WO2010019565A2

WO2010019565A2 - Anti-ephrin b2 antibodies and their use in treatment of disease

Info

Publication number: WO2010019565A2
Application number: PCT/US2009/053397
Authority: WO
Inventors: Partha Chowdhury; Emma T. Bowden; Elizabeth Bruckheimer; Steve Coats; Peter Kiener; Herren Wu
Original assignee: Medlmmune, Llc
Priority date: 2008-08-12
Filing date: 2009-08-11
Publication date: 2010-02-18
Also published as: WO2010019565A3

Abstract

The present invention relates to anti-Ephrin B2 antagonist antibodies. The invention further relates to pharmaceutical compositions, imrmmotherapeiuic compositions, and methods of using such compositions tor the treatment of diseases and disorders, such as. but not limited to, malignancies.

Description

ANTI-EPHRIN B2 ANTIBODIES AND THEIR USE IN TREATMENT OF

DISEASE

INTRODUCTION

[0001] The present invention relates to Ephrin B2 antibody compositions and methods for modulating Ephrin B2 activity. The present invention further relates to methods and compositions designed for the treatment, management, prevention and/or amelioration of various disorders associated with aberrant expression and/or activity of Ephrin B2 and/or one or more of the polypeptides, which interact with Ephrin B2 (e.g., EphB4 and EphA4). In particular, the invention provides methods for the treatment, management, prevention and/or amelioration of a disorder associated with aberrant expression and/or activity(ies) of Ephrin B2 and/or the binding partners which interact with Ephrin B2 (e.g., EphB4 and/or EphA4), the method comprising administering to a subject in need thereof a sufficient amount of one or more Ephrin B2 antibodies of the invention. In addition, the invention provides methods for the treatment or prevention of paramyxovirus (e.g., Nipah virus) infection, the methods comprise the administration to a subject in need thereof a sufficient amount of one or more Ephrin B2 antibodies of the invention.

BACKGROUND

[0002] Ephrin B2 is a 40 kDa transmembrane protein that belongs to the Ephrin ligand family and binds Eph receptors such as EphB2, B3, and B4. The involvement of Ephrin B2 in a range of physiological systems has been described, including vascular, lymphatic, neuronal, and renal development, neurotransmission, synaptic plasticity, and tumor metastasis. See, e.g., Oike, Y. etal. (2002) Blood 100:1326; Makinen, T., etal. (2005) Genes Dev. 19:3 ; Takahashi, T. etal (2001) J. Am. Soc. Nephrol. 12:2673; Martinez, A. & E. Soriano, (2005) Brain Res. Brain Res. Rev. 49:211; Meyer, S. etal (2005) Int. J. Oncol. 27:119; and Fuller, T. etal. (2003) J. Cell Sci. 116:2461. Ephrin B2 and its cognate receptors are expressed in complementary fashion on adjacent cells. Upon ligation, both Ephrin-B2 and Eph receptors initiate signals in their respective cells. The combination of forward and reverse signaling is central to the tissue development and remodeling functions of Ephrin and Eph proteins. [0003] It has further been reported that Ephrin B2 ligand is a molecular marker for the arterial endothelium at the earliest stages of embryonic angiogenesis, while EphB4, an Ephrin B2 receptor reciprocally marks the venous endothelium. It has also been reported that antibodies which disrupt the Ephrin B2-EphB4 interaction can inhibit angiogenesis. See, e.g., published U.S. patent application 20070031435. Moreover, it has been demonstrated that overexpression of specific Ephs and ephrins is associated with a poor prognosis in human tumours.

[0004] Angiogenesis is a critical process in the growth, progression, and metastasis of solid tumors within the host. During physiologically normal angiogenesis, the autocrine, paracrine, and amphicrine interactions of the vascular endothelium with its surrounding stromal components are tightly regulated both spatially and temporally. Additionally, the levels and activities of proangiogenic and angiostaα^'c cytokines and growth factors are maintained in balance. In contrast, the pathological angiogenesis necessary for active tumor growth is sustained and persistent, representing a dysregulation of the normal angiogenic system. Solid and hematopoietic tumor types are particularly associated with a high level of abnormal angiogenesis. It is one goal of the present disclosure to provide agents and therapeutic treatments for inhibiting angiogenesis and tumor growth. [0005] It has also been reported that Ephrin B2 is a functional cellular receptor for

Hendra and Nipah viruses. In particular, Ephrin B2 was recently discovered to be the receptor protein for the Nipah virus envelope attachment glycoprotein (NiV-G). Accordingly, the antibodies described herein may also be useful in preventing viral infection.

SUMMARY

[0006] The present invention relates to antibodies that bind Ephrin B2 and inhibit one or more activities of Ephrin B2 (also referred to herein as Ephrin B2 antagonist antibodies of the invention, Ephrin antibodies of the invention, or Ephrin B2 antagonists of the invention), pharmaceutical compositions comprising such antibodies and therapeutic and diagnostic uses thereof.

[0007] In one aspect, the present invention relates to methods of inhibiting angiogenesis or treating a vascular anomaly in a mammal comprising administering to the mammal an amount of an EphrinB2 antagonist of the invention, which is effective for inhibiting angiogenesis or for treating the vascular anomaly in the mammal. Thus, the present invention provides methods of treating diseases or disorders characterized by undesirable or excessive vascularization, including by way of example tumors (e.g., solid malignant tumors), rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, and chronic inflammation, by administering an effective amount of an Ephrin B2 antagonist antibody of the invention to a patient in need thereof.

[0008] In one embodiment, the invention provides a method of treating cancer, such as a solid malignant tumor, in a mammal comprising administering to the mammal a therapeutically effective amount of an Ephrin B2 antagonist of the invention.

[0009] In one embodiment, Ephrin B2 antibodies of the invention comprise one, two, three, four, five, or all six of the CDRs of an isolated human antibody selected from the group consisting of: E2, Dl, Bl, and Al 1.

[0010] In another embodiment, Ephrin B2 antibodies of the invention comprise a polypeptide which comprises at least one heavy chain variable region, at least one light chain variable region, or both light and heavy chain variable regions of an antibody selected from the group consisting of: E2, Dl, Bl, and Al l.

[0011] The invention further provides isolated polynucleotides comprising a nucleotide sequence encoding an Ephrin B2 antibody of the invention or a portion thereof.

[0012] In yet a further aspect, the invention provides an article of manufacture, comprising: a container; a label; and a composition comprising an active agent contained within the container; wherein the label indicates that the composition can be used to inhibit angiogenesis or to treat a disease or disorder characterized by undesirable or excessive vascularization and the active agent in the composition is an Ephrin B2 antagonist of the invention.

DEFINITIONS

[0013] As used herein, the terms "antibody" and "antibodies" (immunoglobulins) encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecifϊc antibodies (e.g., bispecific 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, disulfide-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. In particular, 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.

[0014] 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. Light chains are classified as either lambda chains or kappa chains based on the amino acid sequence of the light chain variable and constant regions. The variable domain of a kappa light chain may also be denoted herein as VK. The term 'Variable region" may also be used to describe the variable domain of a heavy chain or light 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.

[0015] The term "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). The variable domains of native heavy and light chains each comprise four FW regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-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.

[0016] 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 residues 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. Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and/or those residues from a "hypervariable loop" (e.g., residues 26-32 (Ll ), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)). "Framework" or "FW" residues are those variable domain residues flanking the CDRs. FW residues are present in chimeric, humanized, human, domain antibodies, diabodies, vaccibodies, linear antibodies, and bispecific antibodies.

[0017] The term "monoclonal antibody" as used herein 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. Alternative production methods are known to those trained in the art, for example, a monoclonal antibody may be produced by cells stably or transiently transfected with the heavy and light chain genes encoding the monoclonal antibody. [0018] 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. For example, 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.

[0019] The term "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).

[0020] "Humanized" forms of nonhuman (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. For the most part, 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. In some instances, FW region residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may comprise residues mat are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, 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. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al., Nature, 321 : 522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0021] A "human antibody" can be an antibody derived from a human or an antibody obtained from a transgenic organism mat 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.

[0022] "Effector cells" are leukocytes which express one or more FcRs and perform effector functions. The cells express at least FcγRI, FCγRII, FcγRIII and/or FcγRTV and carry out ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.

[0023] The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the

Fc region of an antibody. In one embodiment, the FcR is a native sequence human FcR. Moreover, in certain embodiments, the FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, FcγRIII, and FcγRTV subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (FTIM) in its cytoplasmic domain. (See, Daeron, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel etal., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)). [0024] "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 VH-VL 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.

[0025] "Affinity" of an antibody for an epitope to be used in the treatments) 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. Values of 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. When antibody affinity is used as a basis for administration of the treatment methods described herein, or selection for the treatment methods described herein, 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.

[0026] As used herein, 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) [0027] 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.

[0028] The term "antibody half-life" as used herein 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 SO 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. [0029] 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. Depending on the amino acid sequence of the heavy chain constant region, a given human antibody or immunoglobulin can be assigned to one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these classes may be further divided into subclasses (isotypes), e.g., IgGl (gamma 1), IgG2 (gamma 2), IgG3 (gamma 3), and IgG4 (gamma 4), and IgAl and Ig A2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The structures and three-dimensional configurations of different classes of immunoglobulins are well-known. Of the various 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. [0030] As used herein, 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).

[0031] As used herein, the term "antigenicity" means that a compound is recognized by an antibody or may bind to an antibody and induce an immune response. [0032] 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. Thus, the terms "treat," "treating" or "treatment of (or grammatically equivalent terms) refer to bom prophylactic and therapeutic treatment regimes.

[0033] As used herein, 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). For example, a "sufficient amount" or "an amount sufficient to" can be an amount that is effective to reduce tumor growth. [0034] 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.

[0035] The term "antagonist" when used herein refers to a molecule which is capable of inhibiting one or more of the biological activities of a target molecule, such as a human Ephrin B2. Antagonists may act by interfering with the binding of a receptor to a ligand and vice versa, by incapacitating or killing cells which have been activated by a ligand, and/or by interfering with receptor or ligand activation (e.g. tyrosine kinase activation) or signal transduction after ligand binding to a cellular receptor. The antagonist may completely block receptor-ligand interactions or may substantially reduce such interactions. An antagonist may have one or more of the following effects: inhibit human EphB4 (hEphB4)-human Ephrin B2 (hEphrin B2) binding, inhibit EphB4 phosphorylation, inhibit Ephrin B2 binding to one or more of its endogenous receptor (e.g., EphB4, EphBl, EphB2, EphB3, EphB6, EphA4, EphA3), inhibit tubule formation (HUAEC, HUVEC or HMEC endothelial cells), inhibit survival of endothelial cells, inhibit survival of tumor cells, inhibit matrigel plug angiogenesis in response to bFGF or VEGF, or inhibit growth of a tumor cells (e.g., tumor cells selected from the group consisting of: breast, renal, colon, bladder, prostate, Kaposi's sarcoma, and melanoma tumor cells. An antagonist may also have the effect of inhibiting human EphB4 (hEphB4)-human Ephrin B2 (hEphrin B2) binding while also increasing EphB4/Ephrin B2 phosphorylation. All such points of intervention by an antagonist shall be considered equivalent for purposes of this invention. Thus, included within the scope of the invention are antagonists (e.g. neutralizing antibodies) that bind to Ephrin B2 or a complex of an Eph receptor and Ephrin B2.

