HK1187352B

HK1187352B - Anti-ephrinb2 antibodies and methods using same

Info

Publication number: HK1187352B
Application number: HK14100195.7A
Authority: HK
Inventors: 严民宏; 吴雁
Original assignee: 健泰科生物技术公司
Priority date: 2006-01-20
Filing date: 2014-01-08
Publication date: 2016-04-01

Description

anti-EPHRINB 2 antibodies and methods of use thereof

The application is a divisional application of an invention application with the application date of 2007, 19.01 and 19, Chinese application No. 200780010106.8 and the invention name of "anti-EPHRINB 2 antibody and a use method thereof".

RELATED APPLICATIONS

This application claims priority from U.S. provisional application No.60/760,891 filed 2006, 1, 20, 35USC § 119, the entire content of which is incorporated herein by reference.

Technical Field

The present invention relates generally to the field of molecular biology. More specifically, the invention concerns anti-EphrinB 2 antibodies and uses thereof.

Background

The development of vascular supply is an essential requirement for many physiological and pathological processes. Actively growing tissues such as embryos and tumors require an adequate blood supply. They meet this need by generating pro-angiogenic factors that promote neovascularization via a process known as angiogenesis. Tube formation of blood vessels is a complex but orderly biological event involving all or many of the following steps: a) proliferating EC from existing Endothelial Cells (ECs) or differentiating EC from progenitor cells; b) EC migrate and coalesce to form cord-like structures; c) vascular chordal then occurs by tubulogenesis (tubulogenesis) to form a blood vessel with a central lumen; d) existing cords or vessels bud to form secondary vessels; e) further remodeling (remodelling) and reshaping (reshaping) of the primary vascular plexus; and f) periendothelial cells (peri-endothelial cells) are recruited to surround the endothelial vessels, providing maintenance and regulatory functions to the blood vessels, including pericytes (pericytes) for small capillaries, smooth muscle cells for large vessels, and cardiomyocytes in the heart. Hanahan, Science277:48-50(1997), Hogan & Kolodziej, nat. Rev. Genet.3:513-23(2002), Lubarsky & Krasnow, Cell112:19-28 (2003).

The pathogenesis of angiogenesis in relation to a variety of conditions is now well established. These include solid tumors and metastases, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular diseases such as proliferative retinopathies like 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), Garner A., "Vascalded diseases" in Pathiology of Ocular disease.A Dynamic Approach, Garner A and Klintworth GK, 2 nd edition, Marcel Dekker, NY,1994, pp 1625-1710.

In the case of tumor growth, angiogenesis appears to be critical for the transition from hyperplasia to neoplasia, and to provide nutrition for tumor growth and metastasis. Folkman et al, Nature339:58 (1989). Neovascularization (neovascularization) allows tumor cells to acquire growth advantages and proliferative autonomy compared to normal cells. Tumors usually start with a single abnormal cell that can only proliferate to a size of a few cubic millimeters due to the distance from the available capillary bed, and that can remain "dormant" for an extended period of time without further growth and spread. Some tumor cells then switch to the angiogenic phenotype to initiate endothelial cells, which proliferate and mature into new capillaries. These newly formed blood vessels allow not only the primary tumor to continue growing, but also the metastatic tumor cells to spread and repopulate (recolonization). Thus, a correlation was observed between microvascular density in tumor sections and patient survival for breast cancer and several other tumors. Weidner et al, N.Engl.J.Med.324:1-6(1991), Horak et al, Lancet340:1120-24(1992), Macchiarini et al, Lancet340:145-46 (1992). The precise mechanism controlling the angiogenic switch is not fully understood, but it is believed that the neovascularization of tumor mass results from the net balance of numerous angiogenic stimulators and inhibitors. Folkman, nat. Med.1(1):27-31 (1995).

The process of vascular development is tightly regulated. To date, a number of molecules, mostly secreted factors produced by peripheral cells, have been shown to regulate EC differentiation, proliferation, migration, and coalescence into cord-like structures. For example, Vascular Endothelial Growth Factor (VEGF) has been identified as a key factor involved in the stimulation of angiogenesis and in the induction of vascular permeability. Ferrara et al, Endocr. Rev.18:4-25 (1997). Even the finding that loss of a single VEGF allele results in embryonic lethality points to the irreplaceable role this factor plays in the development and differentiation of the vascular system. Furthermore, VEGF has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders. Ferrara et al, Endocr. VEGF mRNA is overexpressed in most human tumors examined. Berkman et al, J.Clin.Invest.91:153-59(1993), Brown et al, Human Pathol.26:86-91(1995), Brown et al, Cancer Res.53:4727-35(1993), Mattern et al, Brit.J.cancer73:931-34(1996), Dvorak et al, am.J.Pathol.146:1029-39 (1995).

