MX2008008740A - Anti-ephb4 antibodies and methods using same - Google Patents

Abstract

The invention provides anti-EphB4 antibodies, and compositions comprising and methods of using these antibodies.

Description

ANTI-EPHB4 ANTIBODIES AND METHODS OF USE OF THE SAME FIELD OF THE INVENTION The present invention is generally concerned with the fields of molecular biology. More specifically, the invention is concerned with anti-EphB4 antibodies and uses thereof.

BACKGROUND OF THE INVENTION The development of a vascular supply is a fundamental requirement for many physiological and pathological processes. Actively growing tissues such as embryos and tumors require an appropriate blood supply. They satisfy this need by producing angiogenic pro-factors, which promote the formation of new blood vessels via a process called angiogenesis. Vascular tube formation is a complex but orderly biological event that involves all or many of the following stages: a) endothelial cells (EC) proliferate from existing CDs or differentiate from progenitor cells; b) the ECs migrate and coalesce to form rope-like structures; c) the vascular cords then undergo tubulogenesis to form vessels with a central lumen; d) existing ropes or vessels send shoots to form secondary vessels; e) primitive vascular plexus undergo remodeling and additional reformation; and f) peri-endothelial cells are recruited to enclose the endothelial tubes, providing maintenance functions and modulators to the vessels; such cells include pericytes for small capillaries, smooth muscle cells for larger vessels, and myocardial cells in the heart. Hanahan, Science 277: 48-50 (1997); Hogan & Kolodziej, Na t. Rev. Genet. 3: 513-23 (2002); Lubarsky & Krasnow, Cell 112: 19-28 (2003). It is now well established that angiogenesis is involved in the pathogenesis of a variety of disorders. These include solid tumors and metastases, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, infraocular neovascular diseases such as proliferative retinopathies, for example, diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue. and other tissues, rheumatoid arthritis and psoriasis. Folkman et al., J. Biol. Chem. 267: 10931-34 (1992); Klagsbrun et al., Annu. Rev. Physiol. 53: 217-39 (1991); and Garner A., "Vascular diseases", In: Pathobiology of Ocular Disease. A Dynamic Approach, Garner A., Klintworth GK, eds., 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710. In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia and to provide nutrition for growth and tumor metastasis. Folkman et al., Na ture 339: 58 (1989). Neovascularization allows tumor cells to acquire an advantage of growth and proliferative autonomy compared to normal cells. A tumor usually begins as a single aberrant cell that can proliferate only to a size of a few cubic millimeters due to the distance of available capillary beds and can remain "dormant" without further growth and spread for a long period of time. Some tumor cells change the angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillary blood vessels. These newly formed blood vessels not only allow the continuous growth of the primary tumor, but also the dissemination and recolonization of metastatic tumor cells. Thus, a correlation has been observed between the density of microvessels in tumor sections and the survival of the patient in breast cancer as well as in several other tumors. Weidner et al., N. Engl. J. Med. 324: 1-6 (1991); Horak et al., Lancet 340: 1120-24 (1992); Macchiarini et al., Lancet 340: 145-46 (1992). The precise mechanism controlling the angiogenic change is not well understood, but it is believed that the neovascularization of the tumor mass resulting from the net equilibrium of a multitude of simulators and inhibitors of angiogenesis (Folkman, Na t.Med.1 (1): 27-31 (1995)). The process of vascular development is strongly regulated. To date, a significant number of molecules, most of the secreted factors produced by cells surrounding, have been shown to regulate EC differentiation, proliferation, migration and coalescence to rope-like structures. For example, vascular endothelial growth factor (VEGF) has been identified as the key factor involved in the stimulation of angiogenesis and in inducing vascular permeability. Ferrara et al., Endocr. Rev. 18: 4-25 (1997). The finding that the loss of even a single allele of VEGF results in embryonic mortality points to an irreplaceable role played by this factor in the development and differentiation of the vascular system. In addition, VEGF has been shown to be a key mediator of neovascularization associated with tumors and infra-ocular alterations. Ferrara et al., Endocr. Rev. supra. VEGF mRNA is overexpressed by the majority of human tumors examined. Berkman et al., J. Clin. Invest. 91: 153-59 (1993); Brown et al., Human Pa thol. 26: 86-91 (1995); Brown et al., Cancer Res. 53: 4727-35 (1993); Mattern et al., Brit. J. Cancer 73: 931-34 (1996); Dvorak et al., Am. J. Pa thol. 146: 1029-39 (1995). Also, the concentration levels of VEGF in ocular fluids are highly correlated with the presence of active proliferation of blood vessels in patients with diabetic retinopathies and other ischemia-related retinopathies. Aiello et al., N. Engl. J. Med. 331: 1480-87 (1994). In addition, studies have shown the location of VEGF in choroidal neovascular membranes in patients affected by AMD. López et al., Invest. Ophthalmol. Vis. Sci. 37: 855-68 (1996). Neutralizing anti-VEGF antibodies suppress the growth of a variety of human tumor cell lines in nude mice (Kim et al., Na ture 362: 841-44 (1993); Warren et al., J. Clin. Invest. 95: 1789-97 (1995), Borgstrom et al., Cancer Res. 56: 4032-39 (1996), Melnyk et al., Cancer Res. 56: 921-24 (1996)) and also inhibit intraocular angiogenesis in models of ischemic retinal alterations (Adamis et al., Arch. Ophthalmol 114: 66-71 (1996)). Accordingly, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of tumors and various infraocular neovascular disorders. Such antibodies are described, for example, in EP 817,648, published January 14, 1998; and in WO 98/45331 and WO 98/45332, both published October 15, 1998. An anti-VEGF antibody, bevacizumab, has been approved by the FDA for use in combination with a chemotherapy regimen to treat metastatic colorectal cancer ( CRC). In addition, bevacizumab is being investigated in many ongoing clinical trials for the treatment of several indications of cancer. The EphB4 receptor ("EphB4" or "EphB4R") is a member of the eph receptor family, which constitutes the largest family of tyrosine kinase receptors in the human genome (reviewed in Dodelet, Oncogene, 19: 5614-5619, 2000). Tyrosine receptor kinases of human eph are categorized by sequence identity to Class A and Class B with corresponding type A and type B ligands referred to as efriñas. Signaling can occur in a forward manner, in which the receptor tyrosine kinase is activated by the ligand and in reverse, in which the transmembrane ephrin B ligands are activated by interaction with receptors. Eph receptor ligand interactions have been implicated in a wide range of biological functions including axon guidance, tissue boundary formation, vasculogenesis, and cell mobility (Kullander et al., Nat. Rev. Mol. Cell. Biol. ., 3: 475-486, 2002; Cheng et al., Cytokine Growth Factor Rev., 13: 75-85, 2002; Coulthard et al., Int. J. Dev. Biol., 46: 375-384, 2002). EphB4 binds to ligands such as ephrin-Bl, ephrin-B2 and ephrin-B3. The EphB4 receptor has an extracellular region with a cysteine-rich portion that extends over its amino-terminal half followed by two portions of type II fibronectin. There is an intracellular domain comprising a conserved kinase region and a transmembrane domain. It is clear that there is a need for agents that have clinical attributes that are optimal for development as therapeutic agents. The invention described herein meets this need and provides other benefits.

All references cited herein, in which patent applications and publications are included, are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE INVENTION The invention is based in part on the identification of a variety of EphB4 binding agents (such as immunoconjugates, antibodies and fragments thereof). EphB4 is presented as an important and advantageous therapeutic agent and the invention provides compositions and methods based on the EphB4 binding. EphB4 binding agents of the invention, as described herein, provide important therapeutic and diagnostic agents for use in targeting pathological conditions associated with the expression and / or activity of the EphB4-EphB4 ligand pathways. Thus, the invention provides methods, compositions, equipment and articles of manufacture related to the binding of EphB4. The present invention provides antibodies that bind (such as specifically binding) to EphB4. In one aspect, the invention provides the isolated anti-EphB4 antibody, wherein a full length IgG form of the antibody specifically binds to human EphB4 with a binding affinity of about 50 pM or better. As is well established in art, the binding affinity of a ligand to its receptor can be determined using any of a variety of analysis and expressed in terms of a variety of quantitative values. Thus, in one embodiment, the binding affinity is expressed as Kd values and reflects intrinsic binding affinity (e.g., with minimized avidity effects). In general and preferably, the binding affinity is measured in vitro, either in a cell-free kit or a cell-associated kit. Any of a variety of assays known in the art, including those described herein, can be used to obtain binding affinity measurements, which include, for example, Biacore, radioimmunoassay (RIA) and ELISA. In one aspect, the invention provides an isolated antibody that binds to a ligand binding region of EphB. In some embodiments, the isolated antibody is linked to a polypeptide comprising, consisting of or consisting essentially of the extracellular domain of EphB4. In one aspect, the invention provides an isolated anti-EphB4 antibody that competes with the EphB4 ligand for the EphB4 binding. In one aspect, the invention provides an isolated anti-EphB4 antibody that inhibits, reduces and / or blocks the activity of EphB4. In some embodiments, the autophosphorylation of EphB4 is inhibited, reduced and / or blocked.

In one aspect, the anti-EphB4 antibody of the invention comprises: (a) at least one, two, three, four or five hypervariable region (HVR) sequences selected from the group consisting of: (i) a sequence comprising HVR-L1 Al-All, where Al-All is RASQDVSTAVA (SEQ ID NO: 9) (ii) a sequence of B1-B7, comprising HVR-L2 wherein B1-B7 is SASFLYS (SEQ ID NO: 11) (iii) HVR-L3 comprising the sequence C1-C9, wherein C1-C9 is QESTTTPPT (SEQ ID NO: 15) (iv) HVR-H1 comprising the sequence D1-D10, wherein D1-D10 is GFSISNYYLH ( SEQ ID NO: 2) (v) HVR-H2 comprising the sequence E1-E18, wherein E1-E18 is GGIYLYGSSSEYADSVKG (SEQ ID NO: 4) and (vi) HVR-H3 comprising the sequence F1-F17, in where F1-F17 is ARGSGLRLGGLDYAMDY (SEQ ID NO: 7); and (b) at least one variant HVR, wherein the variant HVR sequence comprises modification of at least one residue of the sequence illustrated in SEQ ID NOs: 1-17. In one aspect, the invention provides an antibody comprising one, two, three, four, five or six HVR, wherein each HVR comprises, consists or consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 1-17 , and where SEQ ID NO: 9 or 10 corresponds to a HVR- Ll, SEQ ID NO: 11 or 12 corresponds to an HVR-L2, SEQ ID NO: 13, 14, 15, 16 or 17 corresponds to an HVR-L3, SEQ ID NO: 1 or 2 corresponds to a HVR-H1, SEQ ID NO: 3, 4, 5 or 6 corresponds to an HVR-H2 and SEQ ID NO : 7 or 8 corresponds to an HVR-H3. In one embodiment, an antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each, in sequence, comprises SEQ ID NO: 9, 11 , 13, 1, 3, 7. In one embodiment, an antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each, in order , comprises SEQ ID NO: 10, 12, 14, 1, 3, 8. In one embodiment, an antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR- H3, wherein each, in sequence, comprises SEQ ID NO: 9, 11, 15, 2, 4, 7. In one embodiment, an antibody of the invention comprises HVR-Ll, HVR-L2, HVR-L3, HVR -Hl, HVR-H2 and HVR-H3, wherein each, in order, comprises SEQ ID NO: 9, 11, 16, 1, 5, 7. In one embodiment, an antibody of the invention comprises HVR-Ll, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each, in sequence, comprises SEQ ID NO: 9, 11, 17, 1, 6, 7.

HVR variants in an antibody of the invention may have modifications of one or more (such as two, three, four, five or more) residues within the HVR. In one embodiment, a variant HVR-Ll comprises 1-5 (1, 2, 3, 4 or 5) substitutions in any combination of the following positions: A6 (V or S), A7 (S or E), A8 (T or I), A9 (A or F) and A10 (V or L). In one embodiment, a variant HVR-L2 comprises 1-2 (1 or 2) substitutions in any combination of the following positions: B4 (F or N) and B6 (Y or E). In one embodiment, a variant HVR-L3 comprises 1-7 (1, 2, 3, 4, 5, 6 or 7) substitutions in any combination of the following positions: C2 (Q, E or K), C3 (S or T), C4 (Y, N, T, E or A), C5 (T, A or Q), C6 (T, V or l), C8 (P, L or E) and C9 (T or S). In one embodiment, a variant HVR-Hl comprises 1-3 (1, 2 or 3) substitutions in any combination of the following positions: D3 (T or S), D6 (G or N) and D9 (I or L). In one embodiment, a variant HVR-H2 comprises 1-5 (1, 2, 3, 4 or 5) substitutions in any combination of the following positions: E5 (P or L), E7 (S or G), E8 (G) or S), ElO (T, S or R) and Eli (D, E or G). In one embodiment, a variant HVR-H3 comprises 1 substitution in the following positions: F3 (G or S).

The letter (s) in parentheses next to each position indicates an illustrative substitution (ie, replacement) amino acid; as would be apparent to one skilled in the art, the desirability of other amino acids as substitution amino acids in the context described herein can be determined systematically using techniques known in the art and / or described herein. In one aspect, the invention provides an antibody comprising an HVR-H1 region comprising the sequence of SEQ ID NO: 1 or 2. In one aspect, the invention provides an antibody comprising an HVR-H2 region comprising the sequence of SEQ ID NO: 3, 4, 5 or 6. In one aspect, the invention provides an antibody comprising a region of HVR-H3 comprising the sequence of SEQ ID NO: 7 or 8. In one embodiment, the invention provides an antibody comprising a region of HVR-Ll comprising the sequence of SEQ ID NO: 9 or 10. In one embodiment, the invention provides an antibody comprising a region of HVR-L2 comprising the sequence of SEQ ID NO: 11 or 12. In one embodiment, the invention provides an antibody comprising an HVR-L3 region comprising the sequence of SEQ ID NO: 13, 14, 15, 16 or 17. In one aspect, the invention provides an antibody that comprises at least one, at least two, or all three of s following: (i) a HVR-H1 sequence comprising the sequence of SEQ ID NO: 2; (ii) a sequence of HVR-H2 comprising the sequence of SEQ ID NO: 4 (iii) a sequence of HVR-H3 comprising the sequence of SEQ ID NO: 7. In one aspect, the invention provides an antibody comprising at least one, at least two, or all three of the following: (i) a HVR-Ll sequence comprising the sequence of SEQ ID NO: 9; (ii) a sequence of HVR-L2 comprising the sequence of SEQ ID NO: 11; (iii) a sequence of HVR-L3 comprising the sequence of SEQ ID NO: 15. The amino acid sequences of SEQ ID NOs: 1-17 are numbered with respect to the individual HVR (ie, Hl, H2 or H3) as indicated in Figure 1, the numbering is consistent with the Kabat numbering system as described hereinafter. In one aspect, the invention provides antibodies comprising heavy chain HVR sequences as illustrated in Figure 1.

In one aspect, the invention provides antibodies comprising light chain HVR sequences as illustrated in Figure 1. Some antibody modalities of the invention comprise a light chain variable domain of humanized 4D5 antibody (huMAb4D5-8) (HERCEPTIN® , Genentech, Inc., South San Francisco, CA, USA) (also referred to in US Patent No. 6, 407, 233 and Lee et al., J. Mol. Biol. (2004), 340 (5): 1073-93) as illustrated in SEQ ID NO: 18 below. 1 Asp He Gln Met Thr Gln Ser Pro Le Ser Ser Leu Ser Val Gly Asp Arg Val Thr He Thr Cys Arq Ala Ser Gln Asp Val Asn Thr Ala Val Wing Trp Tyr Gln Gln Lys Pro Gly Lys Wing Pro Lys Leu Leu He Tyr Be Wing Be Phe Leu Tyr Be Gly Val Pro Be Arg Phe Be Gly Be Arg Be Gly Thr Asp Phe Thr Leu Thr Be Ser Leu Gln Pro Glu Asp Phe Wing Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu He Lys 107 (SEQ ID NO: 18) (the HVR residues are underlined) In one embodiment, the light chain variable domain sequence of huMAb4P5-8 is modified to one or more of 30 positions, 66 and 91 (Asn, Arg and His as indicated in bold / italics above, respectively). In one embodiment, the modified huMAb4P5-8 sequence comprises Ser in position 30, Gly in position 66 and / or Ser in position 91. Thus, in one embodiment, an antibody of the invention comprises a variable domain of light chain comprising the sequence illustrated in SEQ ID NO: 52 below: 1 Asp He Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr He Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Wing Trp Tyr Gln Gln Lys Pro Gly Lys Wing Pro Lys Leu Leu He Tyr Being Wing Being Phe Leu Tyr Being Gly Val Pro Being Arg Phe Being Gly Being Gly Being Gly Thr Asp Phe Thr Leu Thr Being Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu He Lys 107 (SEQ ID NO: 52) (the HVR residues are underlined) The residues substituted with respect to huMAb4D5 -8 are indicated in bold / italics above. The antibodies of the invention may comprise any variable domain sequence of appropriate structure, provided that the binding activity to EphB4 is substantially retained. For example, in some embodiments, the antibodies of the invention comprise a consensus sequence of human subgroup III heavy chain structure. In one embodiment of these antibodies, the structure consensus sequence comprises substitution at positions 71, 73 and / or 78. In some embodiments of these antibodies, position 71 is A, 73 is T and / or 78 is A. In a embodiment, these antibodies comprise heavy chain variable domain sequences of huMAb4D5-8 (HERCEPTIN®, Genentech, Inc., South San Francisco, CA, USA) (also referred to in US Pat. Nos. 6,407,213 and 5,821,337 and Lee et al., J. Mol. Biol. (2004), 340 (5): 1073-93). In one embodiment, these antibodies further comprise a human light chain structure consensus sequence. In one embodiment, these antibodies comprise huMAb4D5-8 light chain HVR sequences as described in U.S. Patent Nos. 6,407,213 and 5,821,337). In one embodiment, these antibodies comprise light chain variable domain sequences of huMAb4D5-8 (HERCEPTIN®, Genentech, Inc., South San Francisco, CA, USA) (also referred to in U.S. Patent Nos. 6,407,213 and 5,821,337 and Lee et al., J. Mol. Biol. (2004), 340 (5): 1073-93). In one embodiment, an antibody of the invention comprises a heavy chain variable domain, wherein the structure sequence comprises the sequence of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36 and / or 37 and HVR H1, H2 and H3 sequences are SEQ ID NOS:, 11 and / or 15, respectively. In one embodiment, an antibody of the invention comprises a light chain variable domain, wherein the structure sequence comprises the sequence of SEQ ID NOS: 38, 39, 40 and / or 41 and the sequence of HVR Ll, L2 and L3 are SEQ ID NOs: 2, 4 and / or 7, respectively. In one embodiment, an antibody of the invention comprises a heavy chain variable domain, wherein the structure sequence comprises the sequence of SEQ ID NOS: 46, 47, 48 and / or 49 and the HVR sequences Hl, H2 and H3 are SEQ ID NOS: 2, 4 and / or 7, respectively. In one embodiment, an antibody of the invention comprises a light chain variable domain, wherein the structure sequence comprises the sequence of SEQ ID NOS: 42, 43, 44 and / or 45 and HVR sequences Ll, L2 and L3 are SEQ ID NOS: 9, 11 and / or 15, respectively. In one embodiment, an antibody of the invention comprises a heavy chain variable domain, wherein the structure sequence comprises the sequences of SEQ ID NOS: 46, 47, 51 and 49 and the sequences of HVR H1, H2 and H3 are SEQ. ID NOS: 2, 4 and / or 7, respectively. In one embodiment, an antibody of the invention comprises a light chain variable domain, wherein the structure sequence comprises the sequences of SEQ ID NOS: 42, 43, 50 and 45 and the sequences of HVR Ll, L2 and L3 are SEQ. ID NOS: 9, 11 and / or 15, respectively. In one embodiment, an antibody of the invention is matured by affinity to obtain the desired target binding affinity. In one example, an affinity-matured antibody of the invention comprises substitution at one or more of amino acid positions H28, H31, H34, H52a, H54, H55, H57, H58, H95, L29, L30, L31, L32, L33, L53, L55, L90, L91, L92, L93, L94, L96 or L97. In one example an affinity-matured antibody of the invention comprises one or more of the following substitutions: (a) in the heavy chain, T28S, G31N, I34L, P52aL, S54G, G55S, T57S or R, D58, E or G, G95S or (b), in the light chain, V29S, S30E, T31I, A32F, V33L, F53N, Y55E, Q90E or K, S91T, Y92N TE or A, T93V or I, T94V or I, P96L or E or T97S. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 53. In one embodiment, an antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO: 54. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 53 and a light chain variable domain comprising the sequence of SEQ ID NO: 54. embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 55. In one embodiment, an antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO: 56. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 55 and a light chain variable domain comprising the SE sequence. Q ID NO: 56. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 57. In one embodiment, an antibody of the invention comprises a light chain variable domain that comprises the sequence of SEQ ID NO: 58. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 57 and a light chain variable domain comprising the SEQ ID sequence. NO: 58. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 59. In one embodiment, an antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO: 60. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 59 and a light chain variable domain comprising the sequence of SEQ ID NO: 60. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 61. In one embodiment, an antibody of the invention comprises a variable domain of cade. A light comprising the sequence of SEQ ID NO: 62. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 61 and a light chain variable domain comprising the sequence of SEQ ID NO: 62. In one aspect, the invention provides an antibody that competes with any of the aforementioned antibodies previously by the link to EphB4. In one aspect, the invention provides an antibody that binds to the same epitope in EphB4 as any of the antibodies mentioned above. As is known in the art and as is described in more detail later herein, the position / amino acid boundary that delineates a hypervariable region of an antibody may vary, depending on the context and the various definitions known in the art (as described later in the present). Some positions within a variable domain can be observed as hybrid hypervariable positions in which these positions can be considered to be within a hypervariable region under a set of criteria insofar as they are considered to be outside a hypervariable region under a different set of criteria . One or more of these positions can also be found in extended hypervariable regions (as defined later herein). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is selected from the group consisting of a chimeric antibody, an affinity matured antibody, a humanized antibody and a human antibody. In some embodiments, the antibody is an antibody fragment. In some modalities, the antibody is a Fab, Fab ', Fab'-SH, F (ab') 2 or scFv. In one embodiment, the antibody is a chimeric antibody, for example, an antibody that comprises antigen binding sequences from a non-human donor grafted to a non-human, human or humanized heterologous sequence (e.g., structure sequences and / or sequences constant domain). In one modality, the non-human donor is a mouse. In one embodiment, an antigen binding sequence is synthetic, for example obtained by mutagenesis (e.g., phage display selection, etc.). In one embodiment, a chimeric antibody of the invention has murine V regions and human C region. In one embodiment, the murine light chain V region is fused to a human kappa light chain. In one embodiment, the murine heavy chain V region is fused to a C region of human IgGl. Humanized antibodies of the invention include those that have amino acid substitutions in the FRs and affinity maturation variants with changes in the grafted CDRs. The amino acids substituted in the CDR or FR are not limited to those present in the donor or receptor antibody. In other embodiments, the antibodies of the invention further comprise changes in amino acid residues in the Fc region leading to improved effector function in which enhanced CDC and / or ADCC function or extermination of B cell. Other antibodies of the invention include those that have specific changes that improve stability. In other embodiments, the antibodies of the invention comprise changes in amino acid residues in the Fc region that lead to decreased effector function, for example decreased CDC and / or ADCC function and / or decreased B cell extermination. In some embodiments, the antibodies of the invention are characterized by decreased binding (such as lack of binding) to the human complement and / or human Fc receptor Clq factor in natural killer (NK) cells. In some embodiments, the antibodies of the invention are characterized by decreased binding (such as lack of binding) to human FcγRI, FcγRIIA and / or FcγRIIIA. In some embodiments, the antibodies of the invention are of the IgG class (eg, IgGl or IgG4) and comprise at least one mutation in E233, L234, G236, D265, D270, N297, E318, K320, K322, A327, A330, P331 and / or P329 (numbering according to the EU index). In some embodiments, the antibodies comprise the L234A / L235A or D265A / N297A mutation. In one aspect, the invention provides anti-EphB4 polypeptides that comprise any of the antigen binding sequences provided herein, wherein the anti-EphB4 polypeptides specifically bind to EphB4. The antibodies of the invention bind (such as specifically binding) to EphB4 and in some embodiments, can modulate one or more aspects of the EphB4-associated effects, which include but are not limited to activation of EphB4, molecular signaling downstream of EphB4, activation of EphB4 ligand, molecular signaling downstream of the EphB4 ligand, disruption of ligand binding (eg, ephrin-Bl, ephrin-B2 and / or ephrin-B3) to EphB4, phosphorylation of EphB4 and / or multimerization of EphB4 and / or phosphorylation of EphB4 ligand and / or disruption of any biological pathway EphB4 and / or biologically relevant EphB4 ligand, inhibition of angiogenesis and / or treatment and / or prevention of a tumor, cell proliferative alteration or cancer; and / or treatment or prevention of an alteration associated with the expression and / or activity of EphB4 (such as increased expression and / or EphB4 activity). In some embodiments, the antibody of the invention binds specifically to EphB4. In some embodiments, the antibody specifically binds to the extracellular domain of EphB4 (ECD). In some embodiments, the antibody specifically binds to a polypeptide that consists of or consists essentially of the extracellular domain of EphB4. In some embodiments, the antibody binds specifically to EphB4 with a KD of approximately 50 pM or stronger. In some embodiments, the antibody of the invention reduces, inhibits and / or blocks the activity of EphB4 in vivo and / or in vitro. In some embodiments, the antibody reduces, inhibits and / or blocks the autophosphorylation of EphB4. In In some embodiments, the antibody competes for binding to the EphB4 ligand (reduces and / or blocks the ligand binding of EphB4 to Ephb4). In one aspect, the invention provides the use of an antibody of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of an alteration, such as cancer, a tumor and / or a cell proliferative disorder. In some modalities, the alteration is neuropathy or neurodegenerative disease. In one aspect, the invention provides the use of an antibody of the invention in the preparation of a medicament for the inhibition of angiogenesis. In one aspect, the invention provides compositions comprising one or more antibodies of the invention and a carrier. In one embodiment, the carrier is pharmaceutically acceptable. In one aspect, the invention provides nucleic acids encoding an anti-EphB4 antibody of the invention. In one aspect, the invention provides vectors comprising a nucleic acid of the invention. In one aspect, the invention provides compositions comprising one or more nucleic acids of the invention and a carrier. In one embodiment, the carrier is pharmaceutically acceptable.

