in the assays as a positive control.
  • the results show that several of the humanized anti-EphB4 Fabs identified display EphB4 binding similar to that of the chimeric Fab of the parental murine antibody.
  • Polynucleotides encoding the VH region of each humanized #47 or #131 antibody are cloned into a vector to facilitate mammalian expression of chimeric antibodies.
  • the vector used comprises eukaryotic transcription control elements operably linked to a polynucleotide encoding the MGDNDIHF AFLSTGVHS VH leader (SEQ ID NO:238) and a human IgH ⁇ l constant region wherein said transcription control elements comprise a CMV immediate early promoter and a SV40 poly A addition signal.
  • Cloning steps are designed to ensure that the polynucleotide sequences encoding the VH leader, VH region and IgH ⁇ l constant region are joined in frame within the final heavy chain expression vector.
  • Polynucleotides encoding the VL region of each humanized #47 or #131 antibodies are cloned into a vector to facilitate mammalian expression of chimeric antibodies.
  • the vector used comprises eukaryotic transcription control elements operably linked to a polynucleotide encoding the MDMRVP AQLLGLLLL WLPGAKC VKI-L 12 leader (SEQ ID NO:239) and a human IgL ⁇ constant region wherein said transcription control elements comprise a CMV immediate early promoter and a SV40 poly A addition signal.
  • Cloning steps are designed to ensure that the polynucleotide sequences encoding the VK leader, VL region and IgL ⁇ constant region are joined in frame within the final heavy chain expression vector.
  • Cloning steps are performed following standard protocols.
  • the ligation product is used to transform DHlOB competent E. coli cells according to the manufacturer's protocols.
  • Colonies containing the plasmid and a correct sized insert can be identified using various methods known in the art ⁇ e.g. restriction digest of vector DNA preparation, PCR amplification of vector sequences).
  • Plasmid clones with correct sized insert are sequenced using dideoxy sequencing reaction (e.g., BigDye® Terminator v3.0 Cycle Sequencing Ready Reaction Kit, ABI).
  • Plasmid DNA is prepared from selected clones using the QIAGEN Mini and Maxi Plasmid Kit according to the manufacturer's protocols.
  • Pairs of expression vector DNA preparations encoding each EphB4 binding humanized VH-VL pair identified in the above described screen are used to co-transfect HEK293 cells.
  • Transfected HEK293 cells are cultured for three days to yield antibody- containing conditioned medium suitable for determining total IgG concentrations and EphB4 binding activity.
  • the secreted, soluble humanized IgGIs were purified from the conditioned media directly on 1 ml HiTrap protein A or protein G columns according to the manufacturer's instructions (APBiotech, Inc., Piscataway, NJ). Purified IgGIs (typically > 95% homogeneity, as judged by SDS-PAGE) were dialyzed against phosphate buffered saline (PBS), flash frozen and stored at -70 0 C.
  • PBS phosphate buffered saline
  • HEK 293 cells are transiently transfected with the various antibody constructs (chimeric or humanized IgGl) in 35 mm, 6-wells dishes using Lipofectamine and standard protocols. Typically, 6 ⁇ l Lipofectamine 2000 (Invitrogen) is added to 250 ⁇ L Optimem medium (Invitrogen) and incubated at room temperature for 5 min. The mixture is then successively combined with 2 ⁇ g of each relevant heavy and light chain-encoding mammalian expression vector (see above), incubated at room temperature for 20 min, and applied to confluent HEK 293 cells in 1.5 mL DMEM medium (Invitrogen). Cells are then incubated at 37 0 C under 5% CO 2 .
  • Supernatants are harvested twice at 72 and 144 h post- transfection (referred to as first and second harvest, respectively).
  • the secreted, soluble IgGIs are then assayed in terms of production yields by ELISA.
  • individual wells of a 96-well Biocoat plate (BD Biosciences, San Jose, CA) coated with a goat anti- human IgG are incubated with samples (transfection supernatants) or standards (human IgG, typically 0.5-100 ng/ml), then with a horse radish peroxydase conjugate of a goat anti-human IgG antibody.