Likewise, the concentration levels of VEGF in ocular fluids are highly correlated with the presence of active vascular proliferation in diabetic and other ischemia-related retinopathy patients. Aiello et al, N.Engl.J.Med.331:1480-87 (1994). In addition, studies demonstrate the localization of VEGF in choroidal neovascular membranes in AMD patients. Lopez et al, invest, Ophthalmol. Vis. Sci.37:855-68 (1996).

anti-VEGF neutralizing antibodies suppress the growth of a variety of human tumor cell lines in nude mice (Kim et al, Nature362:841-44(1993); Warren et al, J.Clin.Invest.95:1789-97(1995);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 disorders (Adams et al, Arch. Ophthalmol.114:66-71 (1996)). Thus, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of tumors and a variety of intraocular neovascular disorders. Such antibodies are described, for example, in EP817,648, published at 14/1/1998 and WO98/45331 and WO98/45332, published at 15/10/1998. An anti-VEGF antibody, bevacizumab (bevacizumab), has been FDA approved for use in the treatment of metastatic colorectal cancer (CRC) in combination with a chemotherapeutic regimen. Moreover, bevacizumab is being investigated in a number of ongoing clinical trials for the treatment of various cancer indications.

EphrinB2 ligand ("Ephrin-B2" or "EphrinB 2") is a member of the Ephrin ligand family, which constitutes a large family of tyrosine kinase receptors in the human genome (for review see Dodelet, Oncogene,19: 5614-. Human ephrin ligand tyrosine kinases are classified into a and B classes based on sequence identity, with corresponding a and B type receptors known as Ephs or eph receptors. Signaling can occur in a forward manner, in which the receptor tyrosine kinase is activated by the ligand, or in a reverse manner, in which the transmembrane ephrinB ligand is activated by interaction with the receptor. eph receptor ligand interactions have been linked to a wide range of biological functions including axonal guidance, tissue boundary formation, fibrogenesis (vasculogenesis), and cell motility (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).

Clearly there remains a need for agents with clinical characteristics that are most suitable for development into therapeutic agents. The invention described herein satisfies this need and provides other benefits.

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

Summary of The Invention

The invention is based, in part, on the identification of various EphrinB2 binding agents (such as antibodies, and fragments thereof). EphrinB2 appears to be an important and advantageous therapeutic target, and the present invention provides compositions and methods based on binding EphrinB 2. As described herein, the EphrinB2 binding agents of the present invention provide important therapeutic and diagnostic agents for use in targeting pathological conditions associated with the expression and/or activity of EphrinB2 ligand pathways. Thus, the present invention provides methods, compositions, kits and articles of manufacture relating to EphrinB2 binding.

The invention provides antibodies that bind (such as specifically bind) to EphrinB 2.

In one aspect, the invention provides an isolated anti-EphrinB 2 antibody, wherein the antibody in full-length IgG format specifically binds human EphrinB2 with a binding affinity of 30pM or better. As is well established in the art, the binding affinity of a ligand for its receptor can be determined using any of a variety of assays and expressed in various quantitative numerical forms. Thus, in one embodiment, binding affinity is expressed as Kd values, reflecting intrinsic binding affinity (e.g. with minimized affinity effect). It is common and preferred that binding affinity is measured in vitro, whether in a cell-free environment or a cell-associated environment. Any of a number of assays known in the art, including those described herein, can be used to obtain a measurement of binding affinity, including, for example, Biacore, Radioimmunoassay (RIA), and ELISA.

In one aspect, the invention provides isolated antibodies that bind to Eph receptor binding regions of EphrinB2 (such as EphB1, EphB2, and/or EphB 3).

In one aspect, the invention provides an isolated antibody that binds to a polypeptide comprising, consisting of, or consisting essentially of the extracellular domain of EphrinB 2.

In one aspect, the invention provides isolated anti-EphrinB 2 antibodies that compete with Eph receptors (such as EphB1, EphB2, EphB 3) for binding to EphrinB 2.

In one aspect, the invention provides isolated anti-EphrinB 2 antibodies that inhibit, reduce, and/or block EphrinB2 activity. In some embodiments, the autophosphorylation of EphrinB2 is inhibited, reduced, and/or blocked.

In one aspect, an anti-EphrinB 2 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) HVR-L1, comprising the sequence A1-A11, wherein A1-A11 is RASQDVSTAVA (SEQ ID NO:6),

(ii) HVR-L2 comprising the sequence B1-B7, wherein B1-B7 is SASFLYS (SEQ ID NO:8),

(iii) HVR-L3, comprising the sequence C1-C9, wherein C1-C9 is EQTDSTPPT (SEQ ID NO:12),

(iv) HVR-H1, comprising the sequence D1-D10, wherein D1-D10 is GFTVSSGWIH (SEQ ID NO:2),

(v) HVR-H2 comprising the sequence E1-E18, wherein E1-E18 is AVIFHNKGGTDYADSVKG (SEQ ID NO:4), and

(vi) HVR-H3 comprising the sequence F1-F14, wherein F1-F14 is ARTSAWAQLGAMDY (SEQ ID NO: 5); and

(b) at least one variant HVR, wherein the variant HVR sequence comprises a modification of at least one residue of a sequence set forth in SEQ ID NOS: 1-12.