[0036] The term "inhibiting angiogenesis" refers to the act of substantially preventing or reducing the development of blood vessels in a treated mammal.

[0037] "Diseases or disorders characterized by undesirable or excessive vascularization" include, by way of example, tumors, and especially solid malignant tumors, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age- related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, and chronic inflammation.

[0038] Examples of "diseases or disorders characterized by undesirable or excessive vascular permeability" include edema associated with brain tumors, ascites associated with malignancies, Meigs¹ syndrome, lung inflammation, nephrotic syndrome, pericardia! effusion (such as that associated with pericarditis), and pleural effusion.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1. The Involvement of Ephrin B2 and EphB4 in the Ontogeny of Blood

Vessels.

[0040] Figure 2. Examples of Interactions Between Eph and Ephrin Family Members.

[0041] Figure 3. EphB4/Ephrin B2 Interact with Each Other to Generate Cellular

Signals

[0042] Figure 4. Disrupting EphB4/Ephrin B2 Interaction [0043] Figure 5. Development of ELISA assay for determining human EphB4 (hEphB4) interaction with EphrinB2

[0044] Figure 6. Phage Clones Inhibit hEphrinB2-EphB4 Binding

[0045] Figure 7. Activity of Anti-Ephrin B2 Fab Antibodies

[0046] Figure 8. Human anti-Ephrin B2 Antibodies Bind to hEphrinBl.HSA, hEphrinB2.His, and hEphrinB2.Fc and Murine EphrinB2.Fc Fusion Proteins

[0048] Figure 9. Activity of Anti-Ephrin B2 IgG Antibodies

[0049] Figure 10. Light Chain Variable Regions of Select Clones

[0050] Figure 11. Heavy Chain Variable Regions of Select Clones

[0051] Figure 12A-H. Human Anti-Ephrin B2 Antibodies Bind to Human

EphrinB2.HSA, EphrinB2.His, and EphrinB2.Fc and Murine EphrinB2.Fc Fusion Proteins

[0052] Figure 13. Ability of Ephrin B2 Antibodies to Bind SCC-15 Cells

[0053] Figure 14. EphB4 Extracellular Domain (ECD) Amino Acid Sequence

[0054] Figure 15. Ephrin B2 Extracellular Domain (ECD) Amino Acid Sequence

DETAILED DESCRIPTION

[0055] The present invention relates to antibodies that bind Ephrin B2 and inhibit one or more activities of Ephrin B2 (also referred to herein as Ephrin B2 antagonist antibodies of the invention, Ephrin antibodies of the invention, or Ephrin B2 antagonists of the invention), pharmaceutical compositions comprising such antibodies and therapeutic and diagnostic uses thereof.

[0056] The antibodies of the invention and compositions comprising the same are useful for many purposes, for example, as therapeutics against a wide range of chronic and acute diseases and disorders including, but not limited to, cancer (e.g., treatment of solid malignant tumors), rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, and chronic inflammation, sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e g., psoriatic arthritis), anaphylactic shock, organ ischemia, reperfusion injury, spinal cord injury and allograft rejection. Antibodies of the Invention

[0057] As indicated above, the present invention relates to antibodies that bind human

Ephrin B2 and inhibit one or more activities of the human (or other mammalian) Ephrin B2 polypeptide (also referred to herein as Ephrin B2 antagonist antibodies of the invention, Ephrin antibodies of the invention, or Ephrin B2 antagonists of the invention). [0058] In one embodiment, an Ephrin B2 antibody of the invention possesses one or more of the following activities: inhibits human EphB4 (hEphB4)-human Ephrin B2 (hEphrin B2) binding; cross reacts with mouse Ephrin B2, inhibits EphB4 phosphorylation; fails to induce/stimulate (or significantly induce/stimulate) Ephrin B2 phosphorylation; inhibits Ephrin B2 binding one or more of its endogenous receptor (e.g., EphB4, EphBl, EphB2, EphB3, EphB6, EphA4, EphA3); inhibits tubule formation (HUAEC, HUVEC or HMEC endothelial cells); inhibits survival of endothelial cells; inhibits survival of tumor cells; inhibits angiogenesis; inhibits matrigel plug angiogenesis in response to bFGF or VEGF; inhibits growth of a tumor cells (e.g., tumor cells selected from the group consisting of: breast, colon, bladder, prostate, renal, Kaposi's sarcoma, and melanoma tumor cells); and induces internalization of Ephrin B2 and/or EphB4.

[0059] In one embodiment, Ephrin B2 antibodies of the invention comprise one, two, three, four, five, or all six of the CDRs of an isolated human antibody selected from the group consisting of: E2, Dl, Bl, and Al 1. In one embodiment, Ephrin B2 antibodies of the invention comprise or essentially consist of an isolated human antibody selected from the group consisting of: E2, Dl, Bl, and Al 1. In one aspect, Ephrin B2 antibodies of the invention comprise one, two, three, four, five, or all six CDRs having at least 70% identity, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% identity to the corresponding CDR of an antibody selected from the group consisting of: E2, Dl, Bl, and Al l. For instance, such antibodies include, but are not limited to an antibody comprising a heavy chain CDRl having at least 90% identity to heavy chain CDRl of Ephrin B2 antibody Al l. In another aspect, Ephrin B2 antibodies of the invention comprise one, two, three, four, five, or all six CDRs of an isolated human antibody selected from the group consisting of: E2, Dl, Bl, and Al l, wherein the each CDR comprises less than 2, less than 3, less than 4, less than 5, leass than 8 or less than 10 amino acid substitutions, deletions or insertions. [0060] It will be understood that the complementarity determining regions (CDRs) residue numbers referred to herein are those of Kabat et al. (1991, N1H Publication 91-3242, National Technical Information Service, Springfield, VA). Specifically, residues 24-34 (CDRl), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain and 31-35 (CDRl), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Note that CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology with the Kabat consensus sequences). Maximal alignment of framework residues frequently requires the insertion of "spacer" residues in the numbering system, to be used for the Fv region. It will be understood that the CDRs referred to herein are those of Kabat et al. supra-. In addition, the identity of certain individual residues at any given Kabat site number may vary from antibody chain to antibody chain due to interspecies or allelic divergence. CDR regions of antibodies Al 1, Bl, Dl and E2 are identified in Figures 9 and 10; SEQ ID NOs corresponding to the CDR residues of antibodies Al 1, Bl, Dl and E2 are listed in table 2.

[0061] In another embodiment, Ephrin B2 antibodies of the invention comprise a polypeptide which comprises at least one heavy chain variable region, at least one light chain variable region, or both light and heavy chain variable regions of an antibody selected from the group consisting of: E2, Dl, Bl, and Al l. In one aspect, Ephrin B2 antibodies of the invention comprise a heavy chain variable region, a light chain variable region, or both light and heavy chain variable regions having 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% sequence identity to the corresponding variable region of an antibody selected from the group consisting of: E2, D 1 , B 1 , and Al l. For instance, such antibodies include, but are not limited to an antibody comprising a heavy chain variable region having 90% identity to the heavy chain variable region of Ephrin B2 antibody Al l .

[0062] In another embodiment, the invention includes antibodies that bind the same epitope as an antibody selected from the group consisting of: E2, Dl, Bl, and Al 1. In one aspect, the invention includes antibodies that bind the same epitope as an antibody selected from the group consisting of: E2, Dl, Bl, and Al l, wherein the affinity for such epitope is equal to or greater than the affinity of antibodies E2 or Dl or Bl or Al 1, respectively, to the eptitope. [0063] In one embodiment, the invention includes Ephrin B2 antibodies that cross react with both mouse and human Ephrin B2. In one aspect, the invention includes Ephrin B2 antibodies that bind mouse Ephrin B2 with an affinity of equal to or greater than the antibody binds human Ephrin B2. In one aspect, the invention includes Ephrin B2 antibodies that bind mouse Ephrin B2 with at least half the affinity as the antibody binds human Ephrin B2. [0064] In one embodiment, antibodies of the invention induce internalization of Ephrin

B2.

[0065] Antibodies of the present invention also encompass antibodies that have half- lives (e.g., serum half-lives) in a mammal, (e.g., a, human), of greater than 5 days, greater than 10 days, greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-lives of the antibodies of the present invention in a mammal, (e.g., a human), results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus, reduces the frequency of the administration of said antibodies or antibody fragments and/or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies having increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International Publication Nos. WO 97/34631; WO 04/029207; U.S. 6,737056 and U.S. Patent Publication No. 2003/0190311 and discussed in more detail below).

[0066] In one embodiment, the antibodies of the invention may comprise modifications/substitutions and/or novel amino acids within their Fc domains such as, for example, 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 U S A 92: 11980-11984; Jefferis et al, 1995, Immunol Lett. 44: 11 1-117; Lund et al., 1995, Faseb J 9: 115-119; Jefferis et al, 1996, Immunol Lett 54: 101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J Immunol 164: 1925- 1933; Xu et al., 2000, Cell Immunol 200: 16-26; Idusogie et al, 2001, J Immunol 166:2571- 2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. Patent Application Nos. 10/370,749 and PCT Publications WO 94/2935; WO 99/58572; WO 00/42072; WO 02/060919, WO 04/029207. Other modifications/substitutions of the Fc domain will be readily apparent to one skilled in the art.

[0067] Antibodies of the invention comprising modifications/substitutions and/or novel amino acid residues in their Fc regions can be generated by numerous methods well known to one skilled in the art. Non-limiting examples include, isolating antibody coding regions (e.g., from hybridoma) and making one or more desired substitutions in the Fc region of the isolated antibody coding region. Alternatively, the variable regions of an antibody of the invention may be subcloned into a vector encoding an Fc region comprising one or modifications/ substitutions and/or novel amino acid residues.

[0068] Antibodies of the invention may also be modified to alter glycosylation, again to alter one or more functional properties of the antibody.

[0069] In one embodiment, the glycosylation of the antibodies of the invention is modified. For example, an aglycosylated 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. For example, 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. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861. [0070] Additionally or alternatively, an antibody of the invention can 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. etal (2002) J. Biol. Chem. Ill -.26133-261 Ad; Umaaaetal. (1999) Nat. Biotech. 17: 176-1, as well as, European Patent No: EP 1,176,195; PCT Publications WO 03/035835; WO 99/54342.