In one aspect, the invention provides host cells comprising a nucleic acid or a vector of the invention. A vector can be of any type, for example a recombinant vector such as an expression vector. Any of a variety of host cells can be used. In one embodiment, a host cell is a prokaryotic cell, for example, E. coli. In one embodiment, a host cell is a eukaryotic cell, for example a mammalian cell such as a Chinese hamster ovary cell (CHO). In one aspect, the invention provides methods for the manufacture of an antibody of the invention. For example, the invention provides methods of making an anti-EphB4 antibody (which, as defined herein, includes full length and fragments thereof) or immunoconjugate, the method comprising expressing in a suitable host cell a recombinant vector of the invention encoding the antibody (or fragment thereof) and recovering the antibody. In one aspect, the invention provides a manufacturing article comprising a container; and a composition contained within the container, wherein the composition comprises one or more anti-EphB4 antibodies of the invention. In one embodiment, the composition comprises a nucleic acid of the invention. In one embodiment, a composition comprising a The antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment, a manufacturing article of the invention further comprises instructions for administering the composition (eg, antibody) to a subject (such as instructions for any of the methods described herein). In one aspect, the invention provides a kit comprising a first container comprising a composition comprising one or more anti-EphB4 antibodies of the invention; and a second container comprising a buffer solution for pH. In one embodiment, the pH buffer is pharmaceutically acceptable. In one embodiment, a composition comprising an antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment, a kit further comprises instructions for administering the composition (e.g., the antibody) to a subject. In one aspect, the invention provides the use of an anti-EphB4 antibody of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of an alteration, such as cancer, tumor and / or cell proliferative alteration. In some modalities, the alteration is a neuropathy or neurodegenerative disease. In some modalities, alteration is a pathological condition associated with angiogenesis.

In one aspect, the invention provides the use of a nucleic acid of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of an alteration, such as cancer, tumor and / or cell proliferative alteration. In some modalities, the alteration is a neuropathy or neurodegenerative disease. In some modalities, alteration is a pathological condition associated with angiogenesis. In one aspect, the invention provides the use of an expression vector of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of an alteration, such as cancer, tumor and / or cell proliferative alteration. In some modalities, the alteration is a neuropathy or neurodegenerative disease. In some modalities, alteration is a pathological condition associated with angiogenesis. In one aspect, the invention provides the use of a host cell of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of an alteration, such as cancer, tumor and / or cell proliferative alteration. In some modalities, the alteration is a neuropathy or neurodegenerative disease. In some modalities, alteration is a pathological condition associated with angiogenesis.

In one aspect, the invention provides the use of an article of manufacture of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of an alteration, such as cancer, tumor and / or cell proliferative alteration. In some modalities, the alteration is a neuropathy or neurodegenerative disease. In some modalities, alteration is a pathological condition associated with angiogenesis. In one aspect, the invention provides the use of an equipment of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of an alteration, such as cancer, tumor and / or cell proliferative alteration. In some modalities, the alteration is a neuropathy or neurodegenerative disease. In some modalities, alteration is a pathological condition associated with angiogenesis. The invention provides methods and compositions useful for modulating disease states associated with the expression and / or activity of EphB4, such as increased and decreased expression and / or expression and / or undesirable activity. In one aspect, the invention provides methods for the treatment or prevention of a tumor, cancer and / or cell proliferative alteration associated with the expression and / or increased activity of EphB4, the methods comprising administering a effective amount of an anti-EphB4 antibody to a subject in need of such treatment. In one aspect, the invention provides methods for killing a cell (such as a cancer or tumor cell), the methods comprising administering an effective amount of an anti-EphB4 antibody to a subject in need of such treatment. In one aspect, the invention provides methods for reducing, inhibiting, blocking or preventing the growth of a tumor or cancer, the methods comprising administering an effective amount of an anti-EphB4 antibody to a subject in need of such treatment. In one aspect, the invention provides methods for the treatment or prevention of a neuropathy or neurodegenerative disease or repair of a damaged nerve cell, the methods comprising administering an effective amount of an anti-EphB4 antibody to a subject in need of such treatment. In one aspect, the invention provides methods for promoting the development, proliferation, maintenance or regeneration of neurons, the methods comprising administering an effective amount of an anti-EphB4 antibody to a subject in need of such treatment. In one aspect, the invention provides methods for inhibiting angiogenesis that comprise administering an amount effective anti-EphB4 antibody to a subject in need of such treatment. In one aspect, the invention provides methods for the treatment of a pathological condition associated with angiogenesis which comprises administering an effective amount of an anti-EphB4 antibody to a subject in need of such treatment. In some embodiments, the pathological condition associated with angiogenesis is tumor, cancer and / or cell proliferative alteration. In some embodiments, the pathological condition associated with angiogenesis is an intraocular neovascular disease. The methods of the invention can be used to affect any appropriate pathological condition. Exemplary alterations are described herein and include a cancer selected from the group consisting of small cell lung cancer, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC) and hepatocellular carcinoma. In one embodiment, a cell that is targeted in a method of the invention is a cancer cell. For example, a cancer cell can be one selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, a papillary carcinoma cell, a colon cancer cell, a pancreatic cancer cell, an ovarian cancer cell, a cell of cervical cancer, a cancer cell of the central nervous system, an ostiogenic sarcoma cell, a renal carcinoma cell, a hepatocellular carcinoma cell, a bladder cancer cell, a gastric carcinoma cell, a squamous cell carcinoma cell head and neck, a melanoma cell, a leukemia cell and a colon adenoma cell. In one embodiment, a cell that is targeted in a method of the invention is a hyperproliferative and / or hyperplastic cell. In one embodiment, a cell that is targeted in a method of the invention is a dysplastic cell. In still another embodiment, a cell that is targeted in a method of the invention is a metastatic cell. The methods of the invention may comprise additional treatment steps. For example, in one embodiment, a method further comprises a step wherein a targeted cell and / or tissue (e.g., a cancer cell) is exposed to radiation treatment or a chemotherapeutic agent. In one aspect, the invention provides methods comprising administering an effective amount of an anti-EphB4 antibody in combination with an effective amount of another therapeutic agent (such as an anti-angiogenesis agent). For example, an anti-EphB4 antibody is used in combinations with anti-cancer agent or an anti-angiogenic agent to treat various neoplastic or non-neoplastic conditions. In one modality, the neoplastic condition or not Neoplastic is a pathological condition associated with angiogenesis. In some embodiments, the other therapeutic agent is an anti-angiogenic agent, anti-neoplastic agent and / or chemotherapeutic agent. The anti-EphB4 antibody can be administered serially or in combination with the other therapeutic agent that is effective for those purposes, either one in the same composition or as separate compositions. The administration of the anti-EphB4 antibody and the other therapeutic agent (eg, anti-cancer agent, anti-angiogenic agent) can be performed simultaneously, for example, as a single composition or as two or more different compositions, using the same route or different administration route. Alternatively or additionally, the administration can be done sequentially, in any order. Alternatively or additionally, the steps may be performed as a combination of both sequentially and simultaneously, in any order. In certain modalities, the intervals fluctuate from minutes to days, to weeks to months, they can be presented between the administrations of the two or more compositions. For example, the anti-cancer agent can be administered first, followed by the anti-EphB4 antibody. However, simultaneous administration or administration of the anti-EphB4 antibody first is also contemplated. Thus, in one aspect, the invention provides methods comprising the administration of a anti-EphB4 antibody, followed by the administration of an anti-angiogenic agent (such as an anti-VEGF antibody, such as bevacizumab). In certain modalities, the intervals that fluctuate from minutes to days, weeks to months, may be present between the administrations of the two or more compositions. In certain aspects, the invention provides a method of treating an alteration (such as a tumor, cancer and / or a cell proliferative disorder) administering effective amounts of an anti-EphB4 antibody and / or angiogenesis inhibitor (s) and one or more chemotherapeutic agents. A variety of chemotherapeutic agents can be used in the combined treatment methods of the invention. An exemplary and non-limiting list of contemplated chemotherapeutic agents is provided herein under "Definitions." The administration of the anti-EphB4 antibody and the chemotherapeutic agent can be done simultaneously, for example, as a single composition or as two or more different compositions, using the same route or different routes of administration. Alternatively or additionally, the administration can be done sequentially, in any order. Alternatively or additionally, the steps may be performed as a combination of both sequentially and simultaneously, in any order. In certain modalities, intervals that fluctuate from minutes to days, weeks to months, may be present between the administrations of the two or more compositions. For example, the chemotherapeutic agent can be administered first, followed by the anti-EphB4 antibody. However, simultaneous administration or administration of the anti-EphB4 antibody first is also contemplated. Thus, in one aspect, the invention provides methods comprising the administration of an anti-EphB4 antibody, followed by the administration of a chemotherapeutic agent. In certain modalities, intervals ranging from minutes to days, weeks to months, may be present between the administrations of the two or more compositions. In one aspect, the invention provides methods for improving the efficacy of an anti-angiogenic agent in a subject having a pathological condition associated with angiogenesis, comprising administering to the subject an effective amount of an anti-EphB4 antibody in combination with the agent anti-angiogenic, thereby improving the inhibitory activity of the anti-angiogenic agent. In another aspect, the invention provides methods for detecting EphB4, the methods comprising detecting the anti-EphB4 EphB4 antibody complex in the sample. The term "detection" as used herein includes qualitative and / or quantitative detection (measuring levels) with or without reference to a control control.

In another aspect, the invention provides methods for diagnosing an alteration associated with the expression and / or activity of EphB4, the methods comprising detecting the EphB4-anti-EphB4 complex in a biological sample from a patient having or suspecting to have the alteration . In some embodiments, the expression of EphB4 is increased expression or abnormal expression. In some embodiments, the alteration is a tumor, cancer and / or cell proliferative alteration. In another aspect, the invention provides any of the anti-EphB4 antibodies described herein, wherein the anti-EphB4 antibody comprises a detectable label. In another aspect, the invention provides a complex of any of the anti-EphB4 antibodies described herein and EphB4. In some modalities, the complex is in vivo or in vitro. In some embodiments, the complex comprises a cancer cell. In some embodiments, the anti-EphB4 antibody is detectably labeled.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1: HVR loop sequences of heavy chain and light chain of anti-EphB4 antibodies. The figure shows the heavy chain HVR sequences, Hl, H2 and H3 and light chain HVR sequences, Ll, L2 and L3. The sequence numbering is as follows: clone 30.35 (HVR-Hl is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 3; HVR-H3 is SEQ ID NO: 7; HVR-Ll is SEQ ID NO: 9; HVR-L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 13); clone 30.35.1D2 (HVR-Hl is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 3; HVR-H3 is SEQ ID NO: 8; HVR-Ll is SEQ ID NO: 10; HVR-L2 is SEQ ID NO: 12; HVR-L3 is SEQ ID NO: 14); clone 30.35.2D8 (HVR-Hl is SEQ ID NO: 2; HVR-H2 is SEQ ID NO: 4; HVR-H3 is SEQ ID NO: 7; HVR-Ll is SEQ ID NO: 9; HVR-L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 15); clone 30.35.2D12 (HVR-H1 is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 5; HVR-H3 is SEQ ID NO: 7; HVR-Ll is SEQ ID NO: 9; HVR-L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 16); and clone 30.35.2D13 (HVR-H1 is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 6; HVR-H3 is SEQ ID NO: 7; HVR-Ll is SEQ ID NO: 9; HVR-L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 17). The amino acid positions are numbered according to the Kabat numbering system as described hereinafter. FIGURES 2A and 2B and 3 illustrate exemplary human acceptor consensus structure sequences for use in carrying out the present invention with sequence identifiers as follows: Variable heavy consensus (VH) structures (Figures 2A and 2B) Kabat CDR less consensus structure of subgroup I of human VH (SEQ ID NO: 19) 2 Interfering extended regions less than human VH subgroup I consensus structure (SEQ ID NOs: 20-22) Kabat CDR minus human VH subgroup II consensus structure (SEQ ID NO: 23) Extended hypervariable regions less structure of human VH subgroup II consensus (SEQ ID NOs: 24-26) Kabat CDR minus human VH subgroup III consensus structure (SEQ ID NO: 27) Extended hypervariable regions less than consensus subgroup III human VH structure (SEQ ID NOs: 28-30) CDR of Kabat minus acceptor structure of human VH (SEQ ID NO: 31) Extended hypervariable regions less of acceptor structure of human VH (SEQ ID NOs: 32-33) CDR of Kabat less structure of human VH acceptor 2 (SEQ ID NO: 34) Extended hypervariable regions minus human VH acceptor 2 structure (SEQ ID NOs: 35-37) Variable light consensus (VL) structures (Figure 3) Subgroup consensus structure I of human VL kappa (SEQ ID NO: 38) Structure Consensus of subgroup II human kappaVL (SEQ ID NO: 39) Consensus structure of subgroup III human Kappa VL (SEQ ID NO: 40) Consensus structure of subgroup IV human Kappa VL (SEQ ID NO: 41) FIGURE 4 illustrates sequences of structure region of light and heavy chains of huMAb4D5-8 . The superscript / bold numbers indicate the amino acid positions according to Kabat. FIGURE 5 illustrates sequences of modified structure region / light chain and heavy chains of huMAb4D5-8. The superscript / bold numbers indicate amino acid positions according to Kabat. FIGURE 6 illustrates the heavy chain variable regions and light chain variable regions of antibody clones 30.35, 30.35.1D2, 30.35.2D8, 30.35.2D12 and 20.25.2D13. FIGURE 7: EphB4 receptor signaling blocked with anti-EphB4 monoclonal antibody in a cell-based assay.

DETAILED DESCRIPTION OF THE INVENTION The invention herein provides anti-EphB4 antibodies, which are useful for, for example, treatment or prevention of disease states associated with the expression and / or activity of EphB4, such as expression and / or activity increased or undesirable expression and / or activity. In some embodiments, the antibodies of the invention are used to treat a tumor, cancer and / or cell proliferative alteration. In another aspect, the anti-EphB4 antibodies of the invention find utility as reagents for detection and / or isolation of EphB4, such as detection of EphB4 in various tissues and cell types. The invention further provides methods of making anti-EphB4 antibodies and polynucleotides that encode anti-EphB4 antibodies. General techniques The techniques and methods described or referred to herein are generally well understood and commonly used using conventional methodology by those experienced in the art, such as, for example, the widely used methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3a. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al., Eds., (2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J.

MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)). Definitions An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Pollutants components of its natural environment are materials that would interfere with the diagnostic or therapeutic uses of the antibody and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) at greater than 95% by weight of antibody as determined by the Lowry method and more preferably more than 99% by weight., (2) to a sufficient degree to obtain at least 15 N-terminal or internal amino acid sequence residues by use of a centrifugation cup sequencer or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably, silver-stained. The isolated antibody includes the antibody itself within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, the isolated antibody will be prepared by at least one purification step. An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. A nucleic acid molecule isolated is different than in the form or interaction in which it is found in nature. Accordingly, the isolated nucleic acid molecules are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody wherein, for example, the nucleic acid molecule is at a chromosomal site different from that of natural cells. The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refer to the numbering system used for variable domains of heavy chain or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of or insertion to FR or variable domain CDRs. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (eg, residues 82a, 82b and 82c, etc. according to Kabat ) after the residue 82 of chain FR. The Kabat residue numbering can be determined for a given antibody by aligning in regions of homology of the antibody sequence with a "standard" Kabat numbered sequence. The phrase "substantially similar", or "substantially the same", as used herein, denotes a sufficiently high degree of similarity between two numerical values (generally one associated with an antibody of the invention and the other associated with a reference antibody / comparator) in such a manner that the one experienced in the art would consider the difference between the two values of little or no biological and / or statistical significance within the context of the biological characteristic measured by said values (for example, Kd values). The difference between said two values is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10% as a function of the value for the antibody reference / comparator. "Linkage affinity" refers generally to the intensity or strength of the total sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen) . Unless indicated otherwise, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can in general be represented by the dissociation constant (Kd). The affinity can be measured by common methods known in the art, in which those described herein are included. The low affinity antibodies in ral bind slowly to the antigen and tend to dissociate easily, while the high affinity antibodies bind in general to the antigen faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which may be used for the purposes of the present invention. Specific illustrative modalities are described in the following. In one embodiment, the "Kd" or "Kd value" according to this invention is measured by a radiolabelled antigen binding (RIA) analysis performed with the Fab version of an antibody of interest and its antigen as described in following analysis that measures the binding affinity in Fab solution by antigen by balancing Fab with a minimum concentration of antigen (125I) -marked in the presence of a series of titration of unlabeled antigen, then capture the antigen bound with a plate coated with anti-Fab antibody (Chen, et al., (1999) J. Mol Biol 293: 865-881). To establish conditions for analysis, microtiter plates (Dynex) are coated overnight with 5 ug / ml of an anti-capture Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked with 2% bovine serum albumin (weight / volume) in PBS for two to five hours at room temperature (approximately 23 ° C). In a non-absorbent plate (Nunc # 269620), 100 pM or 26 pM of [125 I] -antigen are mixed with serial dilutions of a Fab of interest (eg, consistent with the determination of an anti-VEGF antibody, Fab-12 , in Presta et al., (1997) Cancer Res. 57: 4593-4599). Then the Fab of interest is incubated throughout the night; however, the incubation may continue for a longer period (eg, 65 hours) to ensure equilibrium is reached. After this, the mixtures are transferred to the capture plate for incubation at room temperature (for example, for one hour). Then the solution is removed and the plate washed eight times with 0.1% Tween-20 in PBS. When the plates have dried, 150 ul / cavity of scintillation agent (MicroScint-20; Packard) is added and the plates are counted in a gamma counter Topcount (Packard) for ten minutes. The concentrations of each Fab that give less or equal to 20% of maximum bond are chosen for use in competitive link analysis. According to another modality the Kd or Kd value is measured when using analysis of surface plasmon resonance using a BIAcore ™ -2000 or BIAcore ™ -3000 (BIAcore, Inc., Piscataway, NJ) at 25 ° C with immobilized antigen CM5 fragments at ~ 10 response units (RU). Briefly, carboxymethylated dextran biodetector fragments (CM5, BIAcore Inc), are activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the instructions of provider. The antigen is diluted with 10 mM sodium acetate, pH 4.8, at 5 ug / ml (~ 0.2uM) before injection at a flow rate of 5 ul / minute to obtain approximately 10 response units (RU) of protein coupled. Following injection of the antigen, 1M ethanolamine is injected to block the unreacted groups. For kinetic measurements, serial two-fold dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25 ° C at a flow rate of approximately 25 ul / min. Association rates (kencend? Do) and dissociation rates (kapagado) are calculated using a simple one-to-one Langmuir link model (BIAcore version 3.2 evaluation programming elements) by simultaneously adjusting the association and dissociation sensorgram. The equilibrium dissociation constant (Kd) is calculated as the ratio kapagacio / kencend? Do- See, for example, Chen, Y., et al., (1999) J. Mol Biol 293: 865-881. If the ignition speed exceeds 106 M "1 S" 1 by the resonance analysis of anterior surface plasmon, then the ignition velocity can be determined by using a fluorescence quencher that measures the increase or decrease and intensity of fluorescence emission (excitation = 295 nm, emission = 340 nm, bandpass of 16 nm) at 25 ° C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increased antigen concentrations as measured on a spectrometer, such as a stoplight-equipped spectrophotometer (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) with a red agitation cover. An "ignition speed" or "association speed" or "kinasend" do "according to this invention can also be determined with the same surface plasmon resonance technique described above using a BIAcore ™ -2000 or a BIAcore ™ -3000 (BIAcore, Inc., Piscataway, NJ) at 25 ° C with CM5 fragments of immobilized antigen at -10 response units (RU) Briefly, carboxymethylated dextran biodetector fragments (CM5, BIAcore Inc) , are activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions.The antigen is diluted with 10 mM sodium acetate, pH 4.8, at 5 ug / ml (-0.2 uM) before injection at a flow rate of 5 ul / minute to obtain approximately 10 response units (RU) of coupled protein. Following injection of the antigen, 1 M ethanolamine is injected to block the unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25 ° C at a flow rate of approximately 25 ul / min. The association (ignition) and dissociation velocities (kapagado) are calculated using a single-to-one Langmuir link model (BIAcore version 3.2 evaluation programming elements) by simultaneously adjusting the association and dissociation sensorgram. The equilibrium dissociation constant (Kd) was calculated as the ratio of kapagado / kencendido- See, for example, Chen, Y., et al., (1999) J. Mol Biol 293: 865-881. However, if the ignition speed exceeds 10d M "1 S_1 by the anterior surface plasmon resonance analysis, then the firing rate is preferably determined by using a fluorescence quenching technique that measures the increase or decrease in intensity of fluorescence emission (excitation = 295 nm, emission = 340 nm, bandpass of 16 nm) at 25 ° C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increased antigen concentrations , as measured on a spectrometer, such as a spectrophotometer equipped with retention-flow (Aviv Instruments) or an 8000 series SLM-Aminco (ThermoSpectronic) spectrophotometer with a stirred cell. The term "vector" as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid" that refers to a circular double-stranded DNA loop to which additional segments of DNA can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, where additional DNA elements can be linked to the viral genome. Certain vectors are capable of autonomous replication in a host cell to which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell after introduction into the host cell and thereby are reflected together with the host genome. However, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors of utility in recombinant DNA techniques are frequently in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as since the plasmid is the most commonly used vector form.