  • Peroxydase activity is detected with 3,3',5,5'-tetramethylbenzidine and the reaction is quenched with 0.2 M H 2 SO 4 . Plates are read at 450 nm.
  • Fab fragments for pi and DSC experiments are obtained from enzymatic cleavage of the chimeric or humanized IgGl versions of the #47 or #131 antibodies. Digestion is carried out using an ImmunoPure Fab Preparation Kit according to the manufacturer's instructions (Pierce, Rockford, IL). Purification of the Fab region can be performed using different techniques. For example protein A columns (Pierce) or affmityPak protein L columns (Pierce) may be used according to the manufacturer's instructions. Purification of the Fab region can also be achieved using an aminolink column (Pierce) conjugated to a goat-anti-human kappa antibody (Southern Biotech, Birmingham, AL) according to standard protocols.
  • the purified Fabs (typically > 95% homogeneity, as judged by SDS-PAGE) are dialyzed against PBS, flash frozen and stored at -70 0 C.
  • the presence of carbohydrates in Fab may be investigated using a glycoprotein carbohydrate estimation kit as recommended by the manufacturer (Pierce). 7.2.2. Analysis of human EphB4 binding by humanized #47 and #131 antibodies using KinExa
  • Coated beads are then separated from unreacted EphB4-Fc using a gentle pulse spin and blocked for approximately 2 h at room temperature with 1 M Tris, pH 8.0, bovine serum albumin 10 mg/mL. Beads are then resuspended in 30 mL of run buffer (PBS, pH 7.4, 0.02% NaN 3 ) and packed into a column.
  • run buffer PBS, pH 7.4, 0.02% NaN 3
  • chimeric and humanized IgGIs are prepared at concentrations of 25 and 250 pM.
  • Human EphB4-Fc is titrated across these IgG solutions at concentrations ranging from 1 fJVI- 12.5 nM and incubated for 3-6 days at room temperature.
  • Samples are then filtered through the above-mentioned column in order to capture free IgGs by the EphB4-coated beads.
  • the amount of free IgG is assessed from the fluorescence signal obtained after the passing of Cy5 -labeled goat anti-human IgG F(ab') 2 (typically 0.5-2 ⁇ g/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) through the column.
  • Dissociation constants K D S
  • K D S Dissociation constants
  • Binding affinities of humanized anti-EphB4 antibodies determined using a KinExa instrument is listed in Table 3.
  • EphB4-Fc The interaction of soluble antibodies (or fragments thereof) with immobilized human EphB4-Fc may be monitored by surface plasmon resonance detection using a BIAcore 3000 instrument (Biacore International AB, Uppsala, Sweden). EphB4-Fc is coupled to the dextran matrix of a CM5 sensor chip (Pharmacia Biosensor) using an Amine Coupling Kit as described (27) at a surface density of between 26 and 534 RU. IgGs are diluted in 0.01 M HEPES, pH 7.4 containing 0.15 M NaCl, 3 mM EDTA and 0.005% P20.
  • Pre-cast ampholine gels (Amersham Biosciences, Uppsala Sweden; pi range 3.5-9.5) are typically loaded with 8 ⁇ g of protein (Fab or IgG) as determined by their absorbance at 280 nm. Protein samples are dialyzed in 10 mM Histidine- HCl, pH 6.0 prior to loading on the gel. Broad range pi marker standards (Amersham Biosciences, pi range 3-10) are used to determine relative pi for the various antibodies or antibody fragments. Electrophoresis is performed at 1500 V, 50 mA for 105 min. Gels are fixed for 45 min using Sigma fixing solution (Sigma, Saint Louis, MO). Staining is performed overnight at room temperature using Simply Blue stain (Invitrogen).
  • Destaining is carried out with a solution that consists of 25 % ethanol, 8 % acetic acid and 67 % purified water. Isoelectric points are determined using a Bio-Rad GS-800 Densitometer running Quantity One Imaging Software (Bio-Rad, Hercules, CA).