[0071] As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C -terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387. [0072] The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding an Ephrin B2 polypeptide or fragment thereof and/or generating a desired response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derealization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids. [0073] In a specific embodiment, the antibodies of the invention inhibit the binding of

Ephrin B2 to EphB4 or EphA4 (or both) by at least about 10%, or by at least about 20%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90%, or by about 100%.

[0074] Non-limiting examples of Ephrin B2 antagonist antibodies include antibodies that act as (i) agents that bind to an Ephrin B2 and prevent or reduce the interaction between an endogenous Eph receptor (e.g., EphB4, EphBl, EphB2, EphB3, EphB6, EphA4, EphA3) and the Ephrin, and induce Ephrin signal transduction; (ii) agents that bind to an Ephrin B2, prevent or reduce the interaction between an Eph receptor and the Ephrin, and prevent or induce very low to negligible levels of Ephrin signal transduction.

[0075] In a embodiment, the antibodies of the invention specifically bind a polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the amino acid sequence of human Ephrin B2. [0076] The percent identity of two amino acid sequences (or two nucleic acid sequences) can be determined, for example, by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The amino acids or nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100). The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A specific, non-limiting example of such a mathematical algorithm is described in Karlin etal., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.2) as described in Schaffer et al., Nucleic Acids Res., 29:2994-3005 (2001). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN) can be used. See http://www.ncbi.nlm.nih.gov, as available on April 10, 2002. In one embodiment, the database searched is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1. [0077] Another, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys) sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, Comput. Appl. Biosci., 10: 3-5 (1994); and FASTA described in Pearson and Lipman, Proc. Natl. Acad. Sci USA, 85: 2444-8 (1988). [0078] In another embodiment, the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (available at http://www.accelrys.com, as available on August 31, 2001) using either a Blossom 63 matrix or a PAM250 matrix, and agap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet another embodiment, the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (available at http://www.cgc.com), using agap weight of 50 and a length weight of 3. [0079] Another embodiment of present invention are antibodies that specifically bind human Ephrin B2 and antigenic fragments thereof with a dissociation constant or K_d (k_off/k_on) of less than 10 ^-5 M, or of less than 10^-6 M, or of less man 10^-7 M, or of less than 10^-8 M, or of less than 10^-9 M, or of less than 10^-10 M, or of less than 10^-11 M, or of less man 10^-12 M, or of less than 10^-13 M, or of less than 5 x 10^{- 13}M, or of less than 10^{- 14}M, less than 5 x 10^{- 14}M, or of less than 10^{- 15}M, or of less than 5 x 10^{- 15}M. In still another embodiment, an antibody of the invention that specifically binds human Ephrin B2 and antigenic fragments thereof has a dissociation constant or K_d (k_off/k_on) of between about 10^{- 7}M and about 10^{- 8}M, between about 10^{- 8}M and about 10^-9M, between about 10^-9M and about 10^{- 10}M, between about 10^{- 10}M and about 10^{- 11}M, between about 10^{- 11}M and about 10^{- 12}M, between about 10^{- 12}M and about 10^{- 13}M, between about 10^{- 13}M and about 10^{- 14}M. In still another embodiment, an antibody of the invention that specifically binds human Ephrin B2 and antigenic fragments thereof has a dissociation constant or K_d (k_off/k_on) of between 10^{- 7}M and 10^{- 8}M, between 10^{- 8}M and 10^-9M, between 10^{- 9}M and 10^{- 10}M, between 10^{- 10}M and 10^{- 11}M, between 10^{- 11}M and 10^{- 12}M, between 10^{- 12}M and 10^{- 13}M, between 10^{- 13}M and 10^{- 14}M.

[0080] It is well known in the art that the equilibrium dissociation constant (K_d) is defined as k_off/k_on. It is generally understood that a binding molecule (e.g. , and antibody) with a low K_d (i.e., high affinity) is preferable to a binding molecule (e.g., and antibody) with a high K_d (i.e., low affinity). However, in some instances the value of the k_on or k_off may be more relevant than the value of the K_d. One skilled in the art can determine which kinetic parameter is most important for a given antibody application. In certain embodiments, the antibodies of the invention have a lower K_d for one antigen than for others.

[0081] In another embodiment, the antibody binds to human Ephrin B2 and antigenic fragments thereof with a k_off of less than 1x 10^-3 s^-1, or of less than 3 x 10^-3 s^-1. In other embodiments, the antibody binds to Ephrin B2 and antigenic fragments thereof with a k_off of less than 10^-3 s^-1, less than 5x10^-3 s^-1, less than 10^-4 s^-1, less than 5x10^-4 s^-1, less than 10^-5 s^-1, less than 5x10^-5 s^-1, less than 10^-6 s^-1, less than 5x10^-6 s^-1, less than 10^-7 s^-1, less than 5x10^-7 s^-1, less than 10^~8 s^-1, less than 5x 10^-8 s^-1, less than 10^-9 s^-1, less than 5x10^-9 s^-1, or less than 10^-10 s^-l. [0082] In another embodiment, the antibody of the invention binds to human Ephrin B2 and/or antigenic fragments thereof with an association rate constant or k_on rate of at least 10⁵ M^-1s^-1, at least 5x10⁵ M ¹s^-1, at least 10⁶ M^-1s^-1, at least 5 x 10⁶ M^-1s^-1, at least 10⁷ M^-1s^-1, at least 5 x 10⁷ M^-1s^-1, or at least 10⁸ M^-1s^-1, or at least 10⁹ M^-1s^-1.

[0083] 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 al. , 1993, Electrophoresis 14: 1023). In addition, 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. Thus, in certain embodiments antibodies having higher Tm are preferable. Tm of a protein domain (e.g., a Fab domain) can be measured using any standard method known in the art, for example, by differential scanning calorimetry (see, e.g., Vermeer et al., 2000, Biophys. J. 78:394-404; Vermeer et al., 2000, Biophys. J. 79: 2150-2154).

[0084] Accordingly, an additional nonexclusive embodiment of the present invention includes antibodies of the invention that have certain preferred biochemical characteristics such as a particular isoelectric point (pi) or melting temperature (Tm). [0085] More specifically, in one embodiment, the antibodies of the present invention have a pi ranging from 5.5 to 9.5. In still another specific embodiment, the high affinity antibodies of the present invention have 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 aboutβ.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. In other specific embodiments, the high affinity antibodies of the present invention have 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. Even more specifically, the high affinity antibodies of the present invention 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, oral 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. In other specific embodiments, the high affinity antibodies of the present invention 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.

[0086] It is possible to optimize solubility by altering the number and location of ionizable residues in the antibody to adjust the pi. For example 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). Without wishing to be bound by any particular theory, 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. One skilled in the art would understand which amino acid substitutions would be most appropriate for a particular antibody to achieve a desired pi. In one embodiment, a substitution is generated in an antibody of the invention to alter the pi. It is specifically contemplated that the substitution(s) of the Fc region that result in altered binding to FcγR (described supra) may also result in a change in the pi. In another embodiment, substitution(s) of the Fc region are specifically chosen to effect both the desired alteration in FcγR binding and any desired change in pi.

[0087] In one embodiment, the antibodies of the present invention have a Tm ranging from 65°C to 120°C. In specific embodiments, the high affinity antibodies of the present invention have a Tm ranging from about 75°C to about 120°C, or about 75°C to about 85°C, or about 85°C to about 95°C, or about 95°C to about 105°C, or about 105°C to about 115°C, or about 115°C to about 120°C. In other specific embodiments, the high affinity antibodies of the present invention have a Tm ranging from 75°C to 120°C, or 75°C to 85°C, or 85°C to 95°C, or 95°C to 105°C, or 105°C to 115°C, or 115°C to 120°C. In still other specific embodiments, the high affinity antibodies of the present invention have a Tm of at least about 65°C, or at least about 70°C, or at least about 75°C, or at least about 80°C, or at least about 85°C, or at least about 90°C, or at least about 95°C, or at least about 100°C, or at least about 105°C, or at least about 110°C, or at least about 115°C, or at least about 120°C. In yet other specific embodiments, the high affinity antibodies of the present invention have a Tm of at least 65°C, or at least 70°C, or at least 75°C, or at least 80°C, or at least 85°C, or at least 90°C, or at least 95°C, or at least 100°C, or at least 105°C, or at least 110°C, or at least 115°C, or at least 120°C.

[0088] In a specific embodiment, the antibodies of the invention or fragments thereof are human or humanized antibodies.

[0089] The present invention also encompasses variants of E2, D 1 , B 1 , and A 11 comprising one or more amino acid residue substitutions in the variable light (V_L) domain and/or variable heavy (V_H) domain. The present invention also encompasses variants of E2, Dl, Bl, and Al l with one or more substitutions in one or more V_L CDRS and/or one or more V_H CDRS. The antibody generated by introducing substitutions in the V_H domain, V_H CDRS, V_L domain and/or V_L CDRs of E2, D 1 , B 1 , and A 11 can be tested in vitro and in vivo, for example, for its ability to bind to human Ephrin B2 or for its ability to inhibit one or more Ephrin B2 actvities.

[0090] Another embodiment of the present invention includes the introduction of conservative amino acid substitutions in any portion of an anti -Ephrin B2 antibody of interest, described supra. It is well known in the art that "conservative amino acid substitution" refers to amino acid substitutions that substitute functionally-equivalent amino acids. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide. Substitutions that are charge neutral and which replace a residue with a smaller residue may also be considered "conservative substitutions" even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine). Families of amino acid residues having similar side chains have been defined in the art. Several families of conservative amino acid substitutions are shown in Table 2.

[0091] The term "conservative amino acid substitution" also refers to the use of amino acid analogs or variants. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," (1990, Science 247:1306-1310).

Methods of Generating and Screening Antibodies of the Invention

[0092] Antibodies or fragments that specifically bind to an Ephrin B2 polypeptide can be identified, for example, by immunoassays, BI Acore, or other techniques known to those of skill in the art.

[0093] The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a human Ephrin B2 polypeptide can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art. [0094] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, 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-CeIl Hybridomas 563-681 (Elsevier, N.Y., 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0095] A "monoclonal antibody" may comprise, or alternatively consist of, two proteins, i.e., a heavy and a light chain.

[0096] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

[0097] Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention. [0098] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain. [0099] The antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[00100] As described in the above references, after phage selection, 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 in detail below. For example, 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 WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.. Science 240:1041-1043 (1988). [00101] Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240: 1038-1040 (1988).

[00102] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989)./. Immunol. Methods 125: 191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397. Humanized antibodies are antibody molecules from non-human species antibody that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR- grafung (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91 :969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

[00103] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741. [00104] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to mat described above.