"Polynucleotide" or "nucleic acid", as used interchangeably herein, refer to polymers of nucleotides of any length and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogues. If present, the modification to the nucleotide structure can be imparted before or after the assembly of the polymer. The nucleotide sequences can be interrupted by components without nucleotides. A polynucleotide can be further modified after synthesis, such as by conjugation with a label. Other types of modifications include for example "crowns", substitution of one or more of the nucleotides that occur stably in nature with an analogue, internucleotide modifications such as, for example, those with uncharged bonds (e.g. methylphosphonate, phosphotriester, phosphoamidate, carbamate, etc.) and with charged bonds (for example, phosphorothioates, phosphorosithioates, etc.), those containing pendant portions, such as, for example, proteins (for example, nucleases, toxins, antibodies, signal peptides, ply, L-lysine, etc.), those with intercaladotes (for example, acridine, psoralen, etc.), those containing chelators (for example, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified bonds (for example, anomeric alpha nucleic acids, etc.), as well as unmodified forms of the polynucleotide (s). In addition, any of the hydroxyl groups ordinarily present in the sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard or activated protecting groups to prepare additional bonds to additional nucleotides, or can be conjugated to solid or semi-solid supports. The terminal OH 5 'and 3' can be phosphorylated or substituted with amines or organic crown group portions of 1 to 20 carbon atoms. Other hydroxyls can also be derived to standard protecting groups. The polynucleotides may also contain analogous forms of deoxyribose ribose that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-alkyl-, 2'- fluoro- or 2'-azido-ribose, carboxylic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lixoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and basic nucleoside analogues such as methylriboside . One or more links phosphodiester can be replaced by alternative linking groups. These alternative linker groups include but are not limited to, embodiments wherein the phosphate is replaced by P (0) S ("thioate"), P (S) S ("dithioate"), "(0) NR2 (" amidate " ), P (0) R, P (0) 0R \ CO or CH2 ("formacetal"), in which each R or R 'is independently H or substituted or unsubstituted alkyl (1-20C) optionally containing a bond of ether (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl Not all bonds in a polynucleotide need to be identical The preceding description applies to all polynucleotides referred to herein, in the RNA and DNA are included. "Oligonucleotide", as used herein, generally refers to generally synthetic, generally single-stranded, short polynucleotides that are generally but not necessarily less than 200 nucleotides in length. The terms "oligonucleotides" and "polynucleotides" are not mutually exclusive. linucleotides is equal and fully applicable to all oligonucleotides. "percent (%) of amino acid sequence identity" with respect to a sequence of peptides or polypeptides is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the peptide sequence or specific polypeptide, after aligning the sequences and inserting spaces, if necessary, to obtain the maximum percent identity of sequences and not considering any conservative substitution as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be obtained in various ways that are within the skill of the art, for example, by using publicly available computer programming elements such as BLASR, BLAST-2 programming elements. , ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters to measure alignment, in which any logarithms necessary to obtain the maximum alignment in the full length of the sequences being compared are included. For purposes of the present, however, amino acid sequence entity% values are generated using the ALIGN-2 sequence comparison computer program, wherein the complete source code for the ALIGN-2 program is provided in the Table A below. The author of the sequence comparison computer program ALIGN-2 is Genentech, Inv. and the source code shown in Table A below has been presented with user documentation in the United States of America Copyright Office, Washington, D.C., 20559, where it is registered under the United States of America Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South of San Francisco, California. The ALIGN-2 program must be compiled for use in a UNIX operating system, preferably UNIX V 4.0 Digital. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for comparison of amino acid sequences, the% amino acid sequence entity of a given amino acid sequence, a con. 0 against a given sequence B of amino acids (which may alternatively be referred to as a sequence A of given amino acids having or comprising a certain percent identity of amino acid sequence A that with, or against a given B sequence of amino acids) is calculated as: 100 times the X / Y fraction where X is the number of amino acid residues that have fluctuation as identical matches by the ALIGN-2 sequence alignment program in that program alignment of A and B and where Y is the total number of amino acid residues in B. it will be appreciated that where the length of amino acid sequences A is not equal to the length of amino acid sequences B, the percent identity of amino acid sequences from A to B will not be equal to the percent identity of amino acid sequences from B to A.

Unless specifically stated otherwise, all amino acid sequence identity values herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. The term "EphB4" (interchangeably referred to as "EphB4R"), as used herein, refers to, unless otherwise specifically or contextually indicated, any naturally occurring or synthetic EphB4 or variant polypeptide (whether natural or synthetic). The term "natural sequence" specifically encompasses truncated or secreted forms that occur stably in nature (e.g., an extracellular domain sequence), variant forms that occur stably in nature (e.g. alternatively spliced) and allelic variants that occur stably in nature The term "wild type EphB4" generally refers to a polypeptide comprising the amino acid sequences of an EphB4 protein that is stably presented in nature. The term "wild-type EphB4 sequence" generally refers to an amino acid sequence found in an EphB4 that is presented in a stable in nature. The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (for example, antibodies full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (eg, bispecific antibodies as long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein) . An antibody can be human, humanized and / or matured by affinity. The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequences between antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not equally distributed in all the variable domains of antibody. It is concentrated in three elements called regions that determine complementarity (CDR) or hypervariable regions both in the variable domains of light chain and in the variable domains of heavy chain. The most highly conserved portions of variable domains are called the structure (FR). Each of the natural heavy and light chain variable domains comprises four regions of FR, which widely adopt a β-sheet configuration, joined by three CDRs, which form loops that join and in some cases form part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FT regions and with the CDRs of the other chain, contribute to the formation of the antigen binding site of the antibodies (see Kabat et al., Sequences of Proteins of Immunological In terest, 5th edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but rather they exhibit several detector portions, such as participation of the antibody in antibody-dependent cellular toxicity. The digestion of antibody papains produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site and a residual "Fc" fragment, whose name reflects its ability to easily crystallize. The pepsin treatment produces an F (ab ') 2 fragment that has two antigen combining sites and is still capable of antigen crosslinking. "Fv" is the minimum antibody fragment that contains a complete antigen binding and recognition site. In a two-chain Fv species, this region consists of a dimer of a variable domain of heavy chain and light chain in non-covalent, hermetic association. In a single chain Fv species, a heavy chain and light chain variable domain can be covalently linked by a flexible peptide linker, such that light and heavy chains can be associated in a "dimeric" structure analogous to those in a kind of Fv of two chains. In this configuration 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 contain 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, albeit at a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab 'fragments differ from the Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain VH1 domain that includes one or more cysteines from the antibody engozyne region. Fab '-SH is the designation herein for Fab' in which the cysteine residue (s) of the constant domains carry a free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments that have engozne cysteines between them. Other chemical couplings of antibody fragments are also known. The "light chains" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (?), based on the amino acid sequences of their constant domains. Depending on the constant domain amino acid sequence of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM and several of them can be further divided into subclasses (isotypes) eg IgGi, IgG2, IgG3, IgG4, IgAi and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called a, d, e and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. "Antibody fragments" comprise only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, functions normally associated with that portion when present in an intact antibody. Examples of antibody fragments include Fab, FabP F (ab ') 2 and Fv fragments; diabodies; linear antibodies; Single-chain antibody molecules and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind to the antigen. In another modality, a antibody fragment, for example one comprising the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC function and complement link. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half-life substantially similar to an intact antibody. For example, such an antibody fragment may comprise an antigen binding expense linked to an Fc sequence capable of conferring in vivo stability to the fragment. The term "hypervariable region", "HVR" or "HV" when used herein refers to the regions of a variable domain of antibodies that are hypervariable in sequence and / or form spectrally defined loops. In general, the antibodies comprise six hypervariable regions; three in the VH (Hl, H2, H3) and three in the VL (Ll, L2, L3). A variety of hypervariable region delineations are in use and are encompassed in the present. The regions that determine the complementarity of Rabat (CDR) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda , MD. (1991)). Chothia refers instead to the location of the loops structural (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). The hypervariable regions of AbM represent an intermediate solution between the Kabat CDRs and structural loops and Chothia loops used by the antibody modeling elements of Oxford AbM molecular. The variable regions of "contact" are based on an analysis of the available complex crystalline structures. The residues of each of these hypervariable regions are indicated below. Loop Kabat AbM Chothia Contact Ll L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L60 L50-L56 L50-L56 L50-L52 L46-L55 L46 L89-L97 L89-L96 L91-L96 L89-L96 H19-H35B -H35B (Kabat Number) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia Number) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101 hypervariable regions can comprise "Extended hypervariable regions" as follows: 24-36 or 24-34 (Ll), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL and 26-35 (Hl), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH. The variable domain residues are numbered according to Rabat et al. , supra for each of these definitions. Residues of "structure" or "FR" are those variable domain residues different from the hypervariable region residues as defined herein. "Humanized" forms of non-human antibodies (e.g., murine) are generic antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which residues of a hypervariable region of the receptor are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity and capacity. In some instances, the structure region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the performance of the antibody. In general, the humanized antibody will substantially comprise all of at least and commonly two variable domains, in which all or substantially all of the hypervariable loops comprise those of a non-human immunoglobulin and all or substantially all of the FR are those of a sequence of human immunoglobulins. The humanized antibody will optionally also comprise at least one immunoglobulin constant region (Fc), commonly that of a human immunoglobulin. For additional 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). See also the following journal articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994). The "chimeric" antibodies (immunoglobulins) have a portion of the heavy and / or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, the rest of the ) chain (s) is identical with a homologue with corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, also as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent 4,816,567 and Morrison et al., Proc. Na ti, Acad. Sci. USA 81: 6851-6855 (1984)). The humanized antibody as used herein is a subset of chimeric antibodies. "Single chain Fv" or antibody fragments from "scFv" comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In general, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that the scFv forms the desired structure for the antigen binding. For a review of scFv, see Pluckthun, in: The Pharmacology of Monoclonal An tibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). An "antigen" is a predetermined antigen to which an antibody can selectively bind. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other compound that occurs stably in nature or synthetic. Preferably, the target antigen is a polypeptide. The term "diabodies" refers to small antibody fragments with two antigen binding sites, such fragments comprising a heavy chain variable domain (VH) linked to the light chain variable domain (VL) in the same polypeptide chain (VH) -VL). When using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161 and Hollinger et al. , Proc. Na ti. Acad. Sci. USA, 90: 6444-6448 (1993). A "human antibody" is one that possesses an amino acid sequence corresponding to those of an antibody produced by a human and / or has been manufactured using any of the human antibody manufacturing techniques as disclosed herein. The definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues. An "affinity matured" antibody is one with one or more alterations in one or more CDRs thereof that results in an improvement in the affinity of the antibody for antigen, as compared to the original antibody that does not possess that (s) alteration ( is) . Preferred affinity-matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by methods known in the art. Marks et al., Bio / Technology 10: 779-783 (1992) describe the affinity maturation of the VH and VL domain intermixes. The random mutagenesis of CDR and / or structure residues is described by: Barbas et al. Proc Nat. Acad. Sci, USA 91: 3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al., J. Immunol. 154 (7): 3310-9 (1995) and Hawkins et al, J. Mol. Biol. 226: 889-896 (1992). "Effector functions" of antibody refer to those biological activities attributable to the Fc region (a region of Fc of natural sequence or Fc region variant of amino acid sequence) of an antibody and varies with the antibody isotype. Examples of antibody effector functions include: Clq linkages and complement dependent cytotoxicity; Fc receptor link; moderate cytotoxicity by the antibody-dependent cell (ADCC); phagocytosis; down regulation of cell surface receptors (eg, B cell receptor) and activation. of "B cell" "antibody-mediated cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which the secreted Ig bound to Fc receptors (FcRs) present in certain cytotoxic cells (e.g., natural killer cells) (NK), neutrophils and macrophages) allow these cytotoxic effector cells to bind specifically to a target cell that carries antigen and subsequently exterminate the target cell with cytotoxins. The antibodies "weapon" of the cytotoxic cells and are absolutely required for such extermination. Primary cells to moderate ADCC, NK cells, express Fc? RIII only, while monocytes express Fc? RI, Fc? RII and Fc? RIII. The expression FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To determine the ADCC of a molecule of interest, an analysis of ADCC in Vítro can be carried out, such as that described in US Patents 5,500,362 or 5,821,337 or 6,737,056 issued to Presta. Useful effector cells for such analysis include peripheral blood mononuclear cells (PRMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest may be determined in vivo, for example, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998). "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and effect ADCC effector function. Examples of human leukocytes that moderate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, PBMC and NK cells are preferred. Effector cells can be isolated from a natural source, for example, blood. "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a human FcR of natural sequence. In addition, a preferred FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the subclasses Fc? RI, Fc? RII and Fc? RIII, in which allelic variables are included and alternatively spliced forms of these receptors. Fc? RII receptors include Fc? RIIA (an "activation receptor") and Fc? RIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ mainly in the cytoplasmic domains thereof. Activating receptor Fc? RIIA contains an immunoreceptor tyrosine-based activation portion (ITAM) in its cytoplasmic domain. The Fc [gamma] RIIB inhibitory receptor contains a portion of immunoreceptor tyrosine-based inhibitor (ITIM) in its cytoplasmic domain (see review M. in Daron, Annu, Rev. Immunol., 15: 203-234 (1997)). The FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al. , Immunomethods 4: 25-34 (1994) and de Haas et al. , J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, which include those to be identified in the future, are covered by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol., 24: 249 (1994)) and regulates immunoglobulin homeostasis. WO 00/42072 (Presta) describes antibody variants with improved or decreased binding to FcRs. The content of that patent publication is specifically incorporated by reference. See also Shields et al. J. Bi ol. Chem. 9 (2): 6591-6604 (2001). Methods to measure the link to FcRn are known (see for example, Ghetie 1997, Hinton 2004). The binding to human FcRn in vivo and the serum half-life of human FcRn high affinity binding polypeptide can be analyzed, for example, in transgenic mice or transfected human cell lines expressing human FcRn or in primates administered with the Fc polypeptides variants. "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) that are linked to its cognate antigen. To determine complement activation, a CDC analysis can be performed, for example, as described in Gazzano-Santoro et al. , J. Immunol. Methods 202: 163 (1996). Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capacity are described in US Patent 6,194,551 Bl and WO 99/51642. The content of those patent publications is specifically incorporated herein by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000). The term "polypeptide comprising Fc region" refers to a polypeptide, such as an antibody or immunoadhesin (see definitions later in present) comprising a region of Fc. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the purification of the polypeptide or by recombinant designs of the nucleic acid encoding the polypeptide. Thus, for a composition comprising a polypeptide having an Fc region according to this invention, it can comprise polypeptides with K447 with all the K447 removed or a mixture of polypeptides with and without the residue K447. A "blocking" antibody or an antibody "antagonist" is one that inhibits or reduces the biological activity of the antigen to which it binds. Blocking antibodies or preferred antagonist antibodies substantially or completely inhibit the biological activity of the antigen. An "agonist antibody" as used herein, is an antibody that indicates at least one of the functional activities of a polypeptide of interest. An "acceptor human structure" for the purposes of the present is a structure comprising the amino acid sequence of a VL or VH structure derived from a human immunoglobulin structure or a human consensus structure. A human acceptor structure "derived from" can be produced from human immunoglobulin or human consensus structure can comprise the same amino acid sequence thereof or can contain sequence changes of existing amino acids. Where pre-existing amino acid changes are present, preferably no more than five and preferably four or fewer or three or fewer existing amino acid changes are present. Where changes of pre-existing amino acids are present in a VH, preferably it is a change being only in three, two or one of the positions 71H, 73H and 78H; for example, the amino acid residues at those positions can be 71A, 73T and / or 78A. In one embodiment, the human acceptor structure of VL is identical in the presence of the VL human immunoglobulin structure sequence or human consensus structure sequence. A "human consensus structure" is a structure representing the amino acid residue that is most commonly present in a selection of VL or VH structure sequences of human immunoglobulin. In general, the selection of VL or VH sequences of human immunoglobulin is from a group of variable domain sequences. In general, the subgroup of sequences is a subgroup as in Kabat et al. In one modality, for the VL, the subgroup is subgroup kappa I as in Kabat et al. In one modality, for VH, the subgroup is subgroup III as in Kabat et al. A "VH subgroup III consensus structure" comprises the consensus sequence obtained from the amino acid sequences in the variable heavy subgroup III of Kabat et al. a l. In one embodiment, the amino acid sequence of subgroup III consensus structure of VH comprises at least a portion or all of each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 42) -Hl-WVRQAPGKGLEWV (SEQ ID NO: 43) -H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 44) -H3-WGQGTLVTVSS (SEQ ID NO: 5). A "consensus structure of subgroup I of VL" comprises the consensus sequence obtained from the amino acid sequences in the subgroup I of variable light kappa of Kabat et al. In one embodiment, the amino acid sequence of consensus structure of subgroup I of VH comprises at least a portion or all of each of the following sequences: DIQMTQSPSSLSASVGDRVTITC (SEQ ID N0: 46) -L1-WYQQKPGKAPKLLIY (SEQ ID NO : 47) -L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 48) -L3-FGQGTKVEIK (SEQ ID NO: 49). An "alteration" or "disease" is any condition that would benefit from treatment with a substance / molecule or method of the invention. This includes alterations or chronic and acute diseases in which are included those pathological conditions that predispose the mammal to the alteration in question. Non-limiting examples of alterations to be treated include malignant and benign tumors, carcinoma, blastoma and sarcoma. The terms "cell proliferative alteration" and "Proliferative alteration" refers to alterations that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative alteration is cancer. "Tumor", as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign and all precancerous and cancerous cells or tissues. The terms "cancer", "cancerous", "cell proliferative alteration", "proliferative alteration" and "tumor" are not mutually exclusive as referred to herein. The terms "cancer" and "cancerous" refer to or describe the physiological condition in a mammal that is commonly characterized by unregulated cell growth / proliferation. Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, tumor squamous cell carcinoma, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma , cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer , vulvar cancer, thyroid cancer, Hepatic carcinoma, gastric cancer, melanoma and various types of head and neck cancer. The deregulation of angiogenesis can lead to many alterations that can be treated by the compositions and methods of the invention. These alterations include both non-neoplastic and neoplastic conditions. Neoplasms include but are not limited to those described above. Non-neoplastic disorders include but are not limited to undesirable hypertrophy or aberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabetic retinopathies and other proliferative retinopathies in which premature retinopathies are included , retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft neovascularization, rejection of corneal graft, retinal / choroidal neovascularization, angle neovascularization (rubeosis), ocular neovascular disease, vascular restenosis, arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasia (in which Grave's disease is included), corneal transplantation and other tissue transplantation, chronic inflammation, lung inflammation, acute lung injury / ARDS, sepsis, hypertension primary pulmonary n, malignant pulmonary effusions, cerebral edema (e.g., associated with acute stroke / closed head injury / trauma), synovial inflammation, pannus formation in RA, occipital myositis, hypertrophic bone formation, osteoarthritis (OA), refractory ascites, polycystic ovary disease, endometriosis, third spacing of fluid diseases (pancreatitis, compartment syndrome, burns, bowel disease), uterine fibroids, premature labor, chronic inflammation such as IBDF (Crohn's disease and ulcerative colitis), rejection of renal allograft, inflammatory bowel disease, nephrotic syndrome, growth of undesirable or aberrant tissue mass (without cancer), hemophilic joints, hypertrophic scars, hair growth inhibition, Osler's syndrome -Weber, retrolentales fibroplasias biogenic granuloma, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis) and pleural effusion. As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated and may be effected either by prophylaxis or during the course of the clinical pathology. Desirable effects of treatment include prevention of the presence or occurrence of disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, decrease in the rate of disease progression, improvement or alleviation of disease status and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to retard the development of a disease or disorder. The terms "neurodegenerative disease" and "neurodegenerative disorder" are used in the broadest sense to include all alterations of the pathology which involves neuronal degeneration and / or dysfunction, which include, without limitation, peripheral neuropathies; motor neuron disorders, such as amilotropic lateral sclerosis (ALS, Lou Gehrig's disease), Bell's palsy and various conditions involving spinal muscular atrophy or paralysis and other human neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, chorea of Huntington, Down syndrome, nerve deafness and Meniere's disease. "Peripheral neuropathy" is a neurodegenerative disorder that affects the peripheral nerves, most frequently manifested as one or a combination of motor, sensory, sensorimotor or autonomic dysfunction. Peripheral neuropathies may for example be genetically acquired, may result from a systemic disease or may be induced by a toxic agent, such as a neurotoxic drug, for example, antineoplastic agent or contaminant environmental industrial. "Peripheral sensory neuropathy" is characterized by the degeneration of peripheral sensory neurons, which can be idiopathic, they can occur, for example, as a consequence of diabetes (diabetic neuropathy), cytostatic drug therapy in cancer (for example, treatment with chemotherapeutic agents) such as vincristine, cisplatin, methotrexaro, 3'-azidp-3'-deoxythymidine or taxanes, for example, paclitaxel [TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ] and doxetaxel [TAXOTERE®, Rhone-Poulenc Rorer, Antony , France]), alcoholism, acquired immunodeficiency syndrome (AIDS) or genetic predisposition. Genetically acquired peripheral neuropathies include, for example, Refsum's disease, Krabbe's disease, metachromatic leukodystrophy, Fabry's disease, Dejerine-Sottas syndrome, abetalipoproteinemia, and Charcot-Marie-Tooth disease (CMT) (also known as proneal muscle atrophy or hereditary motor sensory neuropathy (HMSN)). Most peripheral neuropathies develop slowly, over the course of several months or years. In clinical practice such neuropathies are called chronic. Sometimes a peripheral neuropathy develops rapidly, over the course of a few days and is called acute. Peripheral neuropathy usually affects the motor and motor nerves together as a mixed sensory and motor neuropathy, but pure and motor sensory neuropathy Pure are also known. An "individual" is a vertebrate, preferably a mammal, more preferably a human. Mammals include but are not limited to farm animals such as cows), sport animals, pets (such as cats, dogs and horses), primates, mice and rats. "Mammal" for treatment purposes refers to any animal classified as a mammal, which includes humans, domestic animals and farms, zoo animals, sports animals or pets, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human. An "effective amount" refers to an effective amount at dosages and for periods of time necessary to obtain the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a substance / molecule of the invention, agonist or antagonist may vary according to factors such as disease state, age, sex and weight of the individual and the ability of the substance / molecule agonist or antagonist to produce a desired response in the individual. A therapeutically effective amount is one in which any toxic or detrimental effects of the substance / molecule, agonist or antagonist are overcome by the therapeutically effects beneficial A "prophylactically effective amount" refers to an effective amount, at dosages and for periods of time necessary to obtain the desired prophylactic result. Commonly, but not necessarily, since a prophylactic dose is used in subjects before or in the stage prior to a disease, the prophylactically effective amount will be less than the therapeutically effective amount. The term "cytotoxic agent" as used herein refers to a substance that inhibits or inhibits the production of cells and / or causes cell destruction. The term is intended to include radioactive isotopes' (for example, AMt- 21 1, TI131, tl25, vY90, R D Ye 86, radioactive), chemotherapeutic agents, for example, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleogylic enzymes , antibiotics and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fangal, plant or animal origin, which include fragments and / or variants thereof and various antitumor or anticancer agents subsequently disclosed herein . Other cytotoxic agents are described hereinafter. A tumoricidal agent causes destruction of tumor cells.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide, alkyl sulfonates such as busulfana, improsulfanas and piposulfanas; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacona; lapacol; Colchicines; botulinum acid; a camptothecin (which includes the synthetic topotecan analog (HYCAMTIN®) M CPT-11 (irinotecana, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9-aminocaptothecin); Bryostatin; Callistatin; CC-1065 (in which its synthetic analogs of adozelesin, carzelesin and bizelesin are included); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (which includes the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictine; spongistatin; nitrogen mustard such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide corhydrate, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, mustard of uracil; nitroureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediin antibiotics (eg, calicheamicin, especially gamma II calicheamicin and omega II calicheamicin (see for example, Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)), dinemicin, in which include dinemicin A, esperamycin, as well as neocarzinostatin chromophore and related antibiotic enediin protein chromosomes), aclacinomisins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, ADRIAMICINA® doxorubicin (which includes morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxidoxorubicin), epirubicin, esorubicin, doxorubicin, comoomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, norgalamicin a, olivomycins, peplomycin, potfiromycin, puromycin, chelamicin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroptein, trimetrexate, purine analogs such as fludarabine, 6-mercaptopurine, tiamiprine, thioguanine, pyrimidine analogues such as ancitabine, azacitidine, 6-azautidine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocythabin, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, meptiostane, testolactone; anti-adrenal such as aminoglutethimide, mitotane, trilostane, folic acid replenisher such as hydrochloric acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabuchil; bisantrene; edatraxate; defofamin; demecolcine; diaziquone; elfornitin; elliptinium and epothilone acetate; etoglucid; gallium nitrate; hydroxyurea; lentinana; lonidainin; maytansinoids such as maytansin and ansamitocins; mitofuazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofirano; spiroger anio; tenuazonic acid; triaziquone; 2, 2 ', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); Dacarbazine; mannomustine; mitobronitol; mitolactol; pipobromana; gacitosina; arabinoside ("Ara-C"); thiotepa; taxoids, for example, TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANETM free of camphor, formulation of designed nanoparticles of albumins of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois) and TAXOTERE® doxetaxel (Rhóne-Poulenc Rorer, Antony, France); chloranbuchil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platini analogues such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovina; vinorelbine (NAVELBINE®); novantrone; edatrexate; Daunomycin; aminopterin; ibandronate; Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; also as combinations of two or more of the foregoing such as CHOP, an abbreviation for a combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone and FOLFOX, an abbreviation for an oxaliplatin treatment regimen (ELOXATIN ™) combined with 5-FU and leucovovine . Also included in this definition are antihormonal agents that act to regulate, reduce, block or inhibit the effects of hormones that can promote cancer growth and are frequently in the form of systemic, or whole-body, treatment. They can be hormones by themselves. Examples include anti-estrogens and selective estrogen receptor moderators (SERM), in which include, for example, tamoxifen (which includes tamoxifen NOLVADEX®), raloxifene EVISTA®, droloxifene, 4-hydroxy tamoxifen, trioxifen, keoxifene, LY117018, onapristone and toremifene FARESTON®; anti-progesterone; descending estrogen receptor (ERD) regulators; agents that function to suppress or close the ovaries, for example, luteinizing hormone releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide and aromatase inhibitors that inhibit the aromatase enzyme, which regulates the production of estrogens in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, megestrol acetate MEGASE ®, AROMASIN® exemestane, formestanie, fadrozole, vorozole RIVISOR®, letrozole FEMARA® and anastrozole ARIMIDEX®. In addition, such a definition of chemotherapeutic agents also includes includes bisphosphonates such as clodronate (eg, BONEFOS® or OSTAC®), etidronate DIDROCAL®, NE-58095, ZOMETA® zoledronic acid / zoledronate, alendronate FOSAMAX®, pamidronate AREDIA®, tiludronate SKELID ® or Risedronate ACTONEL®; also as troxacitabine (a cytosine analogue in the 1,3-dioxalane nucleoside); antisense oligonucleotides, particularly those that inhibit the expression of genes in the signaling pathways involved in proliferation of aberrant cell, eg, PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; Topoisomerase 1 inhibitor LURTOTECAN®; ABARELIX® rmRH; lapatinib ditosylate (a small molecule inhibitor of double tyrosine kinase ErbB-2 and EGFR also known as GW572016) and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. A "growth inhibitory agent" when used herein refers to a compound or composition that inhibits the growth of an 8th cell such as a cell expressing EphB4) either in Vi tro or in vivo. Thus, the growth inhibitory agent can be one that significantly reduces the percentage of cells (such as a cell expressing EphB4) in S phase. Examples of growth inhibitory agents include agents that block the advancement of the cell cycle (in a different place to S phase), such as agents that induce Gl arrest and M phase arrest. Classical M phase blockers include vincas (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. Those agents that stop Gl also spill over the S phase arrest, for example, DNA alkylating agents such as tomoxifene, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. Additional information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13. The taxanes (paclitaxel and docetaxel) are anti-cancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from European yew, is a semi-synthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules of tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells. "Doxorubicin" is an anthracycline antibiotic. The complete chemical name of doxorubixcin is (8S-cis) -10- [(3-amino-2,3,6-trideoxy-aL-lixo-hexapyranosyl) oxy] -7,8,9,10-tetrahydro-6, 8, 11-trihydroxy-8- (hydroxyacetyl) -l-methoxy-5, 12-naphta-cione. The term "anti-neoplastic composition" refers to a composition useful in the treatment of cancer comprising at least one active therapeutic agent, for example, "anti-cancer agent". Examples of therapeutic agents (anti-cancer agents, also referred to herein as "anti-neoplastic agent" herein) include but are not limited to, agents chemotherapeutics, growth inhibitory agents, cytotoxic agents, agents used in radiation therapies, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, toxins and other agents to treat cancer, for example, anti-VEGF neutralizing antibody, VEGF antagonists , anti-HER-2, antho-CD20, an epidermal growth factor receptor (EGFR) antagonist (for example, a tyrosine kinase inhibitor), HER1 / EGFR inhibitor, eriotinib, a COX-2 inhibitor (e.g. , celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the receptors ErbB2, ErbB3, ErbB4 or VEGF, inhibitors for receptor tyrosine kinases for platelet-derived growth factor (PDGF) and / or stem cell factor (SCF) (e.g., imatinib mesylate (Gleevec ® Novartis)), TRAIL / Apo2 and other bioactive agents and organic chemicals, etc. The term "prodrug" as used in this application refers to a precursor or derivative of a pharmaceutically active substance that is less cytotoxic to the tumor cells compared to the original drug and is capable of being activated or converted enzymatically to the form most active original. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (Ed.), Pp. 247-267, Humana Press (1985). Prodrugs of this invention include but are not limited to phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, prodrugs containing beta-lactam, prodrugs that contain optionally substituted phenoxyacetamide or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine prodrugs and other prodrugs of 5-fluorouridine that can be converted to the most active cytotoxic free drug. Examples of cytotoxic drugs that can be derived to a prodrug form for use in this invention include but are not limited to those chemotherapeutic agents described above. An "anti-angiogenesis agent" or "angiogenesis inhibitor" refers to a low molecular weight substance, a polynucleotide, a polypeptide, an isolated protein, a recombinant protein, an antibody or conjugates or fusion proteins thereof, which inhibit angiogenesis, vasculogenesis or undesirable vascular permeability, either directly or indirectly. For example, an anti-angiogenesis agent is an antibody or other antagonist to an angiogenic agent as defined above, eg, antibodies to VEGF, antibodies to VEGF receptors, small molecules that block receptor signaling VEGF (for example, PTK787 / ZK2284, SU6668, SUTENT / SUH248 (sunitinib malate), AMG706). Anti-angiogenesis agents also include natural angiogenesis inhibitors, eg, angiostatin, endostatin, etc. See, for example, Klagsbrun and D'Amore, Annu. Rev. Physiol., 53: 217-39 (1991); Streit and Detmar, Oncogene, 22: 3172-3179 (2003) (for example, the Table 3 lists anti-angiogenic therapy in malignant melanoma); Ferrara and Alitalo, Nature Medicine 5 (12): 1359-1364 (1999); Tonini et al., Oncogene, 22: 6549-6556 (2003) (for example, the Table 2 lists anti-angiogenic factors) and Sato Int. J.