  • Isoelectric points of humanized anti-EphB4 antibodies are listed in Table 4. Table 4. Isoelectric points of various anti-human EphB4 antibodies .
  • Thermal melting temperatures are measured with a VP-DSC instrument (MicroCal, LLC, Northampton, MA) using a scan rate of 1.0 °C/min and a temperature range of 25-110 0 C. A filter period of 8 s is used along with a 15 min pre-scan thermostating. Samples (Fab or IgG) are prepared by dialysis into 10 mM Histidine-HCl, pH 6.0 and used at concentrations typically ranging from 200-400 ⁇ g/ml as determined by their absorbance at 280 nm. Multiple baselines are run in the same buffer in both the sample and reference cell to establish thermal equilibrium. After the baseline is subtracted from the sample thermogram, the data are concentration-normalized and the melting temperatures determined using the "Origin" software (OriginLab Corporation, Northampton, MA).
  • Tm Thermal melting temperatures
  • thermogram peaks For each sample, single Tm values correspond to the major transition seen in the corresponding thermograms. b At least two major discrete transitions were observed in this sample. Values reflect each of these individual thermogram peaks.
  • Human EphB4 activation and degradation analyses may be performed using cells endogenously expressing human EphB4.
  • Sub-confluent monolayers of EphB4 expressing cells (plated at a density of ⁇ 3 X 10 5 /35 mm well) are treated with 10 ⁇ g/ml of the various test antibodies for 15 min up to 48 h.
  • Detergent extracts then are prepared using 100- 200 ⁇ l 1% Triton X-100 (Sigma)/well.
  • a cell lysate sample corresponding to ⁇ 100 ⁇ g of total protein is immunoprecipitated using a mouse anti-human EphB4 antibody ⁇ e.g., clone 3D7F8 from Invitrogen), resolved by SDS-PAGE, transferred to a BioTrace PVDF membrane (VWR, West Chester, PA), and probed successively with a 1/1,000 dilution of a mouse anti-phosphotyrosine antibody (Millipore) and a 1/10,000 dilution of a goat anti- mouse horseradish peroxydase conjugate (Jackson ImmunoResearch). Horseradish peroxydase activity is detected using an ECL kit (Amersham Biosciences).
  • ⁇ 40 ⁇ g of total protein (as determined by the BCA method; equal amounts were used for each sample) is resolved by SDS-PAGE, transferred to a BioTrace PVDF membrane (VWR), and probed successively with 0.5 ⁇ g/ml of a mouse anti-human EphB4 antibody (e.g., clone 3D7F8 from Invitrogen) and a 1 :10,000 dilution of a goat anti-mouse horseradish peroxydase conjugate (Jackson ImmunoResearch). Horseradish peroxydase activity was detected using an ECL kit (Amersham Biosciences). Membranes were then stripped and reprobed with antibodies specific for ⁇ -actin to confirm equal sample loading.
  • a mouse anti-human EphB4 antibody e.g., clone 3D7F8 from Invitrogen
  • Jackson ImmunoResearch horseradish peroxydase activity was detected using an ECL kit (Amersham Biosciences).
  • EphB4 protein activity has been shown to influence cell migration in a number of experimental systems (e.g., US20050249736; US20050084873; Yang, N.Y. et al., J Biol Chem, PMID:16950769 (2006); Xia, G. et al, Clin Cancer Res, l l(12):4305-15 (2005) all of which are incorporated by reference in their entireties).
  • the ability of a humanized anti- EphB4 antibody to inhibit cell migration can be assayed using standard protocols.
  • an anti-EphB4 antibody to inhibit migration of mesithelioma cell lines NCI H28, NCI H2052, NCI H2373, and MSTO 21 IH may be assayed as follows using a modified Boyden chamber assay (Xia, G. et al., Clin Cancer Res, 11(12):4305-15 (2005)). Exponentially growing cells are incubated with various amounts (for example 0.1 to 20 ⁇ g/ml) of anti-EphB4 or negative control antibody. 6 hours later, 0.5 x 10 5 cells are transferred into 8 ⁇ m Matrigel-precoated inserts (BD Bioscience, Palo Alto, CA).