[00105] Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non- human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

[00106] Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art (See, e.g., Greenspan & Bona, FASEBJ. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such antiidiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.

[00107] If the antibody is used therapeutically in in vivo applications, the antibody is preferably modified to make it less immunogenic in the individual. For example, if the individual is human the antibody is preferably "humanized"; where the complementarity determining region(s) of the antibody is transplanted into a human antibody (for example, as described in Jones et al., Nature 321:522-525, 1986; and Tempest et al., Biotechnology 9:266- 273, 1991).

[00108] Phage display technology can also be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-B box antibodies or from naive libraries (McCafferty et al., Nature 348:552-554, 1990; and Marks, et al., Biotechnology 10:779-783, 1992). The affinity of these antibodies can also be improved by chain shuffling (Clackson et al., Nature 352: 624-628, 1991).

[00109] The present invention relates to pharmaceutical compositions comprising human, humanized, or chimeric anti-Ephrin B2 antibodies of the IgGl, IgG2, IgG3 or IgG4 human isotype. The present invention also relates to pharmaceutical compositions comprising human or humanized antibodies of the IgG2 or IgG4 human isotype that may mediate human ADCC. In certain embodiments, the present invention also relates to pharmaceutical compositions comprising monoclonal human, humanized, or chimerized Ephrin B2 antibodies that can be produced by means known in the art. [00110] 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.

[00111] It will be understood that Fc region as used herein includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus 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. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgammal (Cγl) and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-termimis, wherein the numbering is according to the EU index as in Kabat et al. (1991, N1H 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. Note: 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. [00112] The present invention encompasses Fc variant proteins which have altered binding properties for an Fc ligand (e.g., an Fc receptor, C Iq) relative to a comparable molecule (e.g., a protein having the same amino acid sequence except having a wild type Fc region). Examples of 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. It is generally understood that a binding molecule (e.g., a Fc variant protein such as an antibody) with a low K_D may be preferable to a binding molecule with a high K_D. However, in some instances the value of the k_on or k_off may be more relevant than the value of the K_D- One skilled in the art can determine which kinetic parameter is most important for a given antibody application.

[00113] 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-FcγR interactions, i.e., specific binding of an Fc region to an FcγR 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). 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. A detailed description of binding affinities and kinetics can be found in Paul, W.E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. [00114] In one embodiment, the Fc variant protein has enhanced binding to one or more Fc ligand relative to a comparable molecule. In another embodiment, 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. In a specific embodiment, the Fc variant protein has enhanced binding to an Fc receptor. In another specific embodiment, the Fc variant protein has enhanced binding to the Fc receptor FcγRIIIA. 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.

[00115] 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.

[00116] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to 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. Specific high-affinity 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. It is contemplated that, in addition to antibodies, other proteins comprising Fc regions, specifically Fc fusion proteins, having the capacity to bind specifically to an antigen- bearing target cell will be able to effect cell-mediated cytotoxicity. For simplicity, the cell- mediated cytotoxicity resulting from the activity of an Fc fusion protein is also referred to herein as ADCC activity.

[00117] The ability of any particular Fc variant protein to mediate lysis of the target cell by ADCC can be assayed. To assess ADCC activity 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. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Specific examples of in vitro ADCC assays are described in Wisecarver et al., 1985 79:277-282; Bruggemann et al., 1987, J Exp Med 166:1351-1361; Wilkinson et al., 2001, J Immunol Methods 258:183- 191; Patel et al., 1995 J Immunol Methods 184:29-38. 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. Sd. USA 95:652-656.

[00118] In one embodiment, an Fc variant protein has enhanced ADCC activity relative to a comparable molecule. In a specific embodiment, an Fc variant protein has ADCC 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, hi another specific embodiment, an Fc variant protein has enhanced binding to the Fc receptor FcγRIIIA and has enhanced ADCC activity relative to a comparable molecule. In other embodiments, the Fc variant protein has both enhanced ADCC activity and enhanced serum half life relative to a comparable molecule.

[00119] "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. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro βt al., 1996, J. Immunol. Methods, 202:163, may be performed. In one embodiment, an Fc variant protein has enhanced CDC activity relative to a comparable molecule. In a specific embodiment, 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. In other embodiments, the Fc variant protein has both enhanced CDC activity and enhanced serum half life relative to a comparable molecule.

[00120] In one embodiment, 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. Optionally, 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). [00121] In a specific embodiment, 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, 24 IY, 241E, 241 R. 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 25 IF, 252Y, 254T, 255L, 256E, 256M, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 2651, 265L, 265H, 265T, 2661, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 2981, 298T, 298F, 2991, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 3051, 313F, 316D, 325Q, 325L, 3251, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 33OC, 330L, 330Y, 330V, 3301, 330F, 33OR, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q₅ 332T, 332H, 332Y, 332A, 339T₅ 370E, 370N₅ 378D, 392T, 396L₅416G, 419H₅ 421K₅ 440Yand 434W as numbered by the EU index as set forth in Kabat. Optionally, 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).

[00122] In another embodiment, 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. In a specific embodiment, 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. Optionally, the Fc region may further comprise additional 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. In a specific embodiment, 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.

[00123] In one embodiment, 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. AcadSci USA 92: 1 1980-11984; Jefferis et al, 1995, Immunol Lett. 44: 111-117; Lund et al., 1995, Faseb J 9: 115-119; Jefferis et al, 1996, Immunol Lett 54: 101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J Immunol 164: 1925-1933; Xu et al., 2000, Cell Immunol 200: 16-26; Idusogie et al, 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. Patent Publication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO 99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; WO 04/063351. Also encompassed by the present invention are 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.

[00124] Methods for generating non-naturally occurring Fc regions are known in the art. For example, amino acid substitutions and/or deletions can be generated by mutagenesis methods, including, but not limited to, site- directed mutagenesis (Kunkel, P roc. 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)). Preferably, 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 (Higuchi, ibid.) can also be used to introduce any desired mutation(s) into a target sequence (the starting DNA). For example, 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). In the second round of PCR, 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. As the first step of mutagenesis, the starting DNA (e.g., encoding an Fc fusion protein, an antibody or simply an Fc region), is operably cloned into a mutagenesis vector. The primers are designed to reflect the desired amino acid substitution. Other methods useful for the generation of variant Fc regions are known in the art (see, e.g., U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. Patent Publication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO 99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; WO 04/063351).

[00125] In some embodiments, 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. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCTWO 01/292246A1; PCT WO 02/311140Al; PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). See, e.g., WO 00061739; EA01229125; US 20030115614; Okazaki et al, 2004, JMB, 336: 1239-49.

[00126] In still another embodiment, the glycosylation of antibodies utilized in accordance with the invention is modified. For example, 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. For example, 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. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861. One or more amino acid substitutions can also be made that result in elimination of aglycosylation site present in the Fc region (e.g., Asparagine 297 of IgG). Furthermore, aglycosylated antibodies may be produced in bacterial cells which lack the necessary glycosylation machinery.

[00127] 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. Biotech. 17: 176-1, as well as, U.S. Patent No: US 6,946,292; European Patent No: EP 1,176,195; PCT Publications WO 03/035835; WO 99/54342 each of which is incorporated herein by reference in its entirety.

[00128] It may be desirable to modify an antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating diseases. 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). [00129] Other methods of engineering Fc regions of antibodies so as to alter effector functions are known in the art (e.g., U.S. Patent Publication No. 20040185045 and PCT Publication No. WO 2004/016750, both to Koenig et al , which describe altering the Fc region to enhance the binding affinity for FcγRIIB as compared with the binding affinity for FCγRIIA; see, also, PCT Publication Nos. WO 99/58572 to Armour et al, WO 99/51642 to Idusogie et al, and U.S. 6,395,272 to Deo et al; the disclosures of which are incorporated herein in their entireties). Methods of modifying the Fc region to decrease binding affinity to FcγRIIB are also known in the art {e.g., U.S. Patent Publication No. 20010036459 and PCT Publication No. WO 01/79299, both to Ravetch et al., the disclosures of which are incorporated herein in their entireties). Modified antibodies having variant Fc regions with enhanced binding affinity for FcγRIIIA and/or FcγRIIA as compared with a wildtype Fc region have also been described {e.g., PCT Publication Nos. WO 2004/063351, to Stavenhagen et al, the disclosure of which is incorporated herein in its entirety). [00130] In vitro assays known in the art can be used to determine whether antibodies used in compositions and methods of the invention are capable of mediating ADCC, such as those described herein.

Polynucleotides Encoding Antibodies

[00131] The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and 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 an antibody of the invention.

[00132] By "stringent hybridization conditions" is intended overnight incubation at

42.degree. C. in a solution comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium cirate), 50 mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran sulfate, and 20 mu g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. l.times.SSC at about 65.degree. C.

[00133] The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, 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.

[00134] Alternatively, a polynucleotide encoding an antibody may 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 of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5¹ 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 cloned into replicable cloning vectors using any method well known in the art. [00135] Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

[00136] In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains of the antibodies of the invention may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well known in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. MoI. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.

[00137] Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

[00138] In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci. 81 :851-855 (1984); Neuberger et al., Nature 312:604- 608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

[00139] Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et <d.,Proc. Natl. Acad. ScL USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al. Science 242: 1038-1041 (1988)).

Methods of Producing Antibodies of the Invention

[00140] The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

[00141] Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, {e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention 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. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain. [00142] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, 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.

[00143] A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, 3T3, PerC6 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.SK promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with 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); Cockett et al., Bio/Technology 8:2 (1990)). Also see, e.g., U.S. patents 5827739, 5879936, 5981216, and 5658759.

[00144] In bacterial systems, 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 a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, 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 J. 2: 1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, 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). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[00145] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera fhigiperda 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).

[00146] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, 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 in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus mat is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). 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 must be in phase 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 Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[00147] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the 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-translarional 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 foreign protein expressed. To this end, 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, NSO, Per.C6 and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell lines such as, for example, CRL7030 and Hs578Bst.

[00148] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

[00149] 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)), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sd. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc 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 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5): 155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY ( 1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre- Garapin et al., J. MoI. Biol. 150: 1 (1981).

[00150] 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). When a marker in the vector system expressing antibody is amplifiable, 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 al., Mol. Cell. Biol. 3:257 (1983)).

[00151] The host cell may be co-transfected with two expression vectors of the invention, 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 selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:562 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for Hie heavy and light chains may comprise cDNA or genomic DNA.

[00152] Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it 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 antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

[00153] Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In certain embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.

Antibody Conjugates and Derivatives [00154] The antibodies of the invention include derivatives that are modified (e.g., by the covalent attachment of any type of molecule to the antibody). For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non- classical amino acids.