Clin. Oncol., 8: 200-206 (2003) (for example, Table 1 lists anti-angiogenic agents used in clinical studies).

COOPERATIVES OF THE INVENTION AND METHODS FOR MANUFACTURING THEM The present invention encompasses compositions, in which pharmaceutical compositions are included, which comprise an anti-EphB4 antibody and polynucleotides comprising sequences encoding an anti-EphB4 antibody. As used herein, the compositions comprise one or more antibodies that bind to EphB4 and / or one or more polynucleotides that comprise sequences encoding one or more antibodies that bind to EphB4. These compositions may further comprise appropriate carriers, such as pharmaceutically acceptable excipients in which pH-regulating solutions are included, which are well known in the art. The invention also encompasses isolated antibody modalities and polynucleotides. The invention also encompasses substantially pure antibody and polynucleotide modalities. The anti-EphB4 antibodies of the invention are preferably monoclonal. Also encompassed within the scope of the invention are Fab, Fab and Fab'-SH and F (ab ') 2 fragments of the anti-EphB4 antibodies provided herein. These antibody fragments can be created by traditional means, such as enzymatic digestion or they can be generated by recombinant techniques. Such antibody fragments may be chimeric or humanized. These fragments are useful for the diagnosis and therapeutic purposes summarized later in the present. The monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible mutations that occur stably in nature that may be present in minor amounts. Thus, the "monoclonal" modifier indicates the character of the antibody because it is not a mixture of discrete antibodies.

The anti-EphB4 monoclonal antibodies of the invention can be made using the hybridoma method first described by Kohler et al. , Na ture, 256: 495 (1975), or it can be elaborated by recombinant DNA methods (U.S. Patent 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized to produce lymphocytes that produce or are capable of producing antibodies that will bind specifically to the protein used for immunization. Antibodies to EphB4 are generally reared in animals by multiple subcutaneous injections (sc) or intraperitoneal (ip) of EphB $ and an adjuvant. EphB4 can be prepared using methods well known in the art, some of which are described further herein. For example, the recombinant production of EphB4 is described later herein. In one embodiment, the animals are immunized with an EphB4 derivative containing the extracellular domain (ECD) of EphB4 fused to the Fc portion of an immunoglobulin heavy chain. In a preferred embodiment, the animals are immunized with a fusion protein of EphB4-IgGl. Animals are ordinarily immunized against immunogenic conjugates or EphB4 derivatives with monophosphoryl lipid A (MPL) / trehalose dicrinomycolate (TDM) (Ribi Immunochem.Research, Inc., Hamilton, MT) and the solution is injected intradermally in multiple sites Two weeks later the animals are reinforced. 7 to 14 days later the animals are bled and the serum is analyzed for the anti-EphB4 titer. The animals are reinforced up to their title plateau. Alternatively, lymphocytes can be immunized in Vi tro. The lymphocytes are then fused with myeloma cells using an appropriate fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal An tibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). The hybridoma cells thus prepared are seeded and cultured in an appropriate medium which preferably contains one or more substances that inhibit the growth or survival of the original unmyelinated myeloma cells. For example, if the original myeloma cells lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the culture medium for the hybridomas will commonly include hypoxanthine, aminopterin and thymidite (HAT medium) such substances inhibit cell growth. HGPRT-deficient. Preferred myeloma cells are those that efficiently fuse, support the production of high stable volume of antibody by the cells that produce selected antibodies that are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA. Mouse-human heteromyeloma cell lines and human myeloma have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The culture medium in which the hybridoma cells are cultured is analyzed for the production of monoclonal antibodies directed against EphB4. Preferably, the binding specificity of monoclonal antibodies produced in the hybridoma cells is determined by immunoprecipitation or by in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can be determined for example, by the Scatchard analysis of Munson et al. , Anal. Biochem. , 107: 220 (1980). After the hybridoma cells are identified that produce antibodies of the desired specificity, affinity and / or activity, the clones can be subcloned by limiting dilution procedures and cultivated by standard methods (Goding, Monoclonal An tibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include for example the D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo as ascites of tumors in an animal. The monoclonal antibodies secreted by the subclones are appropriately separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification methods, such as for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity. The anti-EphB4 anti-bodies of the invention can be prepared by using combination libraries to select clones of synthetic antibodies with the desired activity or activities. In principle, synthetic antibody clones are selected by selecting phage-containing phage libraries that display several fragments of antibody variable region (Fv) fused to the phage coat protein. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing fragments of Fv capable of binding to the desired antigen are adsorbed to the antigen and thus separated from clones with no binding in the library. Then the link clones are eluted from the antigen and can be further enriched by additional adsorption / antigen elution sites. Any of the anti-EphB4 antibodies of the invention can be obtained by designing the appropriate antigen selection procedure to select the phage clone of interest followed by construction of a full length anti-EphB4 antibody clone using the Fv sequences of the clone of the phage of interest and appropriate constant region (Fc) sequences described in Kabat et al. , Sequences of Proteins of Immunologi cal In terest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), volumes 1-3. The antigen binding domain of an antibody is formed of two variable regions (V) of approximately 110 amino acids, each of the light (VL) and heavy (VH) chains, which have both three hypervariable loops or regions that determine complementarity (CDR) The variable domains can be functionally displayed on phage, either as fragments of single chain Fv (scFv), in which VH and VL are covalently linked by means of a short flexible peptide or as Fab fragments in which they are each one fused to a constant domain and interact non-covalently, as described in Winter et al. , Ann. Rev. Immunol. , 12: 433-455 (1994). As used herein, phage clones encoding scFv and phage clones encoding Fab are collectively referred to as "clones of phage of Fv "or" Fv clones. "Repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries which can then be investigated for antigen binding clones such as is described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994) .The libraries of immunized sources provide high affinity antibodies to the immunogen without the requirement to construct hybridomas.Alternatively, the natural repertoire can be cloned to provide a single source of human antibodies to a wide range of autoantigens and also not autoantigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993) .Finally, natural libraries can also be elaborated synthetically by cloning the C gene segments without fixing from stem cells and using PCR cevadapres that contain random sequence for encode the highly variable CDR3 regions relationships and to effect the rearrangement in Vi tro as described by Hoogenboom and Winter, J. Mol. Biol. , 227: 381-388 (1992). Filamentous phage is used to display antibody fragments by fusion to the coating protein lower pHI. Antibody fragments can be displayed as single chain Fv segments, in the which the VH and VL domains are joined in the same polypeptide chain by a flexible polypeptide spacer, for example, as described by Marks et al. , J. Mol. Biol. , 222: 581-597 (1991), i as Fab fragments, in which one strand is fused to pIII and the other is secreted into the periplasm of bacterial host cell where the assembly of a Fab-coating protein structure it is deployed on the surface of the phage by displacing some of the wild-type coat proteins, for example, as described in Hoogenboom et al. , Nucí. Acids Res. , 19: 4133-4137 (1991). In general, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from animals or humans. If a polarized library in favor of anti-EphB4 clones is desired, the subject is immunized with EphB4 to generate an antibody response and spleen cells and / or circulating B cells other than peripheral blood lymphocytes (PBL) are recovered from the library construction. In a preferred embodiment, a human antibody fragment library condensed in favor of anti-EphB4 clones is obtained by generating an anti-EphB4 antibody response in transgenic mice carrying a genetic array of functional human immunoglobulin (and lacking a of production of functional endogenous antibody) in such a way that the Immunization of EphB4 gives rise to B cells that produce human antibodies against EphB4. The generation of transgenic mice that produce human antibodies is described later herein. Additional enrichment for populations of reactive anti-EphB4 cells can be obtained by using an appropriate selection procedure to isolate B cells expressing antibody bound to EphB4-specific membranes, for example, by cell separation with EphB4 affinity chromatography or EphB4 adsorption. cells to fluorochrome-labeled EphB4 followed by flow activated cell sorting (FACS). Alternatively, the use of spleen cells and / or B cells or other PBLs of an undimerized donor provides a better representation of the possible antibody repertoire and also allows the construction of an antibody library using any animal species (human or non-human) ) in which EphB4 is not antigenic. For libraries that incorporate a construction of the antibody gene in Vi tro, stem cells are harvested from the subject to provide nucleic acids encoding unadjusted antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, such as human, mouse, rat, lagomorph, lupine, canine, feline, porcine, bovine, equine and avian species, etc.

The nucleic acid encoding antibody variable gene segments (in which VH and VL segments are included) are recovered from the cells of interest and amplified. In the case of rearranged CVH and 7 VL gene libraries, the desired DNA can be obtained by isolating genomic DNA or mRNA from lingotites, followed by polymerase chain reaction (PCR) with primers that match the 5 'and 3' ends of the VH and VL genes rearranged as described in Orlandi et al. , Proc. Na ti. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby elaborating various V gene repertoires for expression. The C genes can be amplified from cDNA and genomic DNA, with retracers at the 5 'end of the exon encoding the mature V domain and forward primers based on J segments as described in Orlandi et al. (1989) and Ward et al. , Na ture, 341: 544-546 (1989). However, to amplify from the cDNA, the retroveners can first be based on the leader exon as described in Jones et al. , Biotechnol. , 9: 88-89 (1991) and forward primers within the constant region as described in Sastry et al. , Proc. Na ti. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize complementarity, degeneracy can be incorporated into the primers as described in Orlandi et al. (1989) or Sastry et al. (1989). Preferably, library diversity is maximized by using PCR primers targeted to each V gene family in order to amplify all the available VH and VL arrangements present in the nucleic acid sample of immune cells, for example as described in the method of Marks et al., J. Mol. Biol., 222: 581-597 (1991) or as described in the method of Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of amplified DNA to expression vectors, rare restriction sites can be introduced into the PCR primer as an end tag as described in Orlando et al. (1989) or by additional PCR amplification with a labeled primer as described in Clackson et al., Nature, 352: 624-628 (1991). Repertoires of synthetically rearranged V genes can be derived in vitro from V gene segments. Most of the human V H gene segments have been cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227: 776-798 (1992)) and mapped (reported in Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned segments (in which all the major conformations of the Hl and H2 loop are included) can be used to generate various VH gene repertoires with PCR primers that encode H3 loops of different sequence and length as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). they can also be made with all the sequence diversity focused on the single-length H3 buvle as described in Barbas et al., Proc. Nati. Acad. Sci. USA, 89: 4457-4461 (1992). Segments of VK and V? humans have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used to make synthetic light chain repertoires. Synthetic gene V repertoires, based on a range of VH and VL logues and L3 and H3 lengths, will encode antibodies of considerable structural diversity. Following the amplification of DNA encoding gene V, germline V gene segments can be rearranged in Vi tro according to the methods of Hoogenboom and Winter, J. Mol. Biol. , 227: 381-388 (1992). Repertoires of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire can be created in different vectors and the vectors recombined in Vi tro, for example as described in Hogrefe et al. , Gene, 128: 119-126 (1993), or in vivo by combinatorial infection, for example, the loxP system described in Waterhouse et al. , Nucí. Acids Res. , 21: 2265-2266 (1993). The in vivo recombination procedure takes advantage of the two-chain nature of the Fab fragments to overcome the limit in library size imposed by the efficiency of E. coli transformation. Repertories of natural VH and VL are cloned separately, one to a phagemid and the other to a phage vector. Then the two libraries are combined through phage infection of phagemid-containing bacteria, from such a manwera that each cell contains a different combination and the size of the library is limited only by the number of cells present (approximately 1012 clones). Both vectors contain recombination signals in vivo, in such a way that the VH and VL genes are recombined in a single replicon and are co-packed in phage virions. These huge libraries provide large numbers of various Good Affinity antibodies (Kd "1 of approximately 10" 8 M). Alternatively, the repertoires can be cloned sequentially to the same vector, for example as described in Barbas et al. , Proc. Na ti. Acad. Sci. USA, 88: 7978-7982 (1991) or assembled together by PCR and then cloned, for example as described in Clackson et al. , Na ture, 352: 624-628 (1991). The PCR assembly can also be used to bind VH and VL DNA with DNA encoding a flexible peptide spacer to form repeats of single chain Fv (scFv). -In yet another technique, "PCR enasamble in cells" is used to combine VH and VL genes into lymphocytes by PCR and then linked gene clone repertoires as described in Embleton et al. , Nucí. Acids Res. , 20: 3831-3837 (1992). Antibodies produced by natural or (either natural or synthetic) libraries may be of moderate affinity (Kd "1 of approximately 106 to 107 M'1), but Affinity maturation can also be indicated in vi tro when constructing and deselecting secondary libraries as described in Winter et al. (1994), supra. For example, the mutation can be introduced randomly in Vi tro when using error-prone polymerase (reported in Leung et al., Technique, 1: 11-15 (1989)) in the method of Hawkins et al. , J. Mol. Biol. , 226: 889-896 (1992) or in the method of Gram et al. , Proc. Na ti. Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity modulation can be effected by randomly mutating one or more CDRs, for example using PCR with primers carrying random sequence extension of CDR of interest, in individual Fv clones and selection for higher affinity clones. WO 9607754 (published March 14, 1996) describes a method for inducing mutagenesis in a region that determines the complementarity of an immunoglobulin light chain to create a library of light chain genes. Another effective method is to recombine the selected VH or VL domains by displaying phages with repertoires of V domain variants that occur stably in nature obtained from unimmunised rodents and selection for higher affinity in several rounds of chain re-intermingling as described in Marks et al. , Biotechnol. , 10: 779-783 (1992). This technique allows the production of antibodies and antibody fragments with affinities in the range of 10"9 M.

Nucleic acids of EphB4 and amino acid sequences are known in the art. The nucleic acid sequence encoding EphB4 can be designated using the amino acid sequence of the desired region of EphB4. Alternatively, cDNA sequences (or fragments thereof) of GenBank accession number NM__004444 or disclosed in U.S. Patent 5,635,177, may be used. The DNAs encoding EphB4 can be prepared by a variety of methods known in the art. These methods include but are not limited to chemical synthesis by any of the methods described in Engels et al. , Agnew. Chem. In t. Ed. Engl. , 28: 716-734 (1989), such as the trimester, phosphite, phosphamidite and H-phosphonate methods. In one embodiment, the preferred codons for host cell expression are used in the design of DNA encoding EphB4. Alternatively, the DNA encoding EphB4 can be isolated from a genomic library or cDNA library. Following the construction of the DNA molecule encoding EphB4, the DNA molecule is operably linked to an expression control sequence in an expression vector, such as a plasmid, wherein the control sequence is recognized by a host cell transformed with the vector. In general, plasmid vectors containing control replication sequences that are derived from species compatible with the host cell. He vector originally carries a replication site, also as sequences that encode proteins that are capable of providing phenotypic selection in transformed cells. Vectors suitable for expression in prokaryotic and eukaryotic host cells are known in the art and some are described further herein. Eukaryotic organisms, such as yeast or cells derived from multicellular organisms, such as mammals, can be used. Optionally, the DNA coding in EphB4 is operatively linked to a secretory leader sequence resulting in the preservation of expression products by the host cell to the culture medium. Examples of secretory leader sequences include stll, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase alpha factor. Also suitable for use herein is the 36 amino acid leader sequence of protein A (Abrahmsen et al., EMBO J., 4: 3901 (1985)). The host cells are transfected and preferably transformed with the expression or cloning vectors described above of this invention and cultured in conventional culture media multiplied as appropriate to induce promoters, select transformants or amplify the genes encoding the desired sequences. Transfection refers to the absorption of a vector of expression by a host cell whether any coding sequences are indeed expressed. Numerous methods of transfection known to the ordinarily experienced technician, for example, precipitation, CaP04 and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell. Methods for transfection are well known in the art and some are described further herein. Transformation media that introduce DNA to an organism in such a way that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrator. Depending on the host cell used, the transformation is done using standard techniques appropriate for such cells. Methods for transformation are well known in the art and some are described further herein. The prokaryotic host cells used to produce the EphB4 can be cultured as described generally in Sambrook et al. , supra. The mammalian host cells used to produce the EphB4 can be cultured in a variety of media, which are well known in the art and some of which are described herein. The host cells referred to in this disclosure encompass cells in cultures in Vi tro as well. as cells that are inside a host animal. The purification of EphB4 can be carried out using methods recognized in the art, some of which are described herein. The purified EphB4 can be attached to an appropriate matrix such as random beads, archilamide beads, glass beads, cellulose, various acrylic copolymers, hydroxylmethacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers and the like, for Use in affinity chromatographic separation of phage display clones. The annexation of the EphB4 protein to the matrix can be effected by the methods described in Methods in Enzymology, vol. 44 (1976). A technique commonly used to attach protein ligands to polysaccharide matrices, for example agarose, dextran or cellulose, involves activation of the carrier with cyanogen halides and subsequent coupling of the primary aliphatic or aromatic amines of the peptide ligand to the activated matrix. Alternatively, EphB4 can be used to coat the absorption plate cavities, expressed on fixed host cells to adsorption plates or used in cell sorting or conjugated to biotin for capture with streptavidin-coated beads or used in any other method known in the art. art for panning phage display libraries. Samples of phage libraries are contacted with immobilized EphB4 under conditions appropriate for the binding of at least a portion of the phage particles to the absorbent. Normally, conditions, which include pH, ionic strength, temperature and the like are selected to mimic physiological conditions. Phages bound to the solid phase are washed and then eluted by acid, for example, as described in Barbas et al. , Proc. Na ti. Acad. Sci USA, 88: 7978-7982 (1991) or by alkali, for example as described in Marks et al. , J. Mol. Biol. , 222: 581-597 (1991) or by competition for EphB4 antigen, for example, in a procedure similar to the antigen competition method of Clackson et al. , Na ture, 352: 624-628 (1991). Phages can be enriched 20-1,000 times in a single round of selection. In addition, enriched phages can be cultured in bacterial culture and subjected to additional rounds of selection. The efficiency of the selection depends on many factors, in which the dissociation kinetics are included during the wash and either multiple antibody fragments in a single phage can simultaneously be coupled with the antigen. Antibodies with rapid dissociation kinetics (and weak binding affinities) can be retained by the use of short wash, multivalent phage display and density of high coating of antigen in solid phase. The high density not only stabilizes the phage by means of multivalent interactions, but also favors the re-binding of the phage that has dissociated. The selection of antibodies with slow dissociation kinetics (and good binding affinity) can be promoted from the use of long lacquers and monovalent phage display as described in Bass et al. , Proteins, 8: 309-314 (1990) and in WO 92/09690 and a low coating density of antigen as described in Marks et al. , Biotechnol. , 10: 779-783 (1992). It is possible to select between phage antibodies of different affinities, even with affinities that differ slightly, for EphB4. However, the random mutation of a selected antibody (eg, as done in some of the affinity modulation techniques described above) is likely to give rise to many mutants, most of which are antigen binding and a few with more affinity. high. With limiting EphB4, rare high affinity phage could compete. To retain all of the higher affinity mutants, phages can be incubated with biotinylated EphB4 in excess, but with the biotinylated EphB4 at a modality concentration lower than the target molar affinity constant for EphB4. Then the high affinity binding phages can be recaptured by paramagnetic beads coated with streptavidin. Such "capture in equilibrium "allows the antibodies to be sectioned according to their binding affinities, with sensitivity that allows the isolation of such mutants with as little as a two times higher affinity of a larger excess of phage with lower affinity. the washing of phage bound to a solid phase can also be manipulated to discriminate based on the dissociation kinetics .. Anti-EphB4 cones can be selected by activity In one embodiment, the invention provides anti-EphB4 antibodies which block the binding between a ligand Ephb4 (such as ephrin-Bl, ephrin-B2 and / or ephrin-B3) and EphB4, but do not block the link between a ligand of EphB4 and a second protein (such as EphBl, EphB3, EphB4, EphB5 and / or EphBd Fv clones corresponding to such anti-EphB4 antibodies can be selected by (1) isolating anti-EphB4 clones from a phage library as described above and optionally amplifying the isolated population. gives phage clones when culturing the population in an appropriate bacterial host; (2) select EphB4 and a second protein that with blocking activity and no blocking activity, respectively, if desired; (3) adsorption of the anti-EphB4 phage clones to immobilized EphB4; (4) using an excess of the second protein to elute any undesirable clones that recognize determinants of EphB4 that overlap or are shared with the binding determinants of the second protein and (5) elute clones with remnant adsorbed following step (4). Optionally, clones with the desired blocking / non-blocking properties can be further enriched by repeating the screening procedures described herein once or many times. The DNA encoding the hybridoma-derived monoclonal antibodies or phage display Fv clones of the invention is easily isolated and sequenced using conventional methods (eg, using oligonucleotide primers designed to specifically amplify the heavy and light chain coding reactions of Hybridoma interest or phage DNA template). Once isolated, the DNA can be placed in expression vectors, which are then transfected into host cells such as E cells. coli, simian COS cells, Chinese hamster ovary cells (CHO) or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells. Review articles regarding recombinant expression in bacteria of DNA encoding antibody include Skerra et al. , Curr. Opinion in Immunol. , 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992).