  • the inserts are placed in companion wells containing RPMI 1640 supplemented with 5% FBS and 5 ⁇ g/mL f ⁇ bronectin as a chemoattractant. Following 24-hour incubation, the inserts are removed and the noninvading cells on the upper surface are removed with a cotton swab. The cells on the lower surface of the membrane are fixed in 100% methanol for 15 minutes, air dried, and stained with Giemsa stain for 2 minutes. The cells are counted in five individual high-power fields for each membrane under a light microscope. Assays are done in triplicate for each treatment group.
  • an anti-EphB4 antibody inhibits cell migration.
  • HUVEC Human Umbilical Vein Endothelial Cell
  • normal HUVECs are obtained from Cambrex (BioWhittaker) and maintained in EBM2 medium supplemented with 0.1 mg/ml endothelial growth supplement (crude extract from bovine brain), penicillin (50 U/ml), streptomycin (50 U/ml), 2 mmol/1 glutamine and 0.1 mg/ml sodium heparin.
  • Migration of HUVECs is assessed using "Matrigel" matrix-coated 9-mm cell culture inserts (pore size, 8 ⁇ m; Becton Dickinson, Franklin Lakes, N.J.) set in a 24-well plate.
  • the HUVEC cells are seeded at a density of 5x10 3 cells per well into the upper layer of the culture insert and cultured with serum- free EBM in the presence of the test EphB4 antibodies for 24 h.
  • the control group is cultured in the same media without EphB4 antibodies.
  • 0.5 ml of the human SCC15 cell line conditioned medium is filled into the lower layer of the culture insert as a chemo-attractant.
  • the cells are incubated for 24 h, then the remaining cells in the upper layer are swabbed with cotton and penetrating cells in the lower layer are fixed with 5% glutaraldehyde and stained with Diff Quick.
  • the total number of cells passing through the Matrigel matrix and each 8 ⁇ m pore of the culture insert is counted using optical microscopy. Negative unstimulated control values are subtracted from stimulated control and antibody treated sample values and invasion index (cell number/area) is calculated. A decrease in the invasion index following anti-EphB4 treatment would indicate that the anti- EphB4 antibody inhibits HUVEC migration in response to stimulation.
  • a humanized anti-EphB4 antibody may inhibit angiogenesis
  • EphB4 protein activity has been shown to influence angiogenesis in a number of experimental systems (e.g., US20050249736; US20050084873; Kertesz, N. et al., Blood, 106(7):2330-8 (2006); He, S. et al, Invest Opthalmol Vis Sci, 46(12):4772-9 (2005) all of which are incorporated by reference in their entireties).
  • the ability of a humanized anti- EphB4 antibody to inhibit angiogenesis may be assayed using standard protocols.
  • the ability of anti-EphB4 antibodies to inhibit angiogenesis may be tested using a Matrigel in vivo vascularization assay as described in US20050249736. Briefly, Matrigel supplemented with bFGF and VEGF with and without humanized anti- EphB4 antibody is injected s.c. into Balb/C nu/nu mice, forming semi-solid plugs, for six days. Plugs are removed, fixed in formalin, and embedded in paraffin. Gel sections (10 ⁇ m in thickness) are then stained with hematoxylin and eosin and examined under a microscope. Plugs without growth factors are expected to have virtually no vascularization or vessel structures after 6 days.
  • plugs supplemented with bFGF and VEGF should have extensive vascularization and vessels throughout the plug.
  • Plugs taken from mice treated with anti- EphB4 antibody are expected to have had markedly reduced vascularization of plugs, comparable to plugs without growth factor.