[00155] Antibodies or fragments thereof with increased in vivo half-lives can be generated by attaching to said antibodies or antibody fragments polymer molecules such as high molecular weight polyethyleneglycol (PEG). PEG can be attached to said antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C- terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization mat results in minimal loss of biological activity will be used. The degree of conjugation will 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, e.g., size exclusion or ion-exchange chromatography. [00156] Further, antibodies can be conjugated to albumin in order to make the antibody or antibody fragment 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. The present invention encompasses the use of antibodies or fragments thereof conjugated or fused to one or more moieties, including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.

[00157] In one embodiment, the present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, specifically to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins. In another embodiment, the present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, specifically to a polypeptide of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 or at least about 100 amino acids) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. For example, antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the antibodies to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., International publication No. WO 93/21232; European Patent No. EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Patent No. 5,474,981; Gillies et al., 1992, PNAS 89: 1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452.

[00158] The present invention further includes formulations comprising heterologous proteins, peptides or polypeptides fused or conjugated to antibody fragments. For example, the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof. Methods for fusing or conjugating polypeptides to antibody portions are well known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. ScL USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and ViI et al., 1992, Proc. Natl. Acad. Sci. USA 89: 11337- 11341.

[00159] Additional fusion proteins, e.g. , of antibodies that specifically bind human Ephrin B2 or fragments thereof (eg. , supra), may be generated through the techniques of gene- shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments 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. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2): 76-82; Hansson, et al., 1999, J. MoI. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2): 308- 313. Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. One or more portions of a polynucleotide encoding an antibody or antibody fragment, which portions specifically bind to a C/CLP may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

[00160] Moreover, the antibodies of the invention or fragments thereof can be fused to marker sequences, such as a peptide to facilitate purification. In certain embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) and the "flag" tag. [00161] The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnosu-cally to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include but ar not limited to, ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc, in addition positron emitting metals using various positron emission tomographies, noradioactive paramagnetic metal ions, and molecules that are radiolabelled or conjugated to specific radioisotopes can be conjugated to the antibodies of the invention.

[00162] Further, an antibody of the invention or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³ Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites {e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). A more extensive list of therapeutic moieties can be found in PCT publications WO 03/075957.

[00163] The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), CD40 Ligand, a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-I"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

[00164] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to. glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

[00165] Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody in Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62: 119- 58 (1982).

[00166] The antibodies of the invention can be conjugated to other polypeptides. Methods for fusing or conjugating antibodies to polypeptide moieties are known in the art. See, e.g., U.S. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851, and 5,112,946; EP 307,434; EP 367,166; PCT Publications WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, PNAS USA 88: 10535; Zheng et al., 1995. J Immunol 154:5590; and ViI et al., 1992, PNAS USA 89: 11337. The fusion of an antibody to a moiety does not necessarily need to be direct, but may occur through linker sequences. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res 4:2483; Peterson et al., 1999, Bioconjug Chem 10:553; Zimmerman et al., 1999, Nucl Med Biol 26:943; Garnett, 2002, Adv Drug Deliv Rev 53: 171 [00167] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[00168] An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factors) and/or cytokine(s) can be used as a therapeutic.

Assays for Antibody Binding and Activity

[00169] The antibodies of the invention may be assayed for specific (i.e., immunospecific) binding by any method known in the art. The immunoassays which can be used, include but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement- fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

[00170] Immunoprecipitation protocols generally comprise Iysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4. degree. C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4. degree. C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1. [00171] Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1. [00172] ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1. [00173] The binding affinity and other binding properties (e.g., off-rate of an antibody- antigen interaction) of an antibody to an antigen may be determined by a variety of in vitro assay methods well known in the art including for example, equilibrium methods (e.g. , enzyme-linked irnmunoabsorbent assay (ELISA; or radioimmunoassay (RIA)), or kinetics {e.g., BIACORE® analysis), and other methods such as indirect binding assays, competitive binding assays fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). 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. A detailed description of binding affinities and kinetics can be found in Paul, W.E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound in the presence of increasing amounts of an unlabeled second antibody.

[00174] Prophylactic or therapeutic agents can be tested in suitable animal model systems prior to testing in humans, including but not limited to in rats, mice, chicken, cows, monkeys, rabbits, hamsters, etc.

Antibodies, Antibody Compositions of the Invention and Therapeutic and/or Prophylactic Administration Thereof

[00175] In certain embodiments, the present invention provides methods of inhibiting angiogenesis and methods of treating angiogenesis-associated diseases. In other embodiments, 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 Ephrin B2 antagonists of the invention. These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans.

[00176] As described herein, 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, 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; telangiectasia psoriasis scleroderma, pyogenic granuloma, cororany collaterals, ischemic limb angiogenesis, corneal diseases, rubeosis, arthritis, diabetic neovascularization, fractures, vasculogenesis, hematopoiesis.

[00177] It is understood that methods and compositions of the invention are also useful for treating any angiogenesis-independent cancers (tumors). As used herein, the term "angiogenesis-independent cancer" refers to a cancer (tumor) where there is no or little neovascularization in the tumor tissue.

[00178] In particular, antibodies of the 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.

[00179] In certain embodiments of such methods, of antibodies of the invention can be administered, together (simultaneously) or at different times (sequentially), hi addition, polypeptide therapeutic agents can be administered with another type of compounds for treating cancer or for inhibiting angiogenesis.

[00180] In certain embodiments, the subject methods of the invention can be used alone. Alternatively, the subject methods may be used in combination with other conventional anticancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor). For example, such methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy. The present invention recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of a subject polypeptide therapeutic agent.

[00181] A wide array of conventional compounds have been shown to have antineoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant cells in leukemic or bone marrow malignancies. Although chemotherapy has been effective in treating various types of malignancies, many anti-neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.

[00182] When an antibody of the invention is administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such therapeutic agent is shown to enhance the therapeutic effect of the anti-neoplastic agent or overcome cellular resistance to such anti-neoplastic agent. This allows decrease of dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or restores the effectiveness of an antineoplastic agent in resistant cells.

[00183] Pharmaceutical compounds that may be used for combinatory anti-tumor therapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylsulbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[00184] These chemotherapeutic anti-tumor compounds may be categorized by their mechanism of action into, for example, following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antiritotic agents including natural products such as vinca alkaloids(vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane(paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin(actinomycin D), daunorubicin, doxorubicin(adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin(mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines(hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas(carmustine (BCMJ) and analogs, streptozocin), trazenes- dacarbazinine (DΗC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants(heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus(rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin(adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disrupters.

[00185] An Ephrin B2 antibody may be co-administered with at least one additional anti- angiogenesis agent that inhibits angiogenesis in an additive or synergistic manner with the antibody.

[00186] In another aspect, the invention provides a method of treating cancer, such as a solid malignant tumor, in a mammal comprising administering to the mammal a therapeutically effective amount of an Ephrin B2 antagonist of the invention.

[00187] In one embodiment, the invention includes a method of inhibiting angiogenesis in a mammal comprising administering to a human in need thereof a therapeutically-effective amount of an Ephrin B2 antibody of the invention.

[00188] In another aspect, the invention includes a method of suppressing tumor growth in a mammal comprising administering a therapeutically-efFective amount of an Ephrin B2 antibody of the invention.

[00189] In another aspect, the invention includes a method of killing tumor cells in a mammal comprising administering an effective amount of an Ephrin B2 antibody of the invention.

[00190] In another aspect, the invention includes a method of treating a malignancy in a human comprising administering to a human in need thereof a therapeutically-effective amount of an Ephrin B2 antibody of the invention.

[00191] In certain aspects, the disclosure provides methods for treating a patient suffering from a cancer. A method may comprise administering to the patient an Ephrin B2 antagonist antibody of the invention. Optionally, the cancer comprises cancer cells expressing EphrinB2 and/or EphB4 at a higher level than noncancerous cells of a comparable tissue. The cancer may be a metastatic cancer. The cancer may be selected from the group consisting of colon carcinoma, ovarian tumor, breast tumor, mesothelioma, prostate tumor, squamous cell carcinoma, Kaposi sarcoma, and leukemia. Optionally, the cancer is an angiogenesis- dependent cancer or an angiogenesis-independent cancer. The antibody employed may inhibit clustering or phosphorylation of Ephrin B2 or EphB4. The antibody may be co-administered with one or more additional anti-cancer chemotherapeutic agents that inhibit cancer cells in an additive or synergistic manner with the Ephrin B2 antibody. [00192] Further, antibodies of the invention are useful for therapeutic purposes, more specifically, for the treatment, prevention, management or amelioration of cancers including, but are not limited to, cancer of the head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, colorectal, or other gastrointestinal tract organs, stomach, spleen, renal, skeletal muscle, subcutaneous tissue, metastatic melanoma, endometrial, prostate, breast, ovaries, testicles or other reproductive organs, skin, thyroid, blood, lymph nodes, kidney, liver, pancreas, and brain or central nervous system.

[00193] In another aspect, the invention includes a method of detecting a tumor in a mammal comprising: removing a portion of the tumor from said mammal and contacting an antibody of the invention with said portion of tumor; and detecting the level of antibody that binds said tumor.

[00194] The present invention further provides a method of treating diseases or disorders characterized by undesirable or excessive vascularization, including by way of example tumors, and especially solid malignant tumors, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, and chronic inflammation, by administering an effective amount of an Ephrin B2 antagonist antibody of the invention to a patient in need thereof.

[00195] The present invention further provides a method of treating inflammatory disorders such as immune and non-immune inflammation, thrombosis, acute ischemic stroke, chronic articular rheumatism, psoriasis, disorders associated with inappropriate or inopportune invasion of vessels such as diabetic retinopathy, neovascular glaucoma and capillary proliferation in atherosclerotic plaques by administering an effective amount of an Ephrin B2 antagonist antibody of the invention to a patient in need thereof.

[00196] The present invention further provides a method of treating restenosis and/or osteoporosis by administering an effective amount of an Ephrin B2 antagonist antibody of the invention to a patient in need thereof.

[00197] In one embodiment, the methods and formulations of the invention are used for inhibiting angiogenesis. In a specific embodiment, the methods and formulations of the invention are used for inhibiting angiogenesis in a solid tumor. In another embodiment, the methods and formulations of the invention are used for inhibiting angiogenesis in an inflamed, angiogenic tissue including but not limited to retinal tissues and joint tissues.

[00198] In addition, the invention provides methods for the treatment or prevention of paramyxovirus (e.g., Nipah virus) infection, the methods comprise the administration to a subject in need thereof an effective amount of one or more Ephrin B2 antibodies of the invention.

[00199] The present invention further includes a method of preventing or treating bone metabolism and connective tissue disorders or diseases or disorders (e.g., Paget's disease, abnormal bone remodeling, osteoporosis, Gorham-Stout syndrome, arthritis (e.g., osteoarthritis, rheumatoid arthritis, psoriatic arthritis), brittle bone disease) in a mammal wherein the activity of osteoclasts causes or exacerbates (e.g., causes and/or facilitates bone and/or cartilage destruction or inflammation) said connective tissue disease.