The DNA encoding the Fv clones of the invention can be combined into known DNA sequences that encode heavy chain and / or light chain constant regions (e.g., appropriate DNA sequences can be obtained from Kabat et al., supra) to form clones encoding heavy and / or light partial or full length chains. It will be appreciated that constant regions of any isotype can be used for this purpose, in which constant regions of IgG, IgM, IgA, IgD and IgE are included and that these constant regions can be obtained from any human or animal species. An Fv clone derived from the variable domain DNA of an animal species (such as human) and then fused to constant region DNA from another animal species to form coding sequence (s) for heavy chain and / or full length light chain "hybrid" is included in the definition of "chimeric" and "hybrid" antibody as illustrated herein. In a preferred embodiment, an Fv clone derived from human variable DNA is fused to a human constant region DNA to form coding sequence (s) for full length and / or light human partial or heavy chains. The DNA encoding anti-EphB4 antibody derived from a hybridoma of the invention can also be modified for example by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences derived from the hybridoma clone ( for example, as in the method of Morrison et al., Proc. Na ti. Acad. Sci. USA, 81: 6851-6855 (1984)). DNA encoding a hybridoma or antibody derived from Fv clone or fragment can be further modified by covalently binding to the immunoglobulin coding sequence all or part of the coding sequence by a polypeptide without immunoglobulin. In this manner, the "chimeric" or "hybrid" antibody are preparations having the binding specificity of the Fv clone or clone-hybridoma derivative antibodies of the invention.

Antibody fragments The present invention encompasses antibody fragments. In certain circumstances, there are advantages to using fragments of antibodies instead of whole antibodies. The smaller size of the fragments allows for rapid clearance and can lead to improved access to solid tumors. Several techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and fastened in E. coli thus allowing the easy production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E. coli and chemically coupled to form fragments of F (ab) 2 (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another method, F (ab) fragments can be isolated directly from the recombinant host cell culture. The Fab and F (ab) 2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Patent 5,869,046. Other techniques for the production of antibody fragments will be apparent to the experienced technician. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185, U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and scFv are the only species with intact combination sites that are devoid of constant reactions. Thus, they are appropriate for the reduced non-specific binding during in vivo use. ScFv fusion proteins can be constructed to produce the fusion of an effector protein either at the amino terminus or at the carboxy terminus of a scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment can also be a "linear antibody", by example as described in U.S. Patent 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies The present invention encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced thereto from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are commonly taken from a "import" variable domain. Humaization can be effected essentially following the method of Winter et al. (Jones et al. (1986) Na ture 321: 522-525; Riechmann et al. (1988) Na ture 332: 323-327; Verhoeyen et al. (1988 ) Science 239: 1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent 4,816,567) wherein substantially less than a variable domain of intact human has been replaced by the corresponding sequences of a non-human species. In practice, humanized antibodies are commonly human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues of analogous sites in rodent antibodies. The choice of human variable domains, both light and heavy, to be used in the manufacture of humanized antibodies is very important to reduce antigenicity. According to the so-called "best fit" method, the variable domain sequences of a rodent antibody is selected against the entire library of known human variable domain sequences. The human sequence is closest to that of the rodent and is then accepted as the human structure for the humanized antibody (Sims et al (1993) J. Immunol., 151: 2296; Chothia et al. (1987) J. Mol. Biol. 196: 901. Another method uses a particular structure derived from the consensus sequence of all human antibodies of a particular subgroup of heavy or light chains.The same structure can be used for several different humanized antibodies (Carter et al., 1992). Proc. Na ti, Acad. Sci. USA, 89: 4285; Presta et al. (1993) J. Immunol., 151: 2623. It is also important that the antibodies are humanized with retention of high affinity for antigens and other properties. In order to obtain this objective, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and several conceptual humanized products. using three-dimensional models of the peer and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and show probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. The inspection of these exhibits allows the analysis of the probable role of the residues in the functioning of the candidate immunoglobulin sequence, that is, the analysis of the residues that introduced the ability of the candidate immunoglobulin to bind to its antigen. In this manner, FR residues can be selected and combined with the reactor and import sequences in such a manner that the desired antibody characteristic, such as increased affinity for the target antigen (s), is obtained. In general, the hypervariable region residues are directly and more substantially involved in influencing the antigen binding.

Human Antibodies Human anti-EphB4 antibodies of the invention can be constructed by combining selected Fv clone variable domain sequence (s) selected from human-derived phage display libraries with known human constant domain sequence (s) (s). ) as described above.

Alternatively, monoclonal anti-EphB4 antibodies of the invention can be made by the hybridoma method. Human hybridoma cell lines and mouse-human heterohybridoma cell lines for the production of human monoclonal antibodies have been described in Kozbor J. Immunol. , 133: 3001 (1984); Brodeur et al. , Monoclonal Antibody Production Techniques and Appli cations, pp. 51-63 (Marcel Dekker, Inc., New York, 1987) and Boerner et al. , J. Immunol. , 147: 86 (1991). It is now possible to produce transgenic animals (e.g., mice) that are capable, after immunization, of producing a full repertoire of human antibodies in the absence of indigenous immunoglobulin production. For example, it has been described that homocidal cancellation of the antibody heavy chain binding region (JH) gene in chimeric mice and germline mutant mice results in complete inhibition with reduction of endogenous antibody. The transfer of the human germline immunoglobulin genetic array in such germline mutant mice will result in the production of human antibodies after antigen treatment. See, for example, Jakobovits et al. , Proc. Na ti. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al. , Na ture, 362: 255 (1993); Bruggermann et al. , Year in Immunol. , 7:33 (1993). Gene intermixing can also be used to deriving human antibodies from non-human antibodies, eg, rodents, wherein the human antibody has affinities and specificities similar to the non-human starting antibody. According to this method, which is also called "epitope printing", either in the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques, as described above is replaced with a repertoire of human V domain genes, creating a population of scFv or nonhuman chain / human chain Fab chimeras. Screening with antigens results in isolation of scFv or chimeric non-human chain / human chain Fabs wherein the human chain restores the destroyed antigen binding site at the corresponding non-human chain removal in the primary phage display clone , that is, the epitope governs (prints) the choice of a human chain partner. When the process is repeated in order to replace the remaining non-human chain, an antibody is obtained (see PCT WO 93/06213 published April 1, 1993). Unlike the traditional humanization of non-human antibodies by CDR insertion, this technique provides completely human antibodies, which do not have FR or CDR residues of non-human origin.

Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for EphB4 and the other is for any other antigen. Exemplary bispecific antibodies can bind to two different epitopes of the EphB protein. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing EphB4. These antibodies have an EphB4 binding arm and an arm that binds to the cytotoxic agent (eg, saporin, anti-interferon-a, vinca alkaloid, resin chain A, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (for example, bispecific antibodies F (ab) 2). Methods for processing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two heavy chain / immunoglobulin light chain pairs, where the less heavy samples have different longitudinal axes (Milstein and Cuello, Na ture, 305: 537 (1983 )). Due to the random classifications of heavy or light immunoglobulin cha these hybrids (quadrots) produce a potential mixture of 10 molecules of different antibodies, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather annoying and the coatings of the product are low. Similar procedures are disclosed in WO 93/08829 published May 13, 1993 and in Traunecker et al. , EMBO J., 10: 3655 (1991). According to a different and more preferred method, variable domaof antibody with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the engozone, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1), which contathe site necessary for the light chain linkage, present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are rted into separate expression vectors and are co-transfected into an appropriate host organism. This provides greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in modalities when unequal proportions of the three polypeptide chaused in the construction provide the optimal returns. However, it is possible to rt the coding sequences for two or all three polypeptide chainto an expression vector when the expression of at least two polypeptide chain equal proportions results in high yields or when the proportions are not particular meaning. In a preferred embodiment of this method, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a heavy chain-light chain pair of hybrid immunoglobulin (which provides a second binding specificity) in the other arm. It has been found that this asymmetric structure facilitates the separation of the desired bispecific compound from undesirable immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only half of the bispecific molecule provides an easy way of separation. This method is disclosed in WO 94/04690. For further details to generate bispecific antibodies see, for example, Suresh et al. , Methods in Enzymology, 121: 210 (1986). According to another method, the interface between a pair of antibody molecules can be designed to maximize the percentage of heterodimers that are recovered from the recombinant cell culture. The preferred interface comprises at least part of the CH3 domain of a domain antibody constant. In this method, one or more small interface amino acid side chains of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan). "Compensatory cavities" of identical size or size similar to the large side chain (s) are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (eg, alanine or threonine). This provides a mechanism to increase the performance of the heterodimer with respect to the undesirable end products such as homodimers. Bispecific antibodies include cross-linked or "heterocinjugged" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies, for example, have been proposed to target immune system cells to undesirable cells (U.S. Patent 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/00373 and EP 03089). Heteroconjugate antibodies can be made using any convenient crosslinking methods. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Patent 4,676,980 together with a number of crosslinking techniques. Techniques for generating bispecific antibodies from antibody fragments have also been described in Literature. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al. , Science, 229: 81 (1985) describe a method wherein intact antibodies are proteolytically excised to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the complexing agent of dithiol sodium arsenite to stabilize vicinal dithiols and prevent the formation of intermolecular disulfide. Then the Fab 'fragments are converted to thionitrobenzoate derivatives (TNB). One of the derivatives of Fab '-TNB is then converted to Fab' -thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab '-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immunization of enzymes. Recent progress has facilitated the direct recovery of Gab '-SH fragments from E. coli that can be chemically coupled to form bispecific antibodies. Shalaby et al. , J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanised bispecific F (ab ') antibody molecule. Each fragment of Fab 'was secreted separately from E. coli and subjected to direct chemical coupling in Vi tro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, also as to trigger the lytic activity of human cytotoxic lymphocytes against tumor targets of human weight. Several techniques for making and isolating bispecific antibody fragments directly from revolving cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al. , J. Immunol. , 148 (5): 1547-1553 (1992). The leucine zipper peptides of the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by genetic fusion. The antibody homodimers were reduced in the engozone region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al. , Proc. Na ti. Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for making bioscific antibody fragments. The fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two binding sites of antigen. Another strategy for making bispecific antibody fragments by the use of single chain Fv dimers (scFv) has also been reported. See Gruber et al. , J. Immunol. , 152: 5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).

Multivalent antibodies A multivalent antibody can be internalized (and / or catabolized) faster than a bivalent antibody by a cell that expresses an antigen to which the antibodies bind. The antibodies of the invention can be multivalent antibodies (which are different from the IgM class) with three or more antigen binding sites (eg, tetravalent antibodies) which can be easily produced by recombinant nucleic acid expressions encoding the chains of polypeptide of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or an engozne region. In this scenario, the antibody will comprise an Fc region and three or more amino-terminal antigen binding sites to the Fe region. The preferred multivalent antibody of the present comprises (or consists of) three a approximately eight but preferably four antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain (s) comprises (s) two or more variable domains. For example, the polypeptide chain (s) can comprise VDl- (Xl) n -VD2- (X2) n -Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide and n is 0 or 1. For example, the polypeptide chain (s) can (n) comprise: VH-CH1 -linkers flexible-VH-CH1-Fc region chain or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein further preferably comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody of the present may for example comprise from about 2 to about 8 light chain variable domain polypeptides. The light chain variable domain polypeptides concentrated herein comprise a light chain variable domain and optionally further comprise a CL domain.

Antibody variants In some embodiments, modifications of amino acid sequences of the antibodies described in I presented. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Variant amino acid sequences of the antibody are prepared by introducing appropriate nucleotide changes to the antibody nucleic acid or by peptide synthesis. Such modifications include for example cancellations of and / or insertions to and / or substitutions of, residues within the amino acid sequences of the antibody. Any combination of cancellation, insertion and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid alterations can be introduced into the amino acid sequence of the antibody subject to the time in which the sequence is made. A useful method for identifying certain residues or regions of antibody that are preferred sites for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244: 1081-1085. Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys and glu) and replaced by a neutral amino acid or negatively charged amino acid (more preferably alanine or polyalanine) to affect the interaction of the amino acids with antigens. Those amino acid sites that demonstrate functional sensitivity to substitutions are then removed by introducing additional agents and other variants in, or for, replacement sites. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning mutagenesis or random mutagenesis is carried out at the target codon or target region and the expressed immunoglobulins are selected for the desired activity. Insertions of amino acid sequences include amino- and / or carboxyl-terminal fusions that fluctuate in length from a residue to polypeptides containing 100 or more residues, also as intrasequence insertions of a single amino acid residue or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecules include fusion to the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide that increases the half-life in the serum of the antibody. The glycosylation of polypeptides is commonly either N-linked or O-linked. N-linked refers to the annexation of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic annexation of the carbohydrate moiety to the side chain of asparagine. Thus, the presence of either one of these polypeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxylproline or 5-hydroxylysine can also be used. The addition of glycosylation sites to the antibody is conveniently effected by altering the amino acid sequence such that it contains one or more of the dipeptide sequences described above (for N-linked glycosylation sites). The alteration may also be made by adding or substituting one or more serine or threonine residues to the original antibody sequence (for O-linked glycosylation sites). Where the antibody comprises an Fc region, the carbohydrate attached thereto can be altered. For example, antibodies with a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody are described in U.S. Patent Application No. US 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a bisection N-acetylglucosamine (GlcNAc) in the carbohydrate attached to a region Fc of the antibody are referred to in WO 2003/011878, Jean-Mairet et al. and U.S. Patent 6,602,684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide appended to an antibody Fc region are reported in WO 1997/30087, Patel et al. See also WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) concerning the antibody with altered carbohydrate appended to the Fc region thereof. See also United States Patent 2005/0123546 (Umana et al.) For antigen binding molecules with modified glycosylation. The preferred glycosylation variant herein comprises an Fc region, wherein a carbohydrate structure attached to the Fc region lacks fucose. Such variants have enhanced ADCC function. Optionally, the Fc region also comprises one. or more amino acid substitutions therein which ADCC further improves, for example substitutions at positions 298, 333 and / or 334 of the Fc region (Eu residue numbering). Examples of publications concerning "defucosylated" or "fucose-deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005 / 053742; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al.

Biotech Bioeng. 87: 614 (2004). Examples of cell lines that produce defucosylated antibodies include CHO Lecl3 cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys., 249: 533-545 (1986); US Pat. Application US 2003/0157108 Al, Presta, L and WO 2004/056312 Al, Adams et al., especially in Example 11) and expulsion cell lines, such as the alpha-1,6-fucosyltransferase gene, FUT8, CHO expulsion cells (Yamane-Ohnuki et al. Biotech, Bioeng 87: 614 (2004)). Another type of variant is a variant amino acid substitution. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but alterations of FR are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, termed "exemplary substitutions" in Table 1, or as further described hereinafter with reference to amino acid classes may be introduced into the selected products.

Table 1 Substantial changes in the biological properties of the antibody will be carried out by selecting substitutions that differ significantly in their effect in maintaining (a) the structure of the fundamental chain of the polypeptide in the substitution area, eg, a sheet conformation or helical conformation, (b) the load or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The residues that occur in a stable manner in nature are divided into groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acids: asp, glu; (4) Basics: his, lys, arg; (5) Residues that influence the orientation of the chain: gly, pro and (6) Aromatic: trp, tyr, phe. Non-conservative substitutions will involve exchanging a member of one of these classes for another class. One type of substitutional variant involves replacing one or more hypervariable region residues of an original antibody (eg, a humanized or human antibody). In general, the resulting variant (s) selected for further development will have improved biological properties in relation to the original antibody from which they are generated. A convenient way to generate such substitutional variants involves affinity modulation using phage display. Briefly, several hypervariable region sites (eg, 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are folded from filamentous phage particles as fusions to the genetic product III of M13 packaged within each particle. Then the phage-displayed variants are selected in terms of their biological activity (e.g., binding affinity) as revealed in the present. In order to identify hypervariable region sites in candidates for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that are significantly distributed to the antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to selection as described herein and antibodies with properties superiors in one or more relevant analyzes can be selected for further development. Nucleic acid molecules that encode amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include but are not limited to isolation from a natural source (in the case of variants of amino acid sequences that occur stably in nature) or preparation by oligonucleotide-moderate (or site-directed) mutagenesis, mitagénesis of PCR and mitagénesis of cásete of a variant prepared previously or a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications in an Fc region of the immunoglobulin polypeptides of the invention, thereby generating a variant Fc region. The Fc region variant may comprise a human Fc region sequence (e.g., a region of IgG1, IgG2, IgG3 or human IgG4) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions which include those of a cysteine engozne. In accordance with this disclosure and the teachings of the art, it is contemplated that in some embodiments, an antibody used in the methods of the invention may comprise one or more alterations compared to the wild type counterpart antibody, for example, in the region of Fc. These antibodies would nevertheless retain substantially the same characteristics required for therapeutic utility as compared to their wild-type counterpart. For example, it is thought that certain alterations can be made in the Fc region that would result in altered Clq binding (ie, improved or decreased) and / or altered complement dependent cytotoxicity (CDC), for example as described in WO 99/51642. See also Duncan and Winter Na ture 322: 738-40 (1988); U.S. Patent 5,648,260; U.S. Patent 5,624,821 and W094 / 29351 concerning other examples of Fc region variants. WO00 / 42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with improved or reduced linkage to FcRs. The content of these patent publications is specifically incorporated herein by reference. See also Shields et al. J. Biol. Chem. 9 (2): 6591-6604 (2001). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn) which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol., 24: 249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that enhance the binding of the Fc region to FcRn. Variants of polypeptides or amino acid sequences of Fc region and increased or decreased Clq binding capacity are described in US Pat. No. 6,194,551 Bl, WO 99/51642. The content of that patent application is specifically incorporated by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibody Derivatives The antibodies of the present invention can be further modified to contain additional non-proteinaceous portions that are known in the art and are readily available. Preferably, the appropriate portions for derivatization of the antibody are polymers soluble in water. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3, 6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers) and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g. , glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers appended to the antibody can vary and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations that include but are not limited to the particular properties or functions of the antibody to be improved, if the antibody derivative will be used in a therapy under defined conditions, etc.

Selection of antibodies with desired properties The antibodies of the present invention can be characterized in terms of their physical / chemical properties and biological functions by various assays known in the art. In some embodiments, the antibodies are characterized by either one or more of EphB4 activation reduction or blocking, reduction or blocking of molecular signaling downstream of EphB4, reduction or blocking of EphB4 ligand activation, reduction or blocking of molecular signaling. downstream of the EphB4 ligand, disruption or blocking of ligand binding (e.g., efrin-Bl, ephrin-B2 and / or ephrin-B3) to EphB4, phosphorylation of EphB4 and / or multimerization of EphB4 and / or phosphorylation of ligand of EphB4 - and / or treatment and / or prevention of a tumor, cell proliferative alteration or cancer; and / or treatment or prevention of an alteration associated with the expression and / or activity of EphB4 (such as increased expression and / or EphB4 activity). The purified antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.

In certain embodiments of the invention, the antibodies produced herein are analyzed for their biological activity. In some embodiments, the antibodies of the present invention are tested for their antigen binding activity. Antigen binding assays that are known in the art and can be used herein include without limitation any direct or competitive binding analysis using techniques such as western blots, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), immunoassay " sandwich, immunoprecipitation analysis, fluorescent immunoassays and protein immunoassays A. An illustrative antigen binding assay is provided below in the example section. In yet another embodiment, the invention provides anti-EphB4 monoclonal antibodies that compete with antibody 30.35, 30.35.1D2 and / or 30.35.2D8 for binding to EphB4. Such competing antibodies include antibodies that recognize an EphB4 epitope that is the same as or overlaps with the EphB4 epitope recognized by antibody 30.35, 30.35.1D2 and / or 30.35.2D8. Such competing antibodies can be obtained by screening anti-EphB4 hybridoma supernatants for binding to immobilized EphB4 in competition with labeled antibody 30.35, 30.35.1D2 and / or 30.35.2D8. A supernatant of hybridoma containing antibody The competitor will reduce the amount of bound labeled antibody detected in the subject competition binding mixture compared to the amount of bound, labeled antibody detected in a control binding or control-containing mixture containing (or does not contain) irrelevant antibody. Any of the competency analyzes described herein are appropriate for use in the above procedure. In another aspect, the invention provides an anti-EphB4 monoclonal antibody comprising one or more HVRs (such as 2, 3, 4, 5 and / or 6) of antibody 30.35, 30.35.1D2 or 30.35.2D8. An anti-EphB4 monoclonal antibody comprising one or more HVR of 30.35, 30.35.1D2 and / or 30.35.2D8 can be constructed by grafting one or more HVR of 30.35, 30.35.1D2 and / or 30.35.2D8 onto a sequence of template antibody, for example a human antibody sequence that is closer to the corresponding murine sequence of the original antibody or a consensus sequence of all human antibodies in the particular subgroup of the original antibody heavy or light chain and which expresses the Chimeric light chain and / or heavy variable region sequence with no accompanying constant region sequences, in recombinant host cells as described herein. The anti-EphB4 antibodies of the invention possessing the unique properties described herein can be obtained by selection of anti-EphB4 hybridoma clones for the desired properties by any convenient method. For example, if an anti-EphB4 monoclonal antibody that blocks or does not block the ligand binding of EphB4 to EphB4 is desired, the candidate antibody can be tested in a binding competition analysis, such as a competitive binding ELISA, where the plate cavities are coated with EphB4 and an antibody solution in an excess of the Eph ligand of interest is stratified on the coated plates and the bound antibody is detected enzymatically, for example by contacting the bound antibody with anti-Ig antibody HRP-conjugated or biotinylated anti-Ig antibody and developing the color reaction of HRP, for example by developing plates with streptavidin-HRP and / or hydrogen peroxide and detecting the color reaction of HRP by spectrophotometry at 490 nm with an ELISA plate reader. If an anti-EphB4 antibody that inhibits or activates the activation of EphB4 is desired, the candidate antibody can be tested in a phosphorylation assay of EphB4. Such analyzes are known in the art and one such analysis is described in the examples section. If an anti-EphB4 antibody that inhibits cell growth is desired, the candidate antibody can be tested in in vitro and / or in vivo assays that measure the inhibition of cell growth Such analyzes are known in the art and are further described and exemplified herein. In one embodiment, the present invention contemplates an altered antibody that possesses some but not all effector functions, which makes it a desired candidate for many applications in which the antibody half-life in vivo is important and still the effector functions (such as complement and ADCC) are not necessary or are harmful. In certain embodiments, the Fc activities of the immunoglobulin produced are measured to ensure that only the desired properties are maintained. Cytotoxicity analysis in vitro and / or in vivo can be carried out to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor binding (FcR) analysis can be carried out to ensure that the antibody lacks an FcγR linkage (hence, probably lacks ADCC activity), but retains the FcRn binding ability. . Primary cells to moderate ADCC, NK cells, express FcyRIII only, while monocytes express FcyRI, FcyRII and FcyRIII. The expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). An example of an in vitro assay for determining ADCC activity of a molecule of interest is described in U.S. Patent Nos. 5,500,362 or 5,821,337. Cells effectors useful for such analyzes include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be determined in vivo, for example, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998). Clq binding assays can also be performed to confirm that the antibody is not capable of binding to Clq and hence lacks CDC activity. To determine complement activation, a CDC assay can be performed, for example as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). FcRn binding and clearance determinations / in vivo half life can also be effected using methods known in the art, for example those described in the examples section.