  • a humanized anti-EphB4 antibody may inhibit tumor growth
  • EphB4 protein activity has been shown to influence tumor cell survival in a number of experimental systems (e.g., US20050249736; US20050084873; Masood, R. et al., Int J Cancer, 119(6): 1236-48 (2006); Xia, G. et al, Oncogene, 25(5):769-80 (2005) all of which are incorporated by reference in their entireties).
  • the ability of a humanized anti- EphB4 antibody to inhibit tumor growth may be assayed using standard protocols.
  • the ability of a humanized anti-EphB4 antibody to inhibit tumor growth may be assayed using head and neck squamous cell carcinoma (FINSCC) cell line SCC15 as described in US20050249736. Briefly, 5xlO 6 logarithmically growing SCC15 cells, in the presence or absence of human bFGF, are subcutaneously injected into athymic Balb/c nude mice along with Matrigel (BD Bioscience) synthetic basement membrane (1 :1 v IY). In addition, the injection mixture contains either a humanized anti-EphB4 antibody or a control antibody of irrelevant specificity. Tumor cells are allowed to grow for up to two weeks.
  • FTSCC head and neck squamous cell carcinoma
  • tumors are excised, measured, and processed for histological examination. Histological examination may include but is not limited to assesment of the extent of apoptosis/ necrosis; assessment of vascular densities; assessment of lymphocyte invasion; and assessment of tumor cell proliferation rate following standard protocols. Reduced tumor size, increased tumor cell death and/or reduced tumor cell proliferation in the presence of anti-EphB4 antibody would suggest that the anti-EphB4 antibody inhibits tumor growth.
  • Humanized #131 anti-EphB4 antibodies act as receptor agonists.
  • Humanized #131 anti-EphB4 antibody treatment induces EphB4 protein phosphorylation.
  • PC3 a human cancer cell line was seeded into six-well plates (3 x 10 5 cells per well) containing RPMI- 1640 media with 10% fetal bovine serum and incubated for 24 hours at 37°C.
  • Cells were treated with various concentrations (0.1-50 ⁇ g/ml) of #131, IElO, 3E9, 3G8, 4F2, and 10C4 anti-EphB4 antibodies for a short period of time (5-15 minutes) at 37°C.
  • Control cells treated with no antibody, 10 ⁇ g/ml Ephrin B2-Fc, or 10 ⁇ g/ml isotype control antibody were included in the assay.
  • experimental antibody concentration is fixed, for example at 10 ⁇ g/ml, and the incubation time is varied by one minute increments between 5 and 15 minutes.
  • cells were washed with cold phosphate buffered saline (PBS) and lysed with lysis buffer containing 1% Triton-X 100 (TXlOO) (Boston Bioproducts, Boston, MA) and protease inhibitors.
  • PBS cold phosphate buffered saline
  • TXlOO Triton-X 100
  • Protein concentration of the different cell lysates was measured using standard Coomassie assays (Pierce, Rockford, IL) to ensure that the subsequent immunoprecipitation was performed using equal amounts of input protein.
  • EphB4 protein was immunoprecipitated from cell lysate (100-300 ⁇ g inputi protein) using #47 anti-EphB4 antibody according to standard protocols.
  • the immunoprecipitated material was separated by SDS PAGE, transferred to a membrane and probed with an anti-phospho tyrosine antibody (Upstate, Charlottsville, VA) to asses the phosphorylation status of the immunoprecipitated EphB4 protein.
  • the membranes were subsequently stripped and reprobed with an anti- EphB4 antibody to control for any variation in EphB4 protein between samples.
  • SDS PAGE analysis and Western blotting was performed according to standard protocols. Experimental results are summarized in Table 6.
  • the IElO, 3E9, 3G8, 4F2, and 10C4 humanized anti- EphB4 antibodies are capable of inducing EphB4 protein phosphorylation.
  • Humanized #131 anti-EphB4 antibody mediates EphB4 protein degradation.
  • PC3 a human cancer cell line was seeded into six-well plates (3 x 10 5 cells per well) containing RPMI- 1640 media with 10% fetal bovine serum and incubated for 24 hours at 37°C.