[00200] Another embodiment of the invention includes a method of preventing bone cancer metastasis in a mammal by administering an antibody of the invention.

[00201] Another embodiment of the invention includes a method of modulating osteoclast activity and/or normalizing osteoblast: osteoclast homeostasis by administering an antibody of the invention. In one aspect, an antibody of the invention is administered to inhibit bone resorption or bone formation. In another aspect, an antibody of the invention is administered to increase levels of bone resorption or bone formation.

[00202] Another embodiment of the invention includes a method of treating preeclampsia by administering an antibody of the invention.

[00203] In yet a further aspect, the invention provides an article of manufacture, comprising: a container; a label; and a composition comprising an active agent contained within the container; wherein the label indicates that the composition can be used to inhibit angiogenesis or to treat a disease or disorder characterized by undesirable or excessive vascularization or vascular permeability and the active agent in the composition is an Ephrin

B2 antagonist of the invention.

Pharmaceutical Formulations, Administration And Dosing

[00204] Pharmaceutical formulations of the invention may contain as the active ingredient human, humanized, or chimeric Ephrin B2 antibodies of the invention. 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.

[00205] An Ephrin B2 antibody of the invention may be formulated with a pharmaceutically acceptable carrier. The term "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. When used in medicine, 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. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. The term "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.

[00206] According to certain aspects of the invention, 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 polyvinylpyrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN, PLURONICS™ or polyethylene glycol (PEG).

[00207] Ephrin B2 antibody compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal. [00208] Ephrin B2 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. AU methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, Ephrin B2 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.

[00209] Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of Ephrin B2 antibody 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. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, 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. In certain embodiments, carrier formulation suitable for various routes of administration can be the same or similar to that described for RTTUXAN™. 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. In certain embodiments of the invention, anti- Ephrin B2 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. Those of skill in the art are aware that intravenous injection provides a useful mode of administration due to the thoroughness of the circulation in rapidly distributing antibodies. Intravenous administration, however, is subject to limitation by 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.

[00210] 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. For example, 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.

[00211] The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interracial 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. Such techniques are disclosed in Remington 's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[00212] The formulations to be used for in vivo administration are typically sterile. This is readily accomplished by filtration through sterile filtration membranes. [00213] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing an Ephrin B2 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. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and lβuprolide acetate), and 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. 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°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-disulfide 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. [00214] Ephrin B2 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 -Ephrin B2 antibody disclosed herein) to a human. The components of the liposome are commonly arranged in abilayer 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 etal, Proc. Natl. Acad. Set. USA, 82:3688 (1985); Hwang et al, Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. 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 etal., J. National Cancer Inst., (19)1484 (1989). [00215] Some of the pharmaceutical formulations include, but are not limited to: [00216] (a) a sterile, preservative-free liquid concentrate for intravenous (i.v.) administration of anti- Ephrin B2 antibody, supplied at a concentration of 10 mg/ml in either 100 mg (10 iiiL) or 500 mg (50 ttiL) single-use vials. The product can be formulated for i.v. administration using sodium chloride, sodium citrate dihydrate, polysorbate and sterile water for injection. For example, 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.

[00217] (b) A sterile, lyophilized powder in single-use glass vials for subcutaneous (s.c.) injection. The product can be formulated with sucrose, L-histidine hydrochloride monohydrate, L-histidine and polysorbate 20. For example, each single-use vial can contain 150 mg anti-Ephrin B2 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.

[00218] (c) A sterile, preservative-free lyophilized powder for intravenous (i.v.) administration. The product can be formulated with α-trehalose dihydrate, L-histidine HCl, histidine and polysorbate 20 USP. For example, each vial can contain 440 mg anti-Ephrin B2 antibody, 400 mg α,α-trehalose dihydrate, 9.9 mg L-histidine HCl, 6.4 mg L-histidine, and 1.8 mg polysorbate 20, USP. Reconstitution with 20 ml of bacteriostatic water for injection (BWFI), USP, containing 1.1% benzyl alcohol as a preservative, yields a multi-dose solution containing 21 mg/ml antibody at a pH of approximately 6.

[00219] (d) A sterile, lyophilized powder for intravenous infusion in which an anti- Ephrin B2 antibody is formulated with sucrose, polysorbate, monobasic sodium phosphate monohydrate, and dibasic sodium phosphate dihydrate. For example, 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.

[00220] (e) A sterile, preservative-free solution for subcutaneous administration supplied in a single-use, 1 ml pre-filled syringe. 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.

[00221] For example, each syringe can be formulated to deliver 0.8 ml (40 mg) of drug product. Each 0.8 ml contains 40 mg anti-Ephrin B2 antibody, 4.93 mg sodium chloride, 0.69 mg monobasic sodium phosphate dihydratβ, 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.

[00222] (f) A sterile, preservative-free, lyophilized powder contained in a single-use vial that is reconstituted with sterile water for injection (SWFI), USP, and administered as a subcutaneous (s.c.) injection. The product can be formulated with sucrose, histidine hydrochloride monohydrate, L-histidiπe, and polysorbate. For example, a 75 mg vial can contain 129.6 mg or 112.5 mg of an anti-Ephrin B2 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-Ephrin B2 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.

[00223] (g) A sterile, hyophilized product for reconstitution with sterile water for injection. The product can be formulated as single-use vials for intramuscular (IM) injection using mannitol, histidine and glycine. For example, each single-use vial can contain 100 mg anti- Ephrin B2 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. As another example, each single-use vial can contain 50 mg anti- Ephrin B2 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. [00224] (h) 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. For example, 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. As another example, 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.

[00225] In certain embodiments, apharmaceutical composition of the invention is stable at 4°C. In certain embodiments, a pharmaceutical composition of the invention is stable at room temperature. Antibody Half-Life

[00226] In certain embodiments, the half-life of an anti- Ephrin B2 antibody compositions and methods of the invention is at least about 4 to 7 days. In certain embodiments, the mean half-life of an anti-Ephrin B2 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, l l to 15, 12 to 16, 13 to 17, 14 to 18, 15 to 19, or 16 to 20 days. In other embodiments, the mean half-life of an anti- Ephrin B2 antibody 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. In still further embodiments the half-life of an anti-Ephrin B2 antibody of compositions and methods of the invention can be up to about 50 days. In certain embodiments, 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.

[00227] The serum circulation of anti- Ephrin B2 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. Linear or branched polymer derealization 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. [00228] Further, the antibodies of 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. Administration and Dosing

[00229] Administration of compositions of the invention to a human patient 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. In one embodiment, 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. In particular embodiments, the route of administration is via bolus or continuous infusion over a period of time, once or twice a week. In other particular embodiments, the route of administration is by subcutaneous injection, optionally once or twice weekly. In one embodiment, compositions, and/or methods of the invention are administered on an outpatient basis.

[00230] In certain embodiments, the dose of a composition comprising anti-Ephrin B2 antibody is measured in units of mg/kg of patient body weight. In other embodiments, the dose of a composition comprising anti- Ephrin B2 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- Ephrin B2 antibody is measured in units of mg/m² of patient body surface area. In yet other embodiments, the dose of a composition comprising anti- Ephrin B2 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.

[00231] Those skilled in the art will appreciate that dosages can be selected based on a number of factors including the age, sex, species and condition of the subject (e.g., stage of B cell malignancy), the desired degree of cellular depletion, the disease to be treated and/or the particular antibody or antigen-binding fragment being used and can be determined by one of skill in the art. For example, effective amounts of 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 etal, Blood, 89(8): 2994-2998 (1997)), incorporated by reference herein in its entirety). In certain embodiments, for particular B cell malignancies, therapeutic regimens standard in the art for antibody therapy can be used with compositions and methods of the invention.

[00232] 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.

[00233] Those skilled in the art will appreciate mat dosages are generally higher and/or frequency of administration greater for initial treatment as compared with maintenance regimens.

[00234] In certain embodiments, dosages of the antibody (optionally in a pharmaceutically acceptable carrier as part of a pharmaceutical composition) 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/m² 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/m². In certain embodiments, the dosage is between about 0.0005 to about 200 mg/m², between about 0.001 and 150 mg/m², between about 0.075 and 125 mg/m², between about 0.375 and 100 mg/m², between about 2.5 and 75 mg/m², between about 10 and 75 mg/m², and between about 20 and 50 mg/m². In related embodiments, the dosage of anti- Ephrin B2 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. In certain embodiments, the dose of naked anti- Ephrin B2 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 anti -Ephrin B2 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-Ephrin B2 antibody used is at least about 5, 6, 7, 8, 9, or 10 mg/kg of body weight of a patient. In certain embodiments, 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, 186, 188, 190, 192, 194, 196, 198,

200, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,

240, 242, 244, 246, 248, or 250 micrograms/m². In other embodiments, dose is up to 1 g per single dosage unit.

[00235] All of the above doses are exemplary and can be used in conjunction with compositions and methods of the invention; however where an anti-Ephrin B2 antibody is used in conjunction with a toxin or radiotherapeutic agent the lower doses described above may be preferred.

[00236] In certain embodiments of the invention, the dose can be escalated or reduced to maintain a constant dose in the blood or in a tissue, such as, but not limited to, bone marrow.

In related embodiments, 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.

[00237] In certain embodiments, 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.

[00238] According to another aspect of methods of the invention, 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.

Particular embodiments of the invention

[00239] 1. An isolated monoclonal antibody or fragment thereof that binds Ephrin B2 and comprises at least one CDR of an antibody selected from the group consisting of: E2; Dl;

B l; and Al l

[00240] 2. An isolated monoclonal antibody or fragment thereof that binds Ephrin B2 and comprises at least three CDRs of an antibody selected from the group consisting of: E2;

Dl; Bl; and Al l. [00241] 3. An isolated monoclonal antibody or fragment thereof that binds Ephrin B2 and comprises all six CDRs of an antibody selected from the group consisting of: E2; Dl; Bl; and Al l.

[00242] 4. The antibody of embodiment 1 comprising at least one heavy chain polypeptide of an antibody selected from the group consisting of: E2; Dl; Bl; and Al l.

[00243] 5. The antibody of embodiment 1 comprising at least one light chain polypeptide of an antibody selected from the group consisting of: E2; D 1 ; B 1 ; and Al l.

[00244] 6. The antibody of embodiment 1 comprising both the light and heavy chain polypeptide of an antibody selected from the group consisting of: E2; Dl; Bl; and Al 1.

[00245] 7. An isolated monoclonal antibody or antibody fragment that binds the same epitope on Ephrin B2 as the antibody of embodiment 3.