Vectors, host cells and recombinant methods For the recombinant production of an antibody of the invention, the nucleic acid encoding them is isolated and inserted into a replicable vector for further cloning (DNA amplification) or for expression. The DNA encoding the antibody is easily isolated and sequenced using conventional methods (for example, by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. In general, the preferred host cells are either of prokaryotic or eukaryotic origin (in general mammalian). It will be appreciated that constant regions of any isotype can be used for this purpose, in which constant regions of IgG, IgM, IgA, IgD, and IgE are included, and that such constant regions can be obtained from any human or animal species. to. Generation of antibodies using prokaryotic host cells: i. Construction of vector polynucleotide sequences encoding polypeptide components of the antibody of the invention can be obtained using standard recombinant techniques. Sequences of desired polynucleotides can be isolated and sequenced from cells that produce antibodies such as hybridoma cells. Alternatively, the polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art may be used for the purpose of the present invention. The selection of an appropriate vector will depend primarily on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains several components, depending on its function (amplification or heterologous polypeptide expression or both) and its compatibility with the particular host cell in which it resides. The vector components include in general, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, heterologous nucleic acid insert and a transcription termination sequence. In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in relation to these hosts. The vector ordinarily carries a replication site, also as labeling sequences that are capable of providing phenotypic selection in transformed cells. For example, E. coli is commonly transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes that encode resistance to ampicillin (Amp) and tetracycline (Tet) and thus provide an easy means to identify transformed cells. pBR322, its derivatives or other microbial or bacteriophage plasmids may also contain or be modified to contain, promoters which can be used by the microbial organism for the expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237. In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transformant vectors in relation to these hosts. For example, the bacteriophage such as? GEM.TM.-ll can be used in the manufacture of a recombinant vector that can be used to transform susceptible host cells such as E. coli LE392. The expression vector of the invention may comprise two or more promoter-cistron pairs, which encode each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5 ') to a cistron that modulates its expression. Prokaryotic promoters commonly fall into two classes, inducible and constitutive. The inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, for example the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by a variety of potential host cells are well known. He Selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the source DNA promoter via restriction enzyme digestion and insertion of the promoter sequence isolated to the vector of the invention. Both the natural promoter sequence and many heterologous promoters can be used to direct the amplification and / or expression of the target genes. In some embodiments, heterologous promoters are used, since they generally allow higher transcription and higher yields of the expressed target gene compared to the natural target polypeptide promoter. Promoters suitable for use with prokaryotic hosts include the PhoA promoter, β-galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter or the trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published, thereby enabling an experienced technician to operatively link them to cistrons encoding the target light and heavy chains (Siebenlist et al (1980) Cell 20: 269) using linkers or adapters to deliver any required restriction sites .

In one aspect of the invention, each cistron within the recombinant vector comprises a secretion signal sequence component that directs the translocation of the expressed polypeptides through a membrane. In general, the signal sequence may be a component of the vector or it may be part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the natural signal sequences to the heterologous polypeptides, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group consisting of alkaline phosphatase, penicillinase, Ipp or leader Thermally stable enterotoxin II (STII), LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof. In another aspect, the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell and therefore does not require the presence of secretion signal sequences within each cistron. In this regard, heavy immunoglobulin light chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains (e.g., trxB strains of E. coli) provide cytoplasmic conditions that are favorable for disulfide bond formation, thereby allowing for proper folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159: 203 (1995). Appropriate prokaryotic host cells for expressing antibodies of the invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella. , Rhizobia, Vitreoscilla or Paracoccus. In one embodiment, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts for the invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol.2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; Deposit No. ATCC 27.325) and derivatives of the same, which include strain 33D3 that has the genotype W3110? fhuA (? tonA) ptr3 lac Iq lacL8? ompT? (nmpc-fepE) degP41 kanR (U.S. Patent No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli? 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the aforementioned bacteria having defined genotypes are known in the art and described for example in Bass et al., Proteins, 8: 309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration the replication capacity of the replicon in the cells of a bacterium. For example, the species of E. coli, Serratia or Salmonella can be appropriately used as the host when well known plasmids such as pBR322, pBR325, pACYC177 or pKN410 are used to deliver the replicon. Commonly, the host cell must secrete minimal amounts of proteolytic enzymes and additional protease inhibitors can desirably be incorporated into the cell culture. ii. Production of Antibodies The host cells are transformed with the expression vectors described above and cultured in modified conventional nutrient media as appropriate to induce promoters, select transformants or amplify the genes encoding the desired sequences. Transformation means introducing DNA to the prokaryotic host, in such a way that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, the transformation is done using standard techniques appropriate for such cells. The calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell wall barriers. Another method for transformation employs polyethylene glycol / DMSO. Still another technique used is electroporation. The prokaryotic cells used to produce the polypeptides of the invention are cultured in media known in the art and suitable for the culture of the selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutrient supplements. In some embodiments, the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for the culture of cells expressing ampicillin resistance gene. Any necessary supplements, in addition to carbon, nitrogen and inorganic phosphate sources can also be included at appropriate concentrations introduced alone or as a mixture with another complement or medium such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol. The prokaryotic host cells are cultured at appropriate temperatures. For the cultivation of E. coli, for example, the preferred temperature ranges from about 20 ° C to about 39 ° C, more preferably from about 25 ° C to about 37 ° C, even more preferably at around 30 ° C. . The pH of the medium can be any pH that ranges from about 5 to about 9, depending mainly on the host organism. For E. coli, the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0. If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions appropriate for promoter activation. In one aspect of the invention, PhoA promoters are used to control the transcription of the polypeptides. Thus, the transformed host cells are cultured in a phosphate-limiting medium for induction. Preferably, the phosphate-limiting medium is the C.R.A.P medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263: 133-147). A variety of other inducers can be used, according to the employed vector construct, as is known in the art. In one embodiment, the expressed polypeptides of the present invention are secreted to and recovered from the periplasm of host cells. Protein recovery commonly involves disrupting the microorganism, generally by means such as osmotic shock, sonication or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The proteins can be further purified, for example, by affinity resin chromatography. Alternatively, the proteins can be transported to the culture medium and isolated therein. The cells can be removed from the culture and the culture supernatant be filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods, such as polyacrylamide gel electrophoresis (PAGE) and Western blot analysis. In one aspect of the invention, antibody production is carried out in large quantity by a fermentation process. Several large-scale batch feeding fermentation procedures are available for production of recombinant proteins. Large scale fermentations have at least 1000 liters capacity, preferably around 1,000 to 100,000 liters capacity. These riggers use agitator thrusters to distribute oxygen and nutrients, especially glucose (the coal source / preferred energy). Small-scale fermentation generally refers to fermentation in a fermenter that is not more than about 100 liters in volumetric capacity and can range from about 1 liter to about 100 liters. In a fermentation process, the induction of protein expression is commonly initiated after the cells have been cultured under appropriate conditions at a desired density, for example, an OD550 of about 180-220, stage at which the cells are in a premature stationary phase. A variety of inducers can be used, according to the vector construct employed, as is known in the art and described above. Cells can be cultured for shorter periods before induction. The cells are usually induced for about 12-50 hours, although a longer or shorter induction time can be used. To improve the production and quality performance of the polypeptides of the invention, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of secreted antibody polypeptides, additional vectors overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and DsbG) or FkpA (a cis peptidylpropyl, trans-isomerase with chaperone activity) can be used to co-transform the prokaryotic host cells. Chaperone proteins have been shown to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274: 19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275: 17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275: 17106-17113; Arie et al. (2001) Mol. Microbiol. 39: 199-210. To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient in proteolytic enzymes can be used by the present invention. For example, host cell strains can be checked for genetic mutation (s) in genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some protease-deficient E. coli strains are available and are described in for example, Joly et al. (1998), supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Will et al., Microbial Microbial Drug Resistance, 2: 63-72 (1996). In one embodiment, strains of E. coli deficient by proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins are used as host cells in the expression system of the invention. iii. Purification of antibodies Standard protein purification methods known in the art can be used. The following procedures are exemplary of appropriate purification procedures: fractionation or immunoaffinity or ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, precipitation of ammonium sulfate and gel filtration using for example, Sephadex G-75. In one aspect, protein A immobilized on a solid phase is used for immunoaffinity purification of the full length antibody products of the invention. Protein A is a 41 kD cell wall protein of Staphyl'ococcus áureas that binds with high affinity to the Fc region of the antibodies. Lindmark et al (1983) J. Immunol. Meth. 62: 1-13. The solid phase to which protein A is immobilized is preferably a column consisting of a glass or silica surface, more preferably a controlled pore glass column or a silica column. In some applications, the column has been coated with a reagent, such as glycerol, in an attempt to prevent non-specific adhesion of contaminants. As the first purification step, the preparation derived from the cell culture as described above is applied on protein A immobilized on solid phase to allow specific binding of the antibody of interest to protein A. Then the solid phase is washed to remove contaminants not specifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution. b. Generation of antibodies using eukaryotic host cells: The vector components include in general, but are not limited to one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence. (i) Signal sequence component A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected is preferably one that is recognized or processed (ie, cleaved by a signal peptidase) by the host cell. In the expression of mammalian cells, the mammalian signal sequence also as viral secretory leaders, for example, the gD signal of herpes simplex are available. The DNA for such a precursor region is ligated in reading frame to DNA encoding the antibody. (ii) Origin of replication In general, an origin of replication component is not necessary for mammalian expression vectors. For example, the SV40 origin can be commonly used only because it contains the premature promoter. (iii) Selection of genetic component Expression and cloning vectors may contain a selection gene, also referred to as a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotropic deficiencies where relevant or (c) supply critical nutrients not available from media complex. An example of a selection scheme uses a drug to stop the growth of a host cell.

Those cells that are successfully transformed with a heterologous gene produce a protein that confers resistance to drug and thus survive the selection regime. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin. Another example of suitable selectable markers for mammalian cells are those that allow the identification of cells competent to absorb the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is used is the Chinese Hamster Ovary (CHO) cell line deficient in DHFR activity (eg, ATCC CRL-9096). Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild type DHFR protein or other selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected. by culturing the cell in a medium containing a selection agent by the selectable marker such as an antibiotic aminoglycoside, for example, kanamycin, neomycin or G418. See US Patent No. 4, 965,199. (iv) Composition of promoter Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid of the antibody polypeptide. Promoter sequences are known for eukaryotes. Virtually aleucarionic genes have an AT-rich region located approximately 25-30 bases upstream of the site where transcription begins. Another sequence found 70 to 80 bases upstream of the start of transcription of many genes is a CNCAAT region where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence which may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are properly inserted into eukaryotic expression vectors. The transformation of antibody polypeptides from vectors into mammalian host cells is controlled, for example by promoters obtained from the genomes of viruses such as polyoma virus, variola virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma, cytomegalovirus, a retrovirus, hepatitis B virus and simian virus 40 (SV40), from mammalian promoters heterologous, for example the actin promoter or an immunoglobulin promoter, of heat shock promoters, provided that such promoters are compatible with the host cell systems. The SV40 premature and late promoters are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. the immediate premature promoter of the human cytomegalovirus is conveniently obtained as a restriction fragment of HindIII E. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. Alternatively, the long terminal repeat of Rous Sarcoma virus can be used as the promoter. (v) Composition of the best element The transcription of the DNA encoding the antibody polypeptide of this invention by higher eukaryotes is frequently increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Commonly, however, a eukaryotic cell virus enhancer will be used. Examples include the SV40 enhancer on the back side of the origin of replication (bp 100-270), the cytomegalovirus premature promoter enhancer, the polyoma enhancer on the posterior side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982) regarding the method of enhancement for activation of eukaryotic promoters. The enhancer can be cleaved to the vector at a position 5 'or 3' to the coding sequence of the antibody polypeptide, but is preferably located at a 5 'site of the promoter. (vi) Transcription Termination Component Expression vectors used in eukaryotic host cells will also commonly contain sequences necessary for the termination of transcription and to stabilize the mRNA. Such sequences are commonly available from the 5 'region and occasionally the 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain segments of nucleotides transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody. A useful transcription termination component is the polyadenylation region of bovine growth hormone. See WO94 / 11026 and the expression vector disclosed therein. (vii) Selection and transformation of host cells Suitable host cells for cloning or expression of DNA in the vectors herein include higher eukaryotic cells described herein, in which vertebrate host cells are included. The propagation of vertebrate cells in the culture (tissue culture) has become a routine procedure. Examples of mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Uriaub et al., Proc. Nati, Acad. Sci. USA 77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); Kidney kidney cells (CV1 ATCC CCL 70); kidney cells of African green monkey (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cell (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

The host cells are transformed with the expression or cloning vectors described above for the production of antibody and cultured in modified conventional nutrient media as appropriate to induce promoters, select transformants or amplify the genes encoding the desired sequences. (viii) Cul tive of the host cells The host cells used to produce an antibody of the invention can be cultured in a variety of media. Commercially available media such as Ham 's FIO (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for growing host cells In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), US Patent Nos. 4,767,704; 4,657,866 4,927,762, 4,560,655, or 5,122,469, WO 90/03430, WO 87/00195, or US Patent Re. 30,985 can be used as culture media for host cells, either of which can be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), pH-regulating solutions (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN ™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to the skilled artisan. (ix) Purification of antibody When recombinant techniques are used, the antibody can be produced intracellularly or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the debris in particles, either host cells or lysis fragments are removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, the supernatants for such expression systems are generally concentrated first using a commercially available protein concentration filter, for example an Amicon or Millipore Pellicon filtration unit. A protease inhibitor such as PMSF can be included in any of the above steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, affinity chromatography is the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on heavy chains of human?,? 2 or? 4 (Lindmark et al., J. Immunol., Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human? 3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity ligand is attached is most frequently agarose, but other matrices are available. Mechanically stable matrices, such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be obtained with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX ™ resin (J.T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion exchange column, ethanol precipitation, phase HPLC Inverse, silica chromatography, heparin chromatography SEPHAROSE ™ chromatography or an anionic or cationic exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation are also available depending on the antibody to be recovered. Following any preliminary purification step (s), the mixture comprising the antibody of interest and contaminants can be subjected to hydrophobic interaction chromatography at low pH using a buffer solution at a pH of between about 2.5-4.5. , preferably carried out at low salt concentrations (for example, around 0-0.25 molar salt).

Immunoconjugates The invention also provides immunoconjugates (interchangeably called "antibody-drug conjugates" or "ADCs"), which comprises any of the anti-OX40L antibodies described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin or fragments thereof) or a radioactive isotope (i.e., a radioconjugate).

The use of antibody-drug conjugates for the local administration of cytotoxic or cytostatic agents, that is, drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Investigation 19: 605-614; Niculescu -Duvaz and Springer (1997) Adv. Drg Del. Rev. 26: 151-172; U.S. Patent 4,975,278) allows targeted administration of the drug portion to tumors and intracellular accumulation therein, wherein the systemic administration of these agents of unconjugated drug can result in unacceptable levels of toxicity to normal cells, as well as to the tumor cells to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986): 603-05 Thorpe, (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antibodies? 84: Biological and Clinical Applications, A. Pinchera et al. (Eds.), Pp. 475-506). The maximum efficacy with minimal toxicity is sought by this. Polyclonal antibodies and monoclonal antibodies useful in these strategies have been reported (Rowland et al., (1986) Cancer Immunol. Immunother., 21: 183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., (1986) supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin.

(Mandler et al (2000) Journal of the Nat. Cancer Inst. 92 (19): 1573-1581; Mandler et al (2000) Bioorganic &Med. Chem. Letters 10: 1025-1028; Mandler et al (2002) ) Bioconjugate Chem. 13: 786-791), maytansinoids (EP 1391213, Liu et al., (1996) Proc. Nati, Acad. Sci. USA 93: 8618-8623) and calicheamicin (Lode et al (1998) Cancer Res 58: 2928; Hinman et al (1993) Cancer Res. 53: 3336-3342). Toxins can exert their cytotoxic and cytostatic effects through mechanisms in which tubulin binding, DNA binding or topoisomerase inhibition are included. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands. ZEVALIN® (tiuxetan ibritumomab, Biogen / Idec) is an antibody-radioisotope conjugate composed of a murine IgGl kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and mIn or 90Y radioisotope linked through a linker- thiourea chelator (Wiseman et al (2000) Eur. Jour Nucí Med. 27 (7): 766-77; Wiseman et al (2002) Blood 99 (12): 4336-42; Witzig et al (2002) J Clin Oncol 20 (10): 2453-63; Witzig et al (2002) J. Clin Oncol 20 (15): 3262-69). Although ZEVALIN has activity against non-B-cell Hodgkin lymphoma (NHL), administration results in severe and prolonged cytopenias in the majority of patients. MYLOTARG ™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody-drug conjugate composed of a CD33 hu-linked antibody to calicheamicin, was approved in the year 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25 (7): 686; US Nos. 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Cantuzumab Mertansine (Immunogen, Inc.), an antibody-drug conjugate composed of the huC242 antibody bound via the disulfide linker SPP to the maytansinoid drug portion, DMl, is progressing to Phase II trials for the treatment of cancers expressing CanAg, such as colon, pancreatic, gastric and others. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen, Inc.), a drug-antibody conjugate composed of anti-prostate-specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety, DMl, is under development for the potential treatment of prostate tumors. The auristatin, auristatin E (AE) and monomethylauristatin (MMAE) peptides, synthetic analogs of dolastatin, were conjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Y in carcinomas) and cILO (specific to CD30 in hematological malignancies) (Doronina et al. (2003) Nature Biotechnology 21 (7): 778-784) and are under therapeutic development. Chemotherapeutic agents useful in the generation of immunoconjugates are described herein (e.g., previously) . Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, active fragments without diphtheria toxin binding, exotoxin A chain (from Pseudomonas aeruginosa), castor chain A, abrin chain A, modecina chain A, alpha-sarcina, proteins of Aleurites fordii, diantine proteins, proteins of Phytolaca americana (PAPI, PAPH and PAP-S), inhibitor of momordica charantia, curcin, crotina, inhibitor of sapaonaria officinalis, gelonin, mitogeline, restrictocin, fenomycin , enomycin and the trichothecenes. See, for example, WO 93/21232 published October 2, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90? and 186Re. Antibody and cytotoxic agent conjugates are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as apidimidate) dimethyl HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexandiamine), bis-diazonium derivatives (such as bis- (p. diazonium benzoyl) -ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a Castor immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). 14C-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminpentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotide to the antibody. See WO94 / 11026. Conjugates of an antibody and one or more small molecule toxins, such as calicheamicin, maytansinoids, dolastatins, aurostatin, a tricothecene and CC1065, and derivatives of these toxins having toxin activity, are also contemplated herein. i. Maytansine and Maytansinoids In some embodiments, the immunoconjugate comprises an antibody (full length or fragments) of the invention conjugated to one or more molecules of maytansinoids. Maytansinoids are mitotic inhibitors that act by inhibiting the polymerization of tubulin. Maytansine was isolated for the first time from the African bush Maytenus serrata (US Patent No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and maytansinol C-3 esters (US Patent No. 4,151,042). Synthetic synthetic malate and derivatives and analogs thereof are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533. The maytansinoid drug moieties are attractive drug moieties in antibody drug conjugates because they are: (i) relatively accessible to be prepared by fermentation or chemical modification, derivatization of fermentation products, (ii) prone to derivation with groups functional for conjugation by means of the disulfide-free linkers to antibodies, (iii) stable in plasma and (iv) effective against a variety of tumor cell lines. Immunoconjugates containing maytansinoids, methods of making them and their therapeutic use are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl, the disclosures of which are expressly incorporated herein by reference. present by reference. Liu et al., Proc. Nati Acad. Sci. USA 93: 8618-8623 (1996) describe immunoconjugates comprising a maytansinoid designated as DMl linked to monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic toward cultured colon cancer cells and showed antitumor activity in a tumor growth analysis in vivo. Chari et al., Cancer Research 52: 127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to murine antibody A7 that binds to an antigen on human colon cancer cell lines or to another TA murine monoclonal antibody. l which is linked to the HER-2 / neu oncogene. The cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro in the human breast cancer cell line SK-BR-3, which expresses 3 x 105 HER-2 surface antigens per cell. The drug conjugate obtained a degree of cytotoxicity similar to the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The conjugate of A7-maytansinoid showed low systemic cytotoxicity in mice. Antibody-maytansinoid conjugates are prepared by chemically binding an antibody to a maytansinoid molecule without significantly decreasing the biological activity of either the antibody or the "maytansinoid molecule." See, for example, U.S. Patent No. 5,208,020 (the disclosure of which is expressly incorporated herein by reference.) An average of 3-4 molecules - of maytansinoid conjugates per antibody molecule has shown efficacy in improving the cytotoxicity of target cells without adversely affecting the function or solubility of the antibody, although one would expect a toxin molecule / antibody improve cytotoxicity with respect to the use of the naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Patent No. 5,208,020 and in other patents and non-patent publications referred to hereinbefore. Preferred maytansinoids are maytansinol and maytansinol analogs modified in the aromatic ring or in other positions of the maytansinol molecule, such as various maytansinol esters. There are many linking groups known in the art to make antibody-maytansinoid conjugates, which include, for example, those disclosed in U.S. Patent No. 5,208,020 or EP 0 425 235 Bl, Chari et al., Cancer Research 52 : 127-131 (1992) and US patent application No. 10 / 960,602, filed on October 8, 2004, the disclosures of which are expressly incorporated herein by reference. Antibody-maytansinoid conjugates comprising the SMCC linker component can be prepared as disclosed in U.S. Patent Application No. 10 / 960,602, filed October 8, 2004. Binding groups include disulfide groups, thioether groups, groups labile acids, photolabile groups, labile peptidase groups or labile esterase groups, as disclosed in the patents identified above, disulfide groups and thioether groups are preferred. Additional linking groups are described and exemplified herein. Antibody and maytansinoid conjugates can be manufactured using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridylthio) propionate (SPDP), succinimidyl-4- (N-cyclohexan-1-carboxylate) -maleimidomethyl) (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl aphidimide HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexandiamine), bis-diazonium derivatives (such as bis- (p-diazonium benzoyl) -ethylenediamine), diisocyanates (such as 2,6-toluene diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

Particularly preferred coupling agents include N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) (Carlsson et al., Biochem. J. 173: 723-737 (1978)) and N-succinimidyl-4- (2 pyridylthio) pentanoate (SPP) to provide a disulfide bond. The linker can be attached to the maytansinoid molecule in various positions, depending on the type of linkage. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction can occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group and the C-20 position having a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or a maytansinol analogue. ii. Auristatins and dolastatins In some embodiments, the immunoconjugate comprises an antibody of the invention conjugated to dolastatins or analogues or peptide derivatives of dolostatin, auristatins (U.S. Patent Nos. 5635483; 5780588). It has been shown that dolastatins and auristatins interfere with microtubule dynamics, GTP hydrolysis and nuclear and cell division (Woyke et al (2001) Antimicrob Agents and Chemother 45 (12): 3580-3584) and have anticancer activity ( US 5663149) and antifungal (Pettit et al (1998) Antimicrob Agents Chemother 42: 2961-2965). The drug portion of dolastatin or auristatin can be attached to the antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02/088172). Exemplary auristatin moieties include the monomethylauristatin drug moieties linked to the N DE and DF terminology, disclosed in "Monomethylvaline Compounds.