  • Cells were treated with various concentrations (0.1-50 ⁇ g/ml) of #131, IElO, 3E9, 3G8, 4F2, and 10C4 anti-EphB4 antibodies for several hours (1-8 hrs) at 37°C.
  • Control cells treated with no antibody, 10 ⁇ g/ml Ephrin B2-Fc, or 10 ⁇ g/ml isotype control antibody were included in the assay.
  • experimental antibody concentration is fixed, for example at 10 ⁇ g/ml, and the incubation time is varied by one hour increments between 1 and 8 hours.
  • cells were washed with cold phosphate buffered saline (PBS) and lysed with lysis buffer containing 1% Triton-X 100 (TXlOO) (Boston Bioproducts, Boston, MA) and protease inhibitors.
  • PBS cold phosphate buffered saline
  • TXlOO Triton-X 100
  • Protein concentration of the different cell lysates was measured using standard Coomassie assays (Pierce, Rockford, IL) to ensure that the SDS PAGE analysis was performed using equal amounts of input protein.

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Abstract

L'invention concerne des versions humaines d'anticorps monoclonaux de souris anti-EphB4. L'invention concerne également des compositions pharmaceutiques, des compositions immunothérapeutiques et des procédés d'utilisation d'anticorps thérapeutiques qui se lient à l'antigène humain EphB4 et qui peuvent médier ADCC, CDC, et/ou l'apoptose du traitement de tumeurs malignes humaines et de pathologies et de maladies liées à la vasculogenèse.
PCT/US2007/080270 2006-10-04 2007-10-03 Anticors anti-ephb4 humains et leur utilisation dans le traitement de l'oncologie et de maladies liées a la vasculogenèse WO2008042941A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008112290A2 (fr) * 2007-03-12 2008-09-18 Vasgene Therapeutics, Inc. Utilisation du récepteur ephb4 comme marqueur diagnostique et cible thérapeutique dans le cancer de l'ovaire
WO2009023185A1 (fr) * 2007-08-13 2009-02-19 Vasgene Therapeutics, Inc. Traitement contre le cancer utilisant des anticorps humanisés qui se lient à ephb4
US9128101B2 (en) 2010-03-01 2015-09-08 Caris Life Sciences Switzerland Holdings Gmbh Biomarkers for theranostics
US9469876B2 (en) 2010-04-06 2016-10-18 Caris Life Sciences Switzerland Holdings Gmbh Circulating biomarkers for metastatic prostate cancer

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6719971B1 (en) * 1991-06-14 2004-04-13 Genentech, Inc. Method for making humanized antibodies
US20050249736A1 (en) * 2003-03-12 2005-11-10 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6719971B1 (en) * 1991-06-14 2004-04-13 Genentech, Inc. Method for making humanized antibodies
US20050249736A1 (en) * 2003-03-12 2005-11-10 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008112290A2 (fr) * 2007-03-12 2008-09-18 Vasgene Therapeutics, Inc. Utilisation du récepteur ephb4 comme marqueur diagnostique et cible thérapeutique dans le cancer de l'ovaire
WO2008112290A3 (fr) * 2007-03-12 2008-12-31 Vasgene Therapeutics Inc Utilisation du récepteur ephb4 comme marqueur diagnostique et cible thérapeutique dans le cancer de l'ovaire
WO2009023185A1 (fr) * 2007-08-13 2009-02-19 Vasgene Therapeutics, Inc. Traitement contre le cancer utilisant des anticorps humanisés qui se lient à ephb4
US8975377B2 (en) 2007-08-13 2015-03-10 Vasgene Therapeutics, Inc Cancer treatment using humanized antibodies that bind to EphB4
US9128101B2 (en) 2010-03-01 2015-09-08 Caris Life Sciences Switzerland Holdings Gmbh Biomarkers for theranostics
US9469876B2 (en) 2010-04-06 2016-10-18 Caris Life Sciences Switzerland Holdings Gmbh Circulating biomarkers for metastatic prostate cancer

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