[00246] 8. The antibody of any of the preceeding embodiments, wherein said antibody inhibits EphB4-Ephrin B2 binding and inhibits EphrinB2 activity.

[00247] 9. The antibody of embodiment 8, wherein said antibody cross reacts with mouse

Ephrin B2.

[00248] 10. The antibody of embodiment 8 or 9, wherein said antibody blocks EphB4 phosphorylation.

[00249] 11. The antibody of any of the preceeding embodiments, wherein said antibody possesses one or more activity selected from the group consisting of: inhibition of tubule formation (HUAEC, HUVEC or HMEC endothelial cells); inhibition of cell survival of endothelial cells; inhibition of cell survival of tumor cells; inhibition of matrigel plug angiogenesis in response to bFGF or VEGF; and inhibition of growth of a tumor selected from the group consisting of: breast, colon, bladder, renal, ovarian, prostate, and melanoma.

[00250] 12. A nucleic acid encoding the polypeptide sequence of at least one CDR of an antibody selected from the group consisting of: E2; Dl; Bl; and Al l.

[00251] 13. A vector comprising the nucleic acid of embodiment 12.

[00252] 14. An isolated cell comprising the vector of embodiment 13.

[00253] 15. An isolated cell expressing the antibody as in any of embodiments 1-11.

[00254] 16. A pharmaceutical composition comprising the antibody as in any of embodiments 1-11 in a pharmaceutically-acceptable carrier.

[00255] 17. The pharmaceutical composition of embodiment 16, wherein the antibody is of the IgGl, IgG2, IgG3, or IgG4 human isotype. [00256] 18. A method of inhibiting angiogenesis in a mammal comprising administering a therapeutically-effective amount of the antibody as in any of embodiments 1-11.

[00257] 19. A method of suppressing tumor growth in a mammal comprising administering a therapeutically-efTective amount of the antibody as in any of embodiments 1-

11.

[00258] 20. A method of killing tumor cells in a mammal comprising administering a therapeutically-effective amount of the antibody as in any of embodiments 1-11.

[00259] 21. A method of treating a malignancy in a human comprising administering to a human in need thereof a therapeutically-effective amount of the antibody as in any of embodiments 1-11.

[00260] 22. A method of detecting a tumor in a mammal comprising: removing a portion of the tumor from said mammal and contacting the antibody in any of embodiment 1 with said portion of tumor; and detecting the level of antibody that binds said tumor.

[00261] 23. The antibody of embodiment 8 or 9, wherein said antibody induces Ephrin

B2 phosphorylation.

[00262] 24. The antibody of either of embodiments 1-11 or 23, wherein said antibody blocks the binding of the extracellular domains of Ephrin B2 and EphB4.

[00263] 25. The antibody of either of embodiments 1-11, 23, or 24, wherein said antibody blocks the binding of the extracellular domains of Ephrin B2 and EphA4.

[00264] 26. The antibody of either of embodiments 1-11, 23, 24, or 25, wherein said antibody blocks the binding of the extracellular domains of Ephrin B2 and EphB2.

[00265] 27. The antibody of either of embodiments 1-11, 23, 24, 25, or 26, wherein said antibody blocks the binding of the extracellular domains of Ephrin B2 and EphB3.

[00266] 28. The antibody of any of the proceeding embodiments, wherein said antibody inhibits EphB4-Ephrin B2 binding and inhibits angiogenesis.

[00267] 29. The antibody of any of the proceeding embodiments, wherein said antibody induces internalization of Ephrin B2.

[00268] 30. An isolated antibody that immunospecifically binds to Ephrin B2 and comprises: a) a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 20, 26, 32 or 38; b) a VH CDR2 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 21, 27, 33 or 39; c) a VH CDR3 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 22, 28, 34 or 40; d) a VL CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 17, 23, 29 or 35; e) a VL CDR2 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 18, 24, 30 or 36; and f) a VL CDR3 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 19, 25, 31 or 37.

[00269] 31. An isolated antibody that immunospecifically binds to Ephrin B2 and comprises: a) a VH CDRl having the amino acid sequence of SEQ ID NO: 20, 26, 32 or 38; b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 21 , 27, 33 or 39; c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 22, 28, 34 or 40; d) a VL CDRl having the amino acid sequence of SEQ ID NO: 17, 23, 29 or 35; e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 18, 24, 30 or 36; and

£) a VL CDR3 having the amino acid sequence of SEQ ID NO: 19, 25, 31 or 37. [00270] 32. An isolated antibody that (i) immunospecifically binds Ephrin B2; and (ii) comprises a VH domain comprising three CDRs and a VL domain comprising three CDRs, wherein the three CDRs of the VH domain comprise: a) a VH CDRl comprising the amino acid sequence of SEQ ID NO: 20, 26, 32 or 38; b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 21, 27, 33 or 39; and c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 22, 28, 34 or 40.

[00271] 33. An isolated antibody that (i) immunospecifically binds Ephrin B2; and (ii) comprises a VH domain comprising three CDRs and a VL domain comprising three CDRs, wherein the three CDRs of the VH domain comprise: a) a VL CDRl comprising the amino acid sequence of SEQ ID NO: 17, 23, 29 or 35; b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 18, 24, 30 or 36; and c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 19, 25, 31 or 37.

[00272] 34. An isolated antibody that immunospecifically binds Ephrin B2 and comprises a heavy chain variable domain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:4, 8, 12 or 16 and further comprises a light chain variable domain having at least 90%, at least 95%, at least

97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2, 6, 10 or 14.

[00273] 35. An isolated antibody that immunospecifically binds Ephrin B2, wherein the antibody comprises the heavy chain variable domain of SEQ ID NO: 4, 8, 12 or 16 and the light chain variable domain of SEQ ID NO: 2, 6, 10 or 14.

[00274] 36. The isolated antibody of embodiment 35 comprising the heavy chain variable domain of SEQ ID NO: 4 and the light chain variable domain of SEQ ID NO: 2.

[00275] 37. The isolated antibody of embodiment 35 comprising the heavy chain variable domain of SEQ ID NO: 8 and the light chain variable domain of SEQ ID NO: 6.

[00276] 38. The isolated antibody of embodiment 35 comprising the heavy chain variable domain of SEQ ID NO: 12 and the light chain variable domain of SEQ ID NO: 10.

[00277] 39. The isolated antibody of embodiment 35 comprising the heavy chain variable domain of SEQ ID NO: 16 and the light chain variable domain of SEQ ID NO: 14.

[00278] 40. An isolated anti-Ephrin B2 monoclonal antibody that binds the same epitope on Ephrin B2 as the antibody of any one of embodiments 30 to 39.

[00279] 41. The isolated antibody as in any of embodiments 30 to 39, wherein the antibody inhibits EphB4-Ephrin B2 binding and inhibits an EphrinB2 activity.

[00280] 42. The isolated antibody as in any of embodiments 30 to 39, wherein the antibody inhibits EphB4-Ephrin B2 binding and inhibits angiogenesis.

[00281] 43. The isolated antibody as in any of embodiments 30 to 39, wherein the antibody blocks EphB4 phosphorylation and does not stimulate Ephrin B2 phosphorylation. [00282] 44. The isolated antibody as in any of embodiments 30 to 39, wherein the antibody possesses one or more activity selected from the group consisting of: a) inhibition of survival of endothelial cells; b) inhibition of angiogenesis in a mammal; c) inhibition of cell survival of tumor cells; d) inhibition of matrigel plug angiogenesis in response to bFGF or VEGF; and e) inhibition of growth of a tumor selected from the group consisting of: breast, colon, bladder, renal, ovarian, prostate, and melanoma.

[00283] 45. A nucleic acid encoding the isolated antibody as in any of embodiments 30 to 39.

[00284] 46. A vector comprising the nucleic acid of embodiment 16.

[00285] 47. An isolated cell comprising the vector of embodiment 17.

[00286] 48. An isolated cell expressing the antibody as in any of embodiments 30 to 39.

[00287] 49. A pharmaceutical composition comprising the antibody as in any of embodiments 30 to 39 in a pharmaceutically-acceptable carrier.

[00288] 50. The pharmaceutical composition of embodiment 20, wherein the antibody is of the IgGl, IgG2, IgG3, IgG4, or IgA human isotype.

[00289] 51. A method of inhibiting angiogenesis in a mammal comprising administering a therapeutically-effective amount of the antibody as in any of embodiments 30 to 39.

[00290] 52. A method of suppressing tumor growth in a mammal comprising administering a therapeutically-effective amount of the antibody as in any of embodiments 30 to 39.

[00291] 53. A method of killing tumor cells in a mammal comprising administering a therapeutically-effective amount of the antibody as in any of embodiments 30 to 39.

[00292] 54. A method of treating a malignancy in a human comprising administering to a human in need thereof a therapeutically-effective amount of the antibody as in any of embodiments 30 to 39.

[00293] 55. The antibody as in any of embodiments 30 to 39, wherein said antibody is conjugated to a toxin.

[00294] 56. The antibody of embodiment 26, wherein said toxin is an auristatin.

[00295] 57. A method of detecting a tumor in a mammal comprising: a) removing a portion of the tumor from said mammal and contacting the antibody as in any of embodiments 30 to 39 with said portion of tumor; and b) detecting the level of antibody that binds said tumor.

EXAMPLES

Isolation of Human Anti-Ephrin B2 Antibodies

[00296] A phage display approach was used to isolate fully human Ephrin B2 antibodies. This approach avoids challenges associated with raising mouse antibodies to human Ephrin B2 since mouse and human Ephrin B2 share significant homology. A large panel of human anti- Ephrin B2 antibodies were isolated from a naive human Fab phage display library by several rounds of panning against immobilized human Ephrin B2 extracellular region-Fc (hEphrinB2- ECD-Fc). The amino acid sequence of an Ephrin B2 ECD sequence is provided in Figure 15. The clones were then sequenced to eliminate duplicate clones and the Fab fragments of some clones were ultimately subcloned into an expression vector for the production of full length IgG. The nucleotide and corresponding amino acid sequences of the variable regions of the light and heavy chains of several antibody clones (Al 1, Bl, Dl, E2) are provided in the sequence listing (SEQ ID NOS: 1-16) and Figures 10 and 11. Figures 10 and 11 represent the variable regions of the light and heavy chains, respectively of antibody clones Al 1, Bl, Dl, E2. The CDRs for each antibody depicted in Figure 10 and 11 are indicated. The resulting full-length antibodies were purified and their biochemical characteristics were determined as described below.

[00297] Isolation of Human anti-Ephrin B2 Antibodies. A human Fab phage display library (Fab310, Dyax, Cambridge, MA) was selected for 3 rounds using human EphrinB2- ECD-Fc immobilized by ProteinA/G coated onto a solid surface. The process is briefly described below.