Capable of Conjugation to Ligands ", US Patent Application No. 10 / 983,340, filed on November 5, 2004, the disclosure of which is expressly incorporated by reference in its entirety Commonly, portions of peptide-based drug may be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lubke , "The Peptides", volume 1, pp 76-136, 1965, Academic Press) which is well known in the field of peptide chemistry Portions of auristatin / dolastatin drug can be prepared according to methods of: US 5635483; US 5780588; Pettit et al (1989) J. Am. Chem. Soc. 111: 5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13: 243-277; Pettit, GR, et al., Synthesis, 1996, 719-725, and Pettit et al (1996) J. Chem. Soc. Perkin Trans. 1 5: 859-863. ase also Doronina (2003) Nat Biotechnol 21 (7): 778-784; "Monomethylvaline Compounds Capable of Conjugation to Ligands", US Ser. No. 10 / 983,340, filed November 5, 2004, incorporated herein by reference in its entirety (disclosing, for example, linkers and methods for preparing compounds of monomethylvaline such as MMAE and MMAF conjugated to linkers). iii. Calicheamycin In other embodiments, the immunoconjugate comprises an antibody of the invention conjugated to one or more calicheamicin molecules. The family of calicheamicin antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see US Patents 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all issued to American Cyanamid Company). Structural analogues of calicheamicin that can be used include, but are not limited to, yp, a2I, a3p N-acetyl-Yi1, PSAG and p (Hinman et al., Cancer Research 53: 3336-3342 (1993), Lode et al. al., Cancer Research 58: 2925-2928 (1998) and the aforementioned US patents issued to American Cyanamid). Another anti-tumor drug to which the antibody can be conjugated is QFA which is an antifoliato. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Accordingly, the cellular uptake of these agents by means of moderate internalization by antibody greatly improves their cytotoxic effects. iv. Other cytotoxic agents Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents commonly known as LL-E33288 complex described in US Patents 5,053,394, 5,770,710, also as esperamycins (US patent 5,877,296). Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, active fragments without diphtheria toxin binding, exotoxin A chain (from Pseudomonas aeruginosa), castor chain A, abrin chain A, modecina chain A, alpha-sarcina, Aleurites fordii proteins, diantine proteins, Phytolaca americana proteins (PAPI, PAPH and PAP-S), inhibitor of momordica charantia, curcinia, crotina, inhibitor of sapaonaria officinalis, gelonin, mitogeline, restrictocin, fenomycin, enomycin and the trichothecenes. See, for example, WO 93/21232 published October 28, 1993. The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as deoxyribonuclease; ). For the selective destruction of the tumor, the antibody can comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used to detection, may comprise a radioactive atom from scintigraphic studies, for example tc99m or I123 or a spin marker for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as 123I, again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Radio-markers or other markers can be incorporated into the conjugate in several known ways. For example, the peptide can be biosynthesized or can be synthesized by chemical amino acid synthesis using appropriate amino acid precursors that involve, for example, fluorine-19 instead of hydrogen. Markers- such as tc99m or I123, Re186, Re188 and In111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. "Monoclonal Antibodi in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail. Antibody and cytotoxic agent conjugates can be manufactured using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridylthio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexan- 1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl apidimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexandiamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as 2,6-toluene diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4). -dinitrobenzene). For example, a castorium immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). The l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) labeled with carbon 14 is an exemplary chelating agent for the conjugation of the radionucleotide to the antibody. See WO94 / 11026. The linker can be a "spiked linker" that facilitates the release of the cytotoxic drug into the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992); US Patent No. 5,208,020) may be used. The compounds of the invention expressly contemplate, but are not limited to, ADC prepared with crosslinking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo -EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB (succinimidyl- (4-vinylsulfone) benzoate) which are commercially available (for example, from Pierce Biotechnology, Inc., Rockford, IL., U.S. A). See pages 467-498, 2003-2004 Applications Handbook and Catalog. v. Preparation of antibody drug conjugates In the antibody-drug conjugates (ADCs) of the invention, an antibody (Ab) is conjugated to one or more drug (D) portions, for example about 1 to about 20 portions of drug per antibody, by means of a linker (L). The ADC of formula I can be prepared by several routes, employing reactions of organic chemistry, conditions and reagents known to e skilled in the art, in which is included: (1) reaction of a nucleophilic group of an antibody with a reagent of bivalent linker, to form Ab-L, via a covalent bond, followed by reaction with a portion of drug D; and (2) reaction of a nucleophilic group of a drug moiety with a divalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic moiety of an antibody. Additional mes for preparing ADCs are described herein. Ab- (L-D) p I The linker can be composed of one or more linker components. Components of exemplary linkers include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzoyloxycarbonyl ("PAB"), pentanoate N-succinimidyl 4- (2-pyridylthio) ("SPP"), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC") and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB") "). Additional linker components are known in the art and some are described herein. See also "Monomethylvaline Compounds Capable of Conjugation to Ligands", US patent application Serial No. 10 / 983,340, filed on November 5, 2004, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the linker may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly) • The amino acid residues that comprise an amino acid linker component include e that occur stably in nature, also as minor amino acids and amino acid analogs that do not occur stably in nature, such as citrulline. The amino acid linker components can be designed and optimized in its selectivity for enzymatic cleavage by particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D or a plasmin protease. Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, for example lysine, (iii) side chain thiol groups, for example cysteine and (iv) hydroxyl sugar or amino groups where the antibody is glycosylated. The amine, thiol and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker portions and linker reagents in which are included: (i) active esters such as NHS esters, HOBt esters, haloformates and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl and maleimide groups. Certain antibodies have interchain chain disulfides that can be reduced, for example, cysteine bridges. The antibodies can be reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced to antibodies by the reaction of pots with 2-iminothiolane (Traut's reagent) resulting in the conversion of an amine to a thiol. The reactive thiol groups can be introduced to the antibody (or fragment thereof) by introducing one, two, three, four or more cysteine residues (e.g., preparation of mutant antibodies comprising one or more unnatural cysteine amino acid residues). The antibody-drug conjugates of the invention can also be produced by modifying the antibody to introduce electrophilic portions, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of the glycosylated antibodies can be oxidized, for example with periodate oxidizing reagents, to form aldehyde groups or ketones that can react with the amine group of linker reagents or drug portions. The resulting imine Schiff base groups can form a stable bond or can be reduced, for example by borohydride reagents to form stable amine bonds. In one embodiment, the reaction of the carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium meta-periodate can produce carbonyl groups (aldehyde and ketone) in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in the production of an aldehyde in place of the first amino acid (Geoghegan &Stroh, (1992) Bioconjugate Chem. 3: 138-146; US 5362852). Such an aldehyde can be reacted with a drug or nucleophilic linker moiety. Also, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on portions of linker and linker reagents in which are included: (i) active esters such as NHS esters, HOBt esters, haloformates and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl and maleimide groups. Alternatively, a fusion protein comprising the antibody and cytotoxic agent can be manufactured, for example, by recombinant techniques or peptide synthesis. The length of the DNA may comprise respective regions that encode the two portions of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate. In yet another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) to use in pre-targeting of tumor wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearance agent and then administration of a "ligand" (eg, avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Pharmaceutical Formulations Therapeutic formulations comprising an antibody of the invention are prepared for storage by mixing the antibody having the desired degree of purity with acceptable physiologically optional carriers, excipients or stabilizers - (Remington: The Science and Pra cti ce of Pharmacy 20th ed. 2000)), in the form of aqueous solutions, freeze-dried or other dry formulations. Acceptable carriers, excipients or stabilizers are non-toxic to the receptors at the dosages and concentrations employed and include pH regulating solutions such as phosphate, citrate, histidine and other organic acids; antioxidants in which ascorbic acid and methionine are included; preservatives (such as octadecyl dimethylbenzyl 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 polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates in which glucose, mannose or dextrins are included; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and / or non-ionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG). 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. Such molecules are appropriately present in combination in amounts that are effective for the intended purpose. The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethyl cellulose or gelatin-microcapsule and poly-microcapsule (methyl methacrylate), respectively, in colloidal drug delivery systems (eg example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington: The Science and Practice of Pharmacy 20th edition (2000). The formulations to be used for in vivo administration must be sterile. This is easily carried out by filtration through sterile filtration membranes. Sustained-release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin of the invention, such matrices being in the form of articles formed, eg, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and? Ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ™ microspheres (injectable microspheres composed of lactic acid-glycolic acid and leuprolide acetate copolymer) and polyhydric acid D- (-) -3-hydroxybutyric. Whereas polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time. When the encapsulated immunoglobulins remain in the body for a long time, they can be denatured or added as a result of exposure to moisture at 37 ° C, resulting in losses of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if it is dvered that the aggregation mechanism is removal of intermolecular SS bond by means of thio-disulfide exchange, stabilization can be obtained by modifying the sulfhydryl residues, lyophilization of acid solutions, control of moisture content, use of appropriate additives and development of specific polymer matrix compositions.

Uses An antibody of the present invention can be used for example, in in vitro, ex vivo and in vivo therapeutic methods. In one aspect, the invention provides methods for the treatment or prevention of a tumor, a cancer and / or a cell proliferative disorder associated with the expression and / or increased activity of EphB4, the methods comprising administration of an effective amount of an anti-EphB4 antibody to a subject in need of such treatment. In one aspect, the invention provides methods for reducing, inhibiting or preventing the growth of a tumor or cancer, the methods comprising administering an effective amount of an anti-EphB4 antibody to a subject in need of such treatment. EphB4 has been implicated in motoaxon guidance and neural crest cell migration in the developing embryo. Thus, the antibodies of the invention are also useful in the treatment (including prevention) of disorders the pathology of which involves degeneration or cellular dysfunction, such as treatment of several neurodegenerative (chronic) disorders and acute nerve cell lesions. Such neurodegenerative disorders include, without limitation, peripheral neuropathies; motor neuron disorders, such as amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Bell's palsy and various conditions involving spinal muscular atrophy or paralysis; and other human neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's chorea, Down's syndrome, nerve deafness and Meniere's disease and acute nerve cell injuries, for example due to trauma or spinal cord injury.

The antibodies of the invention are also useful for inhibiting angiogenesis. In some embodiments, the site of angiogenesis is a tumor or cancer. In one aspect, the invention provides methods for inhibiting angiogenesis which comprises administering an effective amount of an anti-EphB4 antibody to a subject in need of such treatment. In one aspect, the invention provides methods for the treatment of a pathological condition associated with angiogenesis comprising administering an effective amount of an anti-EphB4 antibody to a subject in need of such treatment. In some embodiments, the pathological condition associated with angiogenesis is a tumor, a cancer and / or a cell proliferative disorder. In some embodiments, the pathological condition associated with angiogenesis is an intraocular neovascular disease. In addition, at least some of the antibodies of the invention can bind to the antigen of another species. Thus, the antibodies of the invention can be used to bind to specific antigen activity, for example, in a cell culture containing the antigen, in human subjects or in other mammalian subjects having the antigen with which an antibody of the invention reacts cross way (for example, chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse). In one embodiment, the antibody of the invention can be used to inhibit antigen activities by contacting the antibody with the antigen, in such a way that the antigen activity is inhibited. Preferably, the antigen is a human protein molecule. In one embodiment, an antibody of the invention can be used in a method for binding an antigen in a subject suffering from an alteration associated with increased antigen expression and / or activity, which comprises administering to the subject an antibody of the invention, in such a way that the antigen in the subject is linked. Preferably, the antigen is a human protein molecule and the subject is a human subject. Alternatively, the subject may be a mammal expressing the antigen with which an antibody of the invention binds. Still further, the subject may be a mammal to which the antigen has been introduced (for example, by administration of the antigen or by expression of an antigen transgene). An antibody of the invention can be administered to a human subject for therapeutic purposes. In addition, an antibody of the invention can be administered to a non-human mammal expressing an antigen with which the immunoglobulin cross-reacts (eg, a primate, pig or mouse) for veterinary purposes or as an animal model of human disease . With respect to the latter, such animal models can be useful for evaluating the therapeutic efficacy of the antibody of the invention (eg, dosing tests and courses of administration time). The antibodies of the invention can be used to treat, inhibit, retard the progress of, prevent / retard the recurrence of, ameliorate or prevent diseases, alterations or conditions associated with the expression and / or activity of one or more antigen molecules. Exemplary alterations include carcinoma, lymphoma, blastoma, sarcoma and leukemia and lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer in which small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and carcinoma are included. flaky lung, peritoneal cancer, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary system cancer, hepatoma , breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma , melanoma, multiple myeloma and B-cell lymphoma, cancer brain, also as head and neck cancer and associated metastases. In some embodiments, the cancer is selected from the group consisting of small cell lung cancer, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. In certain embodiments, an immunoconjugate comprising an antibody conjugated to one or more cytotoxic agents is administered to the patient. In some embodiments, the immunoconjugate and / or antigen to which it is linked is / are internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in the extermination of the target cell to which it is linked. In one embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the target cell. In one embodiment, the cytotoxic agent targets or interferes with microtubule polymerization. Examples of such cytotoxic agents include any of the chemotherapeutic agents indicated herein (such as a maytansinoid, auristatin, dolastatin, or a calicheamicin), a radioactive isotope, a ribonuclease, or a DNA endonuclease. The antibodies of the invention can be used either alone or in combination with other compositions in a therapy. For example, an antibody of the invention can be co-administered with another antibody, chemotherapeutic agent (s) (in which cocktails are included). chemotherapeutic agents), other cytotoxic agent (s), anti-angiogenic agent (s), cytokines and / or growth inhibitory agent (s). Where an antibody of the invention inhibits tumor growth, it may be particularly desirable to combine it with one or more other therapeutic agent (s) which also inhibits tumor growth. Alternatively or additionally, the patient may receive combination radiation therapy (e.g., external beam irradiation or therapy with a radioactive labeled agent, such as an antibody). Such combination therapies indicated above include the combined administration (wherein the two or more agents are included in the same formulation or separate formulations) and separate administration, in which case, the administration of the antibody of the invention may occur before and / or after administration. the administration of the therapy or adjunct therapies.

Combination Therapies As indicated above, the invention provides combined therapies, in which an anti-EphB4 antibody is administered with another therapy. For example, anti-EphB4 antibodies are used in combinations with anti-cancer therapeutic or anti-neovascularization therapeutics to treat various neoplastic or non-neoplastic conditions. In one modality, the neoplastic or non-neoplastic condition is characterized by a pathological alteration associated with aberrant or undesirable angiogenesis. The anti-EphB4 antibody can be administered serially or in combination with another agent that is effective for those purposes, either in the same composition or as separate compositions. Alternatively or additionally, multiple anti-EphB4 inhibitors can be administered. The administration of the anti-EphB4 antibody can be done simultaneously, for example as a single composition or as two or more different compositions using the same route or different administration routes. Alternatively or additionally, the administration can be done sequentially, in any order. In certain modalities, ranges that range from minutes to days, to weeks to months, may be present between the administrations of the two or more compositions. For example, the anti-cancer agent can be administered first, followed by the anti-EphB inhibitor. However, simultaneous administration or administration of the anti-EphB4 antibody first is also contemplated. The effective amounts of therapeutic agents administered in combination with an anti-EphB4 antibody will be at the discretion of the physician or veterinarian. The administration of dosage and adjustment is made to obtain the maximum handling of the conditions to be treated. The dose will additionally depend on factors such as the type of agent therapeutic to be used and the specific patient that is treated. Appropriate dosages for the anti-cancer agent are those currently used and may be decreased due to the combined action (synergy) of the anti-cancer agent and the anti-EphB4 antibody. In certain embodiments, the combination of the inhibitors enhances the efficacy of a single inhibitor. The term "potency" refers to an improvement in the efficacy of a therapeutic agent such as its common or approved dose. Commonly, anti-EphB4 antibodies and anti-cancer agents are appropriate for the same or similar diseases to block or reduce a pathological condition such as tumor growth or cancer cell growth. In one embodiment, the anti-cancer agent is an anti-angiogenesis agent. Anti-angiogenic therapy in relation to cancer is a cancer treatment strategy aimed at inhibiting the development of tumor blood vessels required to provide nutrients to support tumor growth. Because angiogenesis is involved in both primary tumor growth and metastasis, the anti-angiogenic treatment provided by the invention is capable of inhibiting the neoplastic growth of the tumor at the primary site, as well as preventing the metastasis of tumors at secondary sites, consequently allowing the attack of tumors by other therapeutic ones.

Many anti-angiogenic agents have been identified and are known in the art, which include those listed herein, for example listed in Definitions and for example Carmeliet and Jain, Nature 407: 249-257 (2000); Ferrara et al., Nature Reviews: Drug Discovery, 3: 391-400 (2004) and Sato Int. J. Clin. Oncol., 8: 200-206 (2003). See also, US patent application US20030055006. In one embodiment, an anti-EphB4 antibody is used in combination with a neutralizing anti-VEGF antibody (or fragment) and / or another VEGF antagonist or VEGF receptor antagonist, which include, but are not limited to, example, fragments of soluble VEGF receptor (eg VEGFR-1, VEGFR-2, VEGFR-3, neuropilins (eg, NRP1, NRP2)), aptamers capable of blocking VEGF or neutralizing anti-VEGR antibodies, low molecular weight inhibitors of VEGR tyrosine kinases (RTK), anti-sense VEGF strategies, ribozymes against VEGF or VEGF receptors, VEGF antagonist variants and any combinations thereof, alternatively or additionally, two or more angiogenesis inhibitors can optionally be -administered to the patient in addition to the VEGF antagonist and another agent. In certain embodiments, one or more additional therapeutic agents, for example anti-cancer agents, may be administered in combination with the anti-EphB4 antibody, the VEGF antagonist and an anti-angiogenesis agent.

In certain aspects of the invention, other therapeutic agents useful for tumor therapy combined with an anti-EphB4 antibody include other cancer therapies (e.g., surgery, radiological treatments (e.g., involving irradiation or administration of radioactive substances), chemotherapy, treatment with anticancer agents listed herein and known in the art or combinations thereof). alternatively or additionally, two or more antibodies that bind to the same or two or more different antigens disclosed herein may be coadministered to the patient. Sometimes, it may be beneficial to administer one or more cytokines to the patient.

Chemotherapeutic Agents In certain aspects, the invention provides a method for blocking or reducing the tumor growth or growth of a cancer cell, by administering effective amounts of an EphB4 antagonist and / or angiogenesis inhibitor (s) and one or more chemotherapeutic agents to a patient susceptible to or diagnosed with cancer. A variety of chemotherapeutic agents can be used in combined treatment methods of the invention. An exemplary and non-limiting list of chemotherapeutic agents contemplated herein is provided herein in "Definitions." As will be understood by those of ordinary skill in the art, appropriate doses of chemotherapeutic agents will be in general around those already employed in clinical therapies, wherein the chemotherapeutics are administered alone or in combination with other chemotherapeutics. The variation in dosage will probably be presented depending on the condition being treated. The doctor administering the treatment will be able to determine the appropriate dose for the individual subject.

Relapse tumor growth The invention also provides methods and compositions for inhibiting or preventing the relapse of tumor growth or cancer cell growth relapse. Tumor growth of relapse or relapse cancer cell growth is used to describe a condition in which patients suffering or being treated with one or more currently available therapies (e.g. cancer therapies, such as chemotherapy, radiation, surgery, hormonal therapy and / or biological therapy / immunotherapy, anti-EphB4 antibody therapy, particularly a standard therapeutic regimen for the particular cancer) is not clinically appropriate to treat patients or are no longer receiving any beneficial effects of therapy , such that these patients need additional effective therapy. As used herein, the phrase may also refer to a condition of the patient "unresponsive / refractory", for example describing patients who respond to therapy but still suffer from side effects, develop resistance, do not respond to therapy , do not respond satisfactorily to therapy, etc. In various embodiments, a cancer is tumor relapse growth or relapse cancer cell growth, wherein the number of cancer cells has not been significantly reduced or increased or the tumor size has not been significantly reduced or has increased or fails any additional reduction in size or number of cancer cells. The determination of whether cancer cells are relapse tumor growth or relapse cancer cell growth can be done either in vivo or in vitro by any method known in the art to analyze the effectiveness of treatment in cancer cells, using the meanings accepted in the art of "relapse" or "refractory" or "unresponsive" in such a context. A tumor resistant to anti-VEGF treatment is an example of a relapse tumor growth. The invention provides methods for blocking or reducing tumor growth from relapse or cancer cell growth relapse in a subject by administering one or more anti-EphB4 antibodies to block or reduce tumor growth from relapse or cell growth. relapse cancer in a subject. In certain embodiments, the antagonist may be administered subsequent to the cancer therapeutic. In certain embodiments, the anti-EphB4 antibody is administered concurrently with cancer therapy. Alternatively or additionally, the anti-EphB4 antibody therapy alternates with another cancer therapy, which can be performed in any order. The invention also encompasses methods for administering one or more inhibitory antibodies to prevent the onset or recurrence of cancer in patients predisposed to have cancer. In general, the subject was or is concurrently suffering from cancer therapy. In one embodiment, cancer therapy is treatment with an anti-angiogenesis agent, for example a VEGF antagonist. The anti-angiogenesis agent includes those known in the art and those found in the Definitions herein. In one embodiment, the anti-angiogenesis agent is a neutralizing anti-VEGF antibody or fragment (e.g. A4.6.1 humanized, AVASTATIN® (Genentech, South San Francisco, CA), Genentech, South San Francisco, CA), Y0317, M4 , G6, B20, 2C3, etc.). See for example, U.S. Patents 6,582,959, 6,884,879, 6,703,020; W098 / 45332; WO 96/30046; WO94 / 10202; EP 0666868B1; US patent applications 20030206899, 20030190317, 20030203409 and 20050112126; Popkov et al., Journal of Immunological Methods 288: 149-164 (2004) and WO2005012359. Additional agents can be administered in combination with the VEGF antagonist and an anti? -EphB4 antibody to block or reduce tumor growth from relapse or relapse cancer cell growth, for example, see section entitled Combination Therapies herein. The antibody of the invention (and adjunctive therapeutic agent) is / are administered by any appropriate routes, which include parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal and if desired for local treatment intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, the antibody is administered appropriately by pulse infusion, particularly with declining doses of the antibody. The dosage can be by any appropriate route, for example by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. The antibody composition of the invention was formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular alteration that is treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disturbance, the site of administration of the agent, the method of administration, the administration schedule and other known factors for the experienced doctors. The antibody need not be but is optionally formulated with one or more agents currently used to prevent or treat the disease in question. The effective amount of such other agents depends on the amount of antibodies of the invention present in the formulation, the type of alteration or treatment and other factors discussed above. These are generally used in the same dosages and with routes of administration as used hereinabove or from about 1 to 99% of the dosages hitherto employed. For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with other agents such as chemotherapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, if the antibody is administered for preventive or therapeutic purposes, prior therapy, the patient's medical history and antibody response and the discretion of the attending physician. The antibody is administered appropriately to the patient once or in a series of treatments. Depending on the type and severity of the disease, approximately 1 μg / Kg at 15 μg / Kg (for example 0.1 mg / Kg-10 mg / Kg) of antibody is an initial candidate dosage for administration to the patient, if for example by one or more separate administrations or by continuous infusion.

A typical daily dosage could range from about 1 μg / Kg to 100 mg / Kg or more, depending on the factors mentioned above. For repeated administrations in several days or longer, depending on the condition, the treatment is sustained until the desired suppression of the symptoms of the disease occurs. An exemplary dosage of the antibody would be in the range of about 0.05 mg / Kg to about 10 mg / Kg. Thus, one or more doses of approximately 0.5 mg / Kg, 2.0 mg / Kg 4.0 mg / Kg or 10 mg / Kg (or any combination thereof) can be administered to the patient. Such doses may be administered intermittently, for example every week or every three weeks (for example, such that the patient receives from about two to about twenty, for example about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. An exemplary dosage regimen comprises administering an initial loading dose of about 4 mg / Kg, followed by a weekly maintenance dose of about 2 mg / Kg of the antibody. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and analyzes. The anti-EphB4 antibodies of the invention are useful in assays that detect the expression of EphB4 (such as diagnostic or prognostic analysis) in specific cells or tissues, wherein the antibodies are labeled as described hereinafter and / or are immobilized in an insoluble matrix. In another aspect, the invention provides methods for detecting EphB4, the methods comprising detecting the EphB4-anti-EphB4 antibody complex in the sample. The term "detection" as used herein includes qualitative or quantitative detection (measuring levels) with or without reference to a control or control. In another aspect, the invention provides methods for diagnosing an alteration associated with the expression and / or activity of EphB4, the methods comprising detecting the EphB4-anti-EphB4 antibody complex in a biological sample from a patient or being suspected of having the alteration. . In some embodiments, the expression of EphB4 is increased expression or abnormal (undesirable) expression. In some embodiments, the alteration is a tumor, cancer and / or cell proliferative alteration. In another aspect, the invention provides any of the anti-EphB4 antibodies described herein, wherein the anti-EphB4 antibody comprises a detectable label. In another aspect, the invention provides a complex of any of the anti-EphB4 antibodies described in present and EphB4. In some modalities, the complex is in vivo or in vitro. In some embodiments, the complex comprises a cancer cell. In some embodiments, the anti-EphB4 antibody is detectably labeled. Anti-EphB4 antibodies can be used for the detection of EphB4 in any of a variety of well-known detection analysis methods. For example, a biological sample can be analyzed for EphB4 by obtaining a sample from a desired source, mixing the sample with anti-EphB4 antibody and allowing the antibody to form the antibody / EphB4 complex with any EphB4 present in the mixture and detect any antibody / EphB4 complex present in the mixture. The biological sample can be prepared for analysis by methods known in the art that are appropriate for the particular sample. The methods of mixing the sample with antibodies and methods for detecting the antibody complex / EphB4 are chosen according to the type of analysis used. Such analyzes include immunochemistry, competitive analysis and sandwich analysis and spherical inhibition analysis. Analytical methods for EphB4 all use one or more of the following reagents: labeled EphB4 analog, immobilized EphB4 analog, labeled anti-EphB4 antibody, immobilized anti-EphB4 antibody and spherical conjugates. The marked reagents are also known as "tracers".