[00298] Day 1. Coat an immunotube with Protein A/G at 2.5 μg/ml and another immunotube at 100 μg/ml in PBS (pH 7.4). Leave them in 4 degrees C overnight. [00299] Day 2. Take out tubes from refrigerator. Bring to room temperature. Take out reagent. Wash tubes 3 times with TPBS (PBS with 0.1 % Tween 20) and 3 times with PBS. Block the tubes with 4% BSA in TPBS. Leave them at RT for 1 hour. Take out blocker. Wash the tubes with PBS. Add 1 ml of EphrinB2.Fc at 40 μg/ml in blocker to tube 1. Add ImI of polyclonal human IgG at 4 mg/ml in blocker to tube 2. Seal tubes and keep in refrigerator over night.

[00300] Day 3: Take immunotubes out of refrigerator. Keep at RT for 60. Take 1000 fold of phage as library size and block with 3% BSA for 45' at RT. Take out solution from tube 2, wash tube 2 for 3 times with TPBS and transfer blocked phage to tube 2 and leave at RT for 1 hour for deselection. Take out solution from tube 1, wash tube 1 for 3 times with TPBS and transfer deselected phage library from tube 2 to tube 1 (save 5 ul for titration of input phage). Leave at RT for 1.5 hours. Wash tube 1 for 15 times with TPBS and 15 times with PBS. Elute the bound phage with 1 ml of 100 mM Triethyl amine (TEA) in water for 15' at RT. Immediately neutralize the eluted phage with 500 μl IM tris, pH 7.4. Save 5 μl of the eluted phage for output titration. Use the rest 1495 μl to infect E.coli TGl culture to amplify the eluted phage. Mix 1 volume of eluted neutralized phage with 5 volumes of log phase TGl and 4 volume of 2YT. Incubate at 37 °C for 30 min. Incubate at 37°C for 30 min with shaking at 250 rpm. Harvest the infected cells by centrifugation and resuspend in 2YT and plate on 2YT agar with 100 μg/ml carbenicillin and 2% glucose and incubate overnight at 30°C. [00301] Day 4: Scrape the colonies from the plates in 2YT containing 100 μg/ml carbenicillin and 2% glucose. Inoculate 25 ml of 2YT containing 100 μg/ml carbenicillin and 2% glucose with the scraped colonies to a final OD600 of 0.1. Incubate the culture at 37 °C with shaking at 250 rpm till it reaches an OD600 of 0.5 at which point take 5 ml of the culture and super-infect with helper phage M 13KO7 at an moi (multiplicity of infection) of 20. Incubate the super-infected culture at 37 °C for 30 min and then at 37°C for 30 min with shaking at 250 rpm. Harvest the superinfected cells by centrifugation and resuspend in 50 ml 2YT with 100 μg/ml carbenicillin and 50 μg/ml kanamycin and incubate overnight at 30°C with shaking at 250 rpm.

[00302] Day 5: Remove the cells totally by centrifugation. To the bacterial culture supernatant add 1/5 volume of cold PEG 6000 (20%). Leave on ice for Ih. Spin at 14000 rpm for 10 min. Resuspend in PBS (pH 7.4). Repeat this phage precipitation steps 3 times. Use the purified phage for the next round of panning by following steps above. [00303] After the third round of panning individual bacterial colonies are cultured and phage particles rescued and tested for specific binding to huEphrinB2 ECD.Fc and huEphrinB2 ECD His (ECD Extra-cellular domain of huEphrinB2).

[00304] The ability of the huEphrinB2 ECD.Fc and huEphrinB2 ECD His-reacting clones to inhibit the binding of EphB4 ECD to EphrinB2 ECD was assayed as follows: ELISA plates were coated with hEphB4.Fc at 5 μg in PBS, pH 7.4. Cell culture supernatant containing huEphrinB2 labeled with alkaline phosphatase (huEphrinB2. AP fusion protein) was added to the ELISA wells at a final dilution of 1 : 12 (EC50 of huEphrinB2.AP binding to huEphB4, Figure 5) mixed with or without huEphrinB2 specific phage supernatants. The interaction (or lack thereof) between EphB4 and Ephrin B2 was detected by addition of PNPP (p-Nitrophenyl Phosphate) at 405nm. See, Figure 6. Four inhibitory clones were selected (circled in Figure 6). These Fab clones were also tested for the ability to bind both murine and human EphrinB2 ECD Fc and huEphrinB2 ECD.His.

[00305] The 4 selected clones containing Fabs of interest were converted to full IgG molecules using IgG expression vectors under the control of CMV promoter (pBh 1 vector, Dyax). The plasmid containing the DNA encoding the IgG was transiently transfected into 293H cells and the antibodies were purified from culture supernatant by passing it through Protein A column. The purified antibodies were tested for activity (binding to hEphrinB2 ECD Fc, mEphrinB2 ECD Fc, hEphrinB2 ECD.His, hEphrinB2 ECD HSA and pHuIgG (control Ig) and the ability to inhibit the binding of hEphB4 ECD.Fc to human EphrinB2 ECD AP and mEphB4 ECD.Fc to biotinylated mouse EphrinB2 ECD.Fc. See, Figures 8 and 9. The antibodies were then tested for their binding specificity to Ephrin B2 ECD by comparing their binding to several other Ephrins family member ECD domains and the ability to bind Ephrin B2 on die surface of cells. See, Figures 12A-H and 13. [00306] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. [00307] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, U.S. Provisional Application No 61/088,228 filed August 12, 2008, is hereby incorporated by reference in its entirety for all purposes.

Claims

.What is claimed is:

1. An isolated antibody that immunospecifically binds to Ephrin B2 and comprises: a) a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 20, 26, 32 or 38; b) a VH CDR2 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 21, 27, 33 or 39; c) a VH CDR3 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 22, 28, 34 or 40; d) a VL CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 17, 23, 29 or 35; e) a VL CDR2 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 18, 24, 30 or 36; and f) a VL CDR3 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 19, 25, 31 or 37.

2. An isolated antibody that immunospecifically binds to Ephrin B2 and comprises: a) a VH CDRl having the amino acid sequence of SEQ ID NO: 20, 26, 32 or 38; b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 21, 27, 33 or 39; c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 22, 28, 34 or 40; d) a VL CDRl having the amino acid sequence of SEQ ID NO: 17, 23, 29 or 35; e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 18, 24, 30 or 36; and f) a VL CDR3 having the amino acid sequence of SEQ ID NO: 19, 25, 31 or 37.

3. An isolated antibody that (i) immunospecifically binds Ephrin B2; and (ii) comprises a VH domain comprising three CDRs and a VL domain comprising three CDRs, wherein the three CDRs of the VH domain comprise: a) a VH CDRl comprising the amino acid sequence of SEQ ID NO: 20, 26, 32 or 38; b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 21 , 27, 33 or 39; and c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 22, 28, 34 or 40.

4. An isolated antibody that (i) immunospecifically binds Ephrin B2; and (ii) comprises a VH domain comprising three CDRs and a VL domain comprising three CDRs, wherein the three CDRs of the VH domain comprise: a) a VL CDRl comprising the amino acid sequence of SEQ ID NO: 17, 23, 29 or 35; b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 18, 24, 30 or 36; and c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 19, 25, 31 or 37.

5. An isolated antibody that immunospecifically binds Ephrin B2 and comprises a heavy chain variable domain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 4, 8, 12 or 16 and further comprises a light chain variable domain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:2, 6, 10 or 14.

6. An isolated antibody that immunospecifically binds Ephrin B2, wherein the antibody comprises the heavy chain variable domain of SEQ ID NO: 4, 8, 12 or 16 and the light chain variable domain of SEQ ID NO: 2, 6, 10 or 14.

7. The isolated antibody of claim 6 comprising the heavy chain variable domain of SEQ ID NO: 4 and the light chain variable domain of SEQ ID NO: 2.

8. The isolated antibody of claim 6 comprising the heavy chain variable domain of SEQ ID NO: 8 and the light chain variable domain of SEQ ID NO: 6.

9. The isolated antibody of claim 6 comprising the heavy chain variable domain of SEQ ID NO: 12 and the light chain variable domain of SEQ ID NO: 10.

10. The isolated antibody of claim 6 comprising the heavy chain variable domain of SEQ ID NO: 16 and the light chain variable domain of SEQ ID NO: 14.

11. An isolated anti-Ephrin B2 monoclonal antibody that binds the same epitope on Ephrin B2 as the antibody of any one of claims 1 to 10.

12. The isolated antibody as in any of claims 1 to 10, wherein the antibody inhibits EphB4- Ephrin B2 binding and inhibits an EphrinB2 activity.

13. The isolated antibody as in any of claims 1 to 10, wherein the antibody inhibits EphB4- Ephrin B2 binding and inhibits angiogenesis.

14. The isolated antibody as in any of claims 1 to 10, wherein the antibody blocks EphB4 phosphorylation and does not stimulate Ephrin B2 phosphorylation.

15. The isolated antibody as in any of claims 1 to 10, wherein the antibody possesses one or more activity selected from the group consisting of: a) inhibition of survival of endothelial cells; b) inhibition of angiogenesis in a mammal; c) inhibition of cell survival of tumor cells; d) inhibition of matrigel plug angiogenesis in response to bFGF or VEGF; and e) inhibition of growth of a tumor selected from the group consisting of: breast, colon, bladder, renal, ovarian, prostate, and melanoma.

16. A nucleic acid encoding the isolated antibody as in any of claims 1 to 10.

17. A vector comprising the nucleic acid of claim 16.

18. An isolated cell comprising the vector of claim 17.

19. An isolated cell expressing the antibody as in any of claims 1 to 10.

20. A pharmaceutical composition comprising the antibody as in any of claims 1 to 10 in a pharmaceutically-acceptable carrier.

21. The pharmaceutical composition of claim 20, wherein the antibody is of the IgGl, IgG2, IgG3, IgG4, or IgA human isotype.

22. A method of inhibiting angiogenesis in a mammal comprising administering a therapeutically-efFective amount of the antibody as in any of claims 1 to 10.

23. A method of suppressing tumor growth in a mammal comprising administering a therapeutically-efFective amount of the antibody as in any of claims 1 to 10.

24. A method of killing tumor cells in a mammal comprising administering a therapeutically-efFective amount of the antibody as in any of claims 1 to 10.

25. A method of treating a malignancy in a human comprising administering to a human in need thereof a therapeutically-effective amount of the antibody as in any of claims 1 to 10.

26. The antibody as in any of claims 1 to 10, wherein said antibody is conjugated to a toxin.

27. The antibody of claim 26, wherein said toxin is an auristatin.

28. A method of detecting a tumor in a mammal comprising: a) removing a portion of the tumor from said mammal and contacting the antibody as in any of claims 1 to 10 with said portion of tumor; and b) detecting the level of antibody that binds said tumor.