The label used is any detectable functionality that does not interfere with the binding of EphB4 and anti-EphB4 antibody. Numerous markers are known for use in immunoassays, examples include portions that can be detected directly, such as fluorochrome, chemiluminescent and radioactive markers, as well as portions such as enzymes that must be reacted or derived to be detected. Examples of such labels include: The label used is any detectable functionality that does not interfere with the binding of EphB4 and anti-EphB4 antibody. Numerous markers are known for use in immunoassays, examples include portions that can be detected directly, such as fluorochrome, chemiluminescent and radioactive markers, as well as portions such as enzymes that must be reacted or derived to be detected. Examples of such labels include 32P, 14C, 125I, 3H and 131I radioisotopes, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives dansyl, umbelliferote, luciferases, for example firefly luciferase and bacterial luciferase (patent US 4,737,456), luciferin, 2,3-dihydrophthalazineadiones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozine, saccharide oxidases, for example glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, oxidases heterocyclics such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin / avidite, spin markers, bacteriophage markers, stable free radicals and the like. Conventional methods are available to bind these markers covalently to proteins or polypeptides. For example coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzothidine and the like can be used to label the antibodies with the fluorescent, chemiluminescent and enzymatic labels described above. See, for example, US patents 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al. , Na ture, 144: 945 (1962); David et al. , Biochemistry, 13: 1014-1021 (1974); Pain et al. , J. Immunol. Methods, 40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem. , 30: 407-412 (1982). Preferred markers herein are enzymes such as horseradish peroxidase and alkaline phosphatase. The conjugation of such a marker, including enzymes, to the antibody is a standard handling procedure for the ordinarily experienced in immunoassay techniques. See, for example, O'Sullivan et al. , "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J.J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166. The immobilization of reagents is required for certain methods of analysis. Immobilization comprises separating the anti-EphB4 antibody from any EphB4 that remains free in solution. This is conventionally effected either by insolubilizing the ahti-EphB4 antibody or EphB4 analog before the analysis procedure, such as by adsorption to a water or surface insoluble matrix (Bennich et al., US Patent 3,720,760) or by insolubilizing the anti-EphB4 antibody or EphB4 analog after this, for example by immunoprecipitation. The expression of proteins in a sample can be examined using immunohistochemistry and dyeing protocols. It has been demonstrated that immunohistochemical staining of tissue sections is a reliable method to determine or detect the presence of proteins in a sample. Immunohistochemical techniques ("IHC") use an antibody to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods. For sample preparation, a tissue or sample of a mammalian cell (commonly a human patient) can be used. Examples of samples include, but are not limited to, cancer cells such as cancer cells of colon, breast, prostate, ovaries, lung, stomach, pancreas, lymphoma and leukemia. The The sample can be obtained by a variety of methods known in the art, including, but not limited to, surgical excision, aspiration or biopsy. The tissue can be new or frozen. In one embodiment, the sample is fixed and imbibed in paraffin or the like. The tissue sample can be fixed (that is, preserved) by conventional methodology. Those of ordinary skill in art will appreciate that the choice of a fixative is determined by the purpose for which the sample is to be stained histologically or analyzed in another way. Those of ordinary skill in the art will also appreciate that the duration of fixation depends on the size of the tissue sample and the fixing agent used. The IHC can be performed in combination with additional techniques, such as morphological staining and / or fluorescence in situ hybridization. Two general methods of IHC are available: direct and indirect analyzes. According to the first analysis, the binding of the antibody to the target antigen (for example EphB4) is determined directly. This direct analysis uses a labeled reagent, such as a fluorescent label or an enzyme-labeled primary antibody, which can be visualized without additional antibody interaction. In a typical indirect assay, the primary antibody is to conjugate is bound to the antigen and then a labeled secondary antibody is bound to the primary antibody. Where the secondary antibody is conjugated to an enzymatic label, a chromogenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody. The primary and / or secondary antibody used for immunohistochemistry will commonly be labeled with a detectable portion. Numerous markers are available that can be grouped in general into the following categories: In addition to the sample preparation procedures discussed above, additional treatment of the tissue section before, during or after IHC may be desirable. For example, epitope recovery methods, such as heating the tissue sample in a citrate pH buffer can be carried out (see, for example, Leong et al., Appl. Immunohistochem 4 (3): 201 (nineteen ninety six) ) . Following an optional blocking step, the tissue section is exposed to the primary antibody for a sufficient period of time and under appropriate conditions, such that the primary antibody binds to the target protein antigen in the tissue sample. Appropriate conditions for obtaining this can be determined by routine experimentation. The extent of antibody binding to the sample is determined by using any of the detectable markers discussed above. Preferably, the label is an enzymatic label (e.g., HRPO) that catalyzes a chemical alteration of the chromogenic substrate such as a 3,3 '-diaminobenzidine chromogen. Preferably, the enzyme label is conjugated to antibody that specifically binds to the primary antibody (e.g. the primary antibody is rabbit polyclonal antibody and the secondary antibody is goat anti-rabbit antibody). The samples thus prepared can be assembled and covered by coverslips. The evaluation of the slide is then determined for example using a microscope and dye intensity criteria used systematically in the art can be employed. The criteria of intensity of dyeing can be evaluated as follows: Table 2 Commonly, a staining pattern score of approximately 2+ or higher in an HIC analysis is diagnostic and / or prognostic. In some modalities, a dye pattern score of approximately 1+ or higher that diagnosis or prognosis. In other modalities, a dyeing pattern score of about 3+ or higher that diagnosis or prognosis. It will be understood that when the cells and / or tissue of a colon tumor or adenoma are examined using IHC, staining is generally determined in the tumor and / or tissue cell (as opposed to stromal tissue or the surrounding area which may be present in the sample). Other methods of analysis, known as competitive or sandwich analysis, are well established and widely used in the commercial diagnostic industry. Competitive analyzes depend on the ability of an EphB4 tracer analog to compete with the EphB4 of the test sample for a limited number of anti-EphB4 antibody antigen binding sites. The anti-EphB4 antibody is generally insolubilized before or after competition and then the tracer and EphB4 bound to the anti-EpriB4 antibody are separated from the tracer and EphB4 unbound. This separation is carried out by decanting (where the binding pattern was pre-insolubilized) or by centrifugation (where the binding partner was precipitated after the competitive reaction). The amount of the EphB4 test sample is inversely proportional to the amount of bound tracer, as measured by the amount of the marking substance. Curves of dose-response with known amounts of EphB4 are prepared and compared with the test results to quantitatively determine the amount of EphB4 present in the test sample. These tests are called ELISA systems when enzymes are used as the detectable markers. Another kind of competitive analysis, called "homogeneous" analysis, does not require phase separation. Here, a conjugate of an enzyme with EphB4 is prepared and used, such that when the anti-EphB4 antibody binds to EphB4 in the presence of the anti-EphB4 antibody, it modifies the enzymatic activity. In this case, the EphB4 or its immunologically active fragments are conjugated with a bifunctional organic bridge to an enzyme such as peroxidase. The conjugates are selected for use with the anti-EphB4 antibody, such that the binding of the anti-EphB4 antibody inhibits or enhances the enzymatic activity of the label. This method is per se practiced widely under the name of EMIT. Spherical conjugates are conjugated in spherical impediment methods for homogeneous analysis. These conjugates are synthesized by covalently linking a low molecular weight hapten to a small EphB4 fragment, such that the antibody to the hapten is substantially inept to bind to the conjugate at the same time as the anti-EphB4 antibody. Under this method of analysis, the EphB4 present in the test sample will bind to the anti-EphB4 antibody, thereby allowing the anti-hapten to bind to the conjugate, resulting in a change in the character of the conjugated hapten, for example a change in fluorescence when the hapten is a fluorophore. Sandwich assays are particularly useful for the determination of EphB4 or anti-EphB4 antibodies. In the sequential sandwich analysis an immobilized anti-EphB4 antibody is used to adsorb the EphB4 from the test sample, the test sample is removed such as by washing, the bound EphB4 is used to adsorb a labeled second anti-EphB4 antibody and then the bound material is separated from the residual tracer. The amount of bound tracer is directly proportional to the EphB4 test sample. In "simultaneous" sandwich analyzes, the test sample is not separated before adding the labeled anti-EphB4. A sequential sandwich analysis using an anti-EphB4 monoclonal antibody as an antibody and a polyclonal anti-EphB4 antibody as the other is useful for testing samples for EphB4. The above are only exemplary detection analyzes for EphB4. Other methods developed now or later that use anti-EphB4 antibody for the determination of EphB4 are included within the scope of the present, including the bioanalyses described herein.

Articles of manufacture In another aspect of the invention there is provided an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the alterations described above. The article of manufacture comprises a container and a package label or insert in or associated with the container. Suitable containers include, for example, bottles, bottles, syringes, etc. The containers can be formed from a variety of materials such as glass or plastic. The container contains a composition that is by itself or when combined with other composition (s) effective to treat, prevent and / or diagnose the condition and may have a sterile access gate (for example, the container may be a bag or bag of intravenous solution having a plug pierced by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the condition of choice, such as cancer. In addition, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention and (b) a second container with a composition contained therein, wherein the The composition comprises an additional cytotoxic agent. The article of manufacture in this modality of the invention may further comprise a package insert which indicates that the first and second antibody compositions can be used to treat a particular condition, for example cancer. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable pH buffer solution, such as bacteriostatic water for injection (BWFI), pH regulated phosphate outlet solution, Ringer's solution and dextrose solution. It may also include other materials from a commercial and user's point of view, which include other pH-regulating solutions, diluents, filters, needles and syringes. The following are examples of the methods and compositions of the invention. It will be understood that several other modalities can be put into practice, given the general description given above.

EXAMPLES Example 1: Generation of anti-EphB4 antibodies Anti-EphB4 antibodies were generated using phage display, using EphB4-His protein to select the phage. A variety of methods are known in the art to generate phage display libraries of which an antibody of interest can be obtained. A method for generating antibodies of interest is through the use of a phage antibody library as described in Lee et al., J. Mol. Biol. (2004), 340 (5): 1073-93. Selected clones using phage display were screened against EphB4-His protein using ELISA phage display. Single clones were selected for further characterization by phage competition ELISA and a blocking analysis to determine if the phage antibody clone could block EphB4-efrinaB2 interactions. Clone 30.35 performed favorably and was selected for further analysis. To improve the affinity of clone 30.35, phage display libraries were generated in the background of YW30: 35, each pointing to HVR selected for soft or hard randomization. Selected clones were selected by phage ELISA and then expressed as Fab protein and their affinity determined using Biacore. The selected clones were re-formatted as full-length IgG and their affinity was determined using Biacore. The sequences of the original clone 30.35 and affinity-matured clones are shown in Figure 1. a Example 2: Characterization of anti-EphB4 antibodies To determine the binding affinity of Mab anti-EphB4, surface plasmon resonance measurement (SRP) was used. ) with a BIAcore ™ -3000 (BIAcore, Inc., Piscataway, NJ). Briefly, carboxymethylated dextran detector chips (CM5, BIAcore Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The anti-EphB4 antibody was diluted with 10 mM sodium acetate, pH .8, at 5 ug / ml before injection at a flow rate of 5 ul / minute to obtain approximately 500 response units (RU) of coupled antibody . Next, 1 M ethanolamine was injected to block the unreacted groups. For kinetic measurements, serial two-fold dilutions of either human or murine EphB4-His molecules (0.7nM to 500nM) were injected into PBs with 0.05% Tween 20 at 25 ° C at a flow rate of 25 ul / min. The association (ignition) and dissociation velocities (kapagado) were calculated using a single-to-one Langmuir link model (BIAcore evaluation programming elements, version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio kapa? Ado / kencendido- The results of this experiment are shown in table 3. "NA" means that the measurement was not carried out.

Table 3: Affinity of binding and kinetics of anti-EphB4 antibodies to human and mouse EphB4 antigen murine human antigen Anti-EphB4 kenc / 105 kopag / 10"4 Kd (nM) kenclO5 kapag / 10'4 Kd (nM) YW30.35 0.31 0.75 2.4 0.03 0.96 32 YW30.35.1D2 1.28 <0.05 <0.04 0.36 <0.05 <0.14 YW30.35.2D8 0.92 <0.05 <0.05 0.4 <0.05 <0.12 YW30.35.2D12 NA NA NA 0.27 <0.05 <0.19 YM30.35.2D13 NA NA NA 0.45 < 0.05 < 0.11 In different experiments, the cross-reactivity of clone 30.35 of the anti-EphB4 antibody was tested against other EphB4 receptors. Briefly, C0S7 cells were transiently transfected with plasmids expressing full length human EphBl, human EphB2, human EphB4 or human EphBd. 24 hours after transfection, the cells were subjected to FACS analysis to detect the binding by anti-EphB4 antibodies, if any. Clone 30.35 anti-EphB4 did not cross-react with human Ephbl, human EphB2, human EphB3 or human EphB6.

Example 3: Signaling of the EpbH4 receptor blocked by anti-EphB4 antibody in a cell-based analysis In order to demonstrate the ability of the Anti-EphB4 antibodies to block the interaction of membrane-bound EfrinaB2 and EphB4, a cell-based analysis was carried out in which EphB4 and EfrinaB2 were presented by different cell types. 3T3 cells overexpressing human EfrinaB2 were used to stimulate HUVEC cells expressing high level of EphB4 but low level of EfrinaB2 and the ability of the anti-EphB4 antibody to inhibit the activation of EphB4 were tested. 3T3 cells overexpressing human EfrinaB2 were prepared as follows: EfrinaB2 full length human was cloned into the vector pcDNA5 / FRT (Invitrogen) and subsequently used to generate the stable cell line with 3T3 cells. Flp (Invitrogen) according to the manufacturer's manual. 3T3 cells overexpressing human EfrinaB2 were deposited on HUVEC cells for 15 or 30 minutes, in the presence or absence of anti-EphB4 antibody. Activation of the EphB4 receptor was determined by immunoprecipitation of EphB4 protein, then detection of the presence or absence of receptor tyrosine phosphorylation using an anti-phospho-tyrosine antibody (4G10 antibody, Upstate) using western blot. Briefly, the cells were lysed with pH-regulating solution RIPA. Cells used were clarified by centrifugation and anti-EphB4 35.2D8 antibody was added at 5 ug / sample. After incubation to 4 ° C for two hours, the immunocomplex was pulled using Random Protein A. Phosphorylation of EphB4 was analyzed by Western blot using anti-phosphotyrosine antibody 4G10 (Upstate) at a concentration of 1 ug / ml. The results of this experiment are shown in Figure 7. The superposition of 3T3 cells on HUVEC cells caused spectacular tyrosine phosphorylation of EphB4 (lanes 4 and 5). A 30 minute preincubation of HUVEC with anti-EphB4 antibody (clone 35.2D8 at 5 ug / 1) effectively abolished the tyrosine phosphorylation of EphB4 induced by overlapping 3T3-EfrinaB2 cells (lane 6). By contrast, treatment of HUVEC cells with anti-EphB5 alone did not cause activation of EphB4 in HUVEC (lanes 2 and 3) and untreated HUVEC cells did not demonstrate activation of EphB4. These results established that the anti-EphB4 antibody blocked the ligand-receptor interaction in the context of direct cell-cell contact. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.

Claims (46)

1. CLAIMS 1. An isolated anti-EphB4 antibody, characterized in that it comprises: (a) at least one, two, three, four or five or hypervariable region (HVR) sequences selected from the group consisting of: (i) an HVR- Ll comprising the sequence Al-All, wherein Al-All is RASQDVSTAVA (SEQ ID NO: 9); (ii) an HVR-L2 comprising the sequence B1-B7, wherein B1-B7 is SASFLYS (SEQ ID NO: 11); (iii) an HVR-L3 comprising the sequence C1-C9, wherein C1-C9 is QESTTTPPT (SEQ ID NO: 15); (iv) a HVR-H1 comprising the sequence D1-D10, wherein D1-D10 is GFSISNYYLH (SEQ ID NO: 2); (v) an HVR-H2 comprising the sequence E1-E18, wherein E1-E18 is GGIYLYGSSSEYADSVKG (SEQ ID NO: 4) and (vi) an HVR-H3 comprising the sequence F1-F17, wherein F1-F17 is ARGSGLRLGGLDYAMDY (SEQ ID NO: 7) and (b) at least one variant HVR, wherein the variant HVR sequence comprises modifications of at least one residue of the sequence illustrated in SEQ ID Nos: 1-17. The antibody according to claim 1, characterized in that a variant of HVR-Ll comprises 1-5 (1, 2, 3, 4 or 5) substitutions in any combination of the following positions: A6 (V or S), A7 (S or E), A8 (T or I), A9 (A or F) and A10 (V or L). The antibody according to claim 1, characterized in that a variant of HVR-L2 comprises 1-2 (1 or 2) substitutions in any combination of the following positions: B4 (F or N) and B6 (Y or E) . The antibody according to claim 1, characterized in that a variant of HVR-L3 comprises 1-7 (1, 2, 3, 4, 5, 6 or 7) substitutions in any combination of the following positions: C2 (Q , E, or K), C3 (S or T), C4 (Y, N, T, E, or A), C5 (T, A, or Q), C6 (T, V, or í), C8 ( P, L, or E) and C9 (T or S). 5. The antibody according to claim 1, characterized in that a variant of HVR-Ll comprises 1-3 (1, 2 or 3) substitutions in any combination of the following positions: D3 (T or S), D6 (G or N) and D9 (I or L). 6. The antibody according to claim 1, characterized in that a variant of HVR-L2 comprises 1-5 (1, 2, 3, 4 or 5) substitutions in any combination of the following positions: E5 (P, or L) , E7 (S or G), E8 (G or S), ElO (T, S, or R) and Eli (D, E, or G). 7. The antibody according to claim 1, characterized in that a variant of HVR-L3 comprises 1 substitution in the following positions: F3 (G or S). 8. An isolated anti-EphB4 antibody, characterized in that it comprises one, two, three, four, five or six HVR, in wherein each HVR comprises, consists or consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 1-17 and wherein SEQ ID NO: 9 or 10 corresponds to a HVR-Ll, SEQ ID NO: 11 or 12 corresponds to an HVR-L2, SEQ ID NO: 13, 14, 15, 16, or 17 correspond to an HVR-L3, SEQ ID NO: 1 or 2 correspond to an HVR-H1, SEQ ID NO: 3, 4, 5, or 6 correspond to an HVR-H2 and SEQ ID NO: 7 or 8 correspond to an HVR-H3. 9. The antibody according to claim 8, characterized in that the antibody comprises HVR-Ll, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein, each in order comprises SEQ ID NO: 9, 11, 13, 1, 3 and 7. The antibody according to claim 8 , characterized in that the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein, each in order comprises SEQ ID NO: 10, 12, 14, 1, 3 and 8. The antibody according to claim 8, characterized in that the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein, each The order comprises SEQ ID NO: 9, 11, 15, 2, 4 and 7. The antibody according to claim 8, characterized in that the antibody comprises HVR-Ll, HVR-L2, HVR-L3, HVR-Hl, HVR-H2 and HVR-H3, wherein, each in order comprises SEQ ID NO: 9, 11, 16, 1, 5 and 7. 13. The antibody according to claim 8, characterized in that the antibody comprises HVR-Ll, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each in order comprises SEQ ID NO: 9, 11, 17, 1 , 6 and 7. The antibody according to any of claims 1-13, characterized in that at least a portion of the structure sequence is a sequence of human consensus structure. 15. The antibody according to claim 1, characterized in that the modification is a substitution, insertion or cancellation. 16. The antibody according to any of claims 1-15, characterized in that the antibody comprises the consensus structure sequence of the human K subgroup. 17. The antibody according to any of claims 1-15, characterized in that the antibody comprises the consensus structure sequence of human heavy chain subgroup III. The antibody according to claim 17, characterized in that the antibody comprises a substitution at one or more positions 73 or 78. 19. The antibody according to claim 18, characterized in that the substitution e is one or more of R71A, N73T or N78A. 20. A polynucleotide, characterized in that it encodes an antibody according to any of claims 1-19. 21. A vector, characterized in that it comprises the polynucleotide according to claim 20. 22. The vector according to claim 21, characterized in that the vector is an expression vector. 23. A host cell, characterized in that it comprises the vector according to claim 21 or 22. 24. The host cell according to claim 23, characterized in that the host cell is prokaryotic. 25. The host cell according to claim 23, characterized in that the host cell is eukaryotic. 26. The host cell according to claim 25, characterized in that the host cell is mammalian. 27. A method for the manufacture of an anti-EphB4 antibody, the method is characterized in that it comprises: (a) expressing the vector according to claim 22 in an appropriate host cell and (b) recovering the antibody. 28. A method for manufacturing an anti-EphB4 immunoconjugate, the method is characterized in that it comprises (a) expressing the vector in accordance with claim 22 in an appropriate host cell and (b) recover the antibody. 29. The method according to claim 27 or 28, characterized in that the host cell is prokaryotic. 30. The method according to claim 27 or 28, characterized in that the host cell is eukaryotic. 31. A method for the detection of EphB4, the method is characterized in that it comprises detecting the EphB4-anti-EphB4 antibody complex in a biological sample, wherein the anti-EphB4 antibody in the complex is anti-EphB4 antibody in accordance with any of claims 1-19. 32. A method for diagnosing an alteration associated with the expression of EphB4, the method is characterized in that it comprises detecting the EphB4 -antibody anti-EphB4 complex in a biological sample of a patient suffering or suspected of having the disorder, wherein the The anti-EphB4 antibody in the complex is the anti-EphB4 antibody according to any of claims 1-19. 33. The method according to any of claims 31-32, characterized in that the anti-EphB4 antibody is detectably labeled. 34. A composition, characterized in that it comprises the anti-EphB4 antibody in accordance with any of the Claims 1-19. 35. A composition, characterized in that it comprises the polynucleotide according to any of claims 20-22. 36. The composition according to claim 34 or 35, characterized in that the composition further comprises a carrier. 37. The anti-EphB4 antibody according to any of claims 1-19, characterized in that it is used in the preparation of a medicament for inhibiting angiogenesis in a subject in need of such treatment. 38. The use according to claim 37, characterized in that the medicament is for use in combination with an anti-angiogenic agent. 39. The use according to claim 38, characterized in that the anti-angiogenic agent is for administration before or subsequent to the administration of the anti-EphB4 antibody. 40. The use according to claim 38, characterized in that the anti-angiogenic agent is for concurrent administration with the anti-EphB4 antibody. 41. The use according to any of claims 38-40, characterized in that the anti-angiogenic agent is an antagonist of vascular endothelial cell growth factor (VEGF). 42. The use according to claim 41, characterized in that the VEGF antagonist is an anti-VEGF antibody. 43. The use according to claim 42, characterized in that the anti-VEGF antibody is bevacizumab. 44. The use according to any of claims 37-43, characterized in that the medicament is for use in combination with a chemotherapeutic agent. 45. The use of the anti-EphB4 antibody according to any of claims 1-19, characterized in that it is used in the preparation of a medicament for the therapeutic and / or prophylactic treatment of an alteration. 46. The use according to claim 45, characterized in that the alteration is a cancer, a tumor and / or a cell proliferative alteration.