In one aspect, the invention provides an antibody comprising one, two, three, four, five or six HVRs, wherein each HVR comprises, consists of or consists essentially of a sequence selected from SEQ ID NOS 1-12, and wherein SEQ ID NOS 6 or 7 corresponds to HVR-L1, SEQ ID NOS 8 or 9 corresponds to HVR-L2, SEQ ID NOS 10, 11 or 12 corresponds to HVR-L3, SEQ ID NOS 1 or 2 corresponds to HVR-H1, SEQ ID NOS 3 or 4 corresponds to HVR-H2, and SEQ ID NOS 5 corresponds to HVR-H3.

In one embodiment, an antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each comprising, in order, SEQ ID NOS 6, 8, 10, 1, 3, 5.

In one embodiment, an antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each comprising, in order, SEQ ID NOs 7, 9, 11, 1, 3, 5.

In one embodiment, an antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each comprising, in order, SEQ ID NOS 6, 8, 12, 2, 4, 5.

A variant HVR in an antibody of the invention can have a modification of one or more (such as two, three, four, five, or more) residues within the HVR.

In one embodiment, a HVR-L1 variant comprises 1-4 (1, 2, 3, or 4) substitutions in any combination of the following positions: a7 (S or D), A8 (T or S), A9 (A or S), and A10 (V or L).

In one embodiment, a HVR-L2 variant comprises 1-3 (1, 2, or 3) substitutions in any combination of the following positions: b1 (S or a), B4 (F or N), and B6 (Y or E).

In one embodiment, a HVR-L3 variant comprises 1-6 (1, 2, 3, 4, 5, or 6) substitutions in any combination of the following positions: c1 (Q or E), C3 (S or T), C4 (Y or D), C5 (T, D or S), C6 (T or N), C8 (P or F).

In one embodiment, a HVR-H1 variant comprises 1-4 (1, 2, 3, or 4) substitutions in any combination of the following positions: d4 (I or V), D5 (T or S), D6 (G or S), and D7 (S or G).

In one embodiment, a HVR-H2 variant comprises 1-4 (1, 2, 3, or 4) substitutions in any combination of the following positions: e4 (Y or F), E5 (P or H), E7 (N or K), and E9 (A or G).

In one embodiment, the HVR-H3 variant comprises 1-14 substitutions in the following positions: f1 (a), F2 (R), F3 (T), F4 (S), F5 (a), F6 (W), F7 (a), F8 (Q), F9 (L), F10 (G), F11 (a), F12 (M), F13 (D), and F14 (Y).

The letters in parentheses after each position indicate exemplary substitute (i.e., replacement) amino acids; it will be apparent to those skilled in the art that the suitability of other amino acids as substitute amino acids in the context described herein can be routinely assessed using techniques known in the art and/or described herein.

In one aspect, the invention provides an antibody comprising an HVR-H1 region, said 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, said HVR-H2 region comprising the sequence of SEQ ID NO. 3 or 4. In one aspect, the invention provides an antibody comprising an HVR-H3 region, wherein the HVR-H3 region comprises the sequence of SEQ ID NO: 5. In one embodiment, the invention provides an antibody comprising a HVR-L1 region, said HVR-L1 region comprising the sequence of SEQ ID NO 6 or 7. In one embodiment, the invention provides an antibody comprising a HVR-L2 region, said HVR-L2 region comprising the sequence of SEQ ID NO 8 or 9. In one embodiment, the invention provides an antibody comprising a HVR-L3 region, said HVR-L3 region comprising the sequence of SEQ ID NO 10, 11, or 12.

In one aspect, the invention provides an antibody comprising at least one, at least two, or all three of the following sequences:

(i) HVR-H1 sequence comprising the sequence of SEQ ID NO. 2;

(ii) HVR-H2 sequence comprising the sequence of SEQ ID NO. 4;

(iii) HVR-H3 sequence comprising the sequence of SEQ ID NO 5.

(i) HVR-L1 sequence comprising the sequence of SEQ ID NO 6;

(ii) HVR-L2 sequence comprising the sequence of SEQ ID NO. 8;

(iii) HVR-L3 sequence comprising the sequence of SEQ ID NO. 12.

As shown in FIG. 1, the amino acid sequences of SEQ ID NOS: 1-12 are numbered for each HVR (i.e., H1, H2, or H3) in a manner consistent with the Kabat numbering system as described below.

In one aspect, the invention provides an antibody comprising the heavy chain HVR sequences shown in figure 1.

In one aspect, the invention provides an antibody comprising the light chain HVR sequences shown in figure 1.

Some embodiments of the antibodies of the invention comprise humanized 4D5 antibody (huMAb4D5-8) (SEQ ID NO:13, infra)Genentech, inc., South San Francisco, CA, USA) (see also U.S. patent No.6,407,213 and Lee et al, j.mol.biol. (2004),340(5): 1073-93).

1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser GlyVal Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys107(SEQ ID NO:13)

(HVR residues underlined)

In one embodiment, the huMAb4D5-8 light chain variable domain sequence has a modification at one or more of positions 30, 66 or 91 (Asn, Arg and His, respectively, in bold/italics above). In one embodiment, the modified huMAb4D5-8 sequence comprises a Ser at position 30, a Gly at position 66, and/or a Ser at position 91. Thus, in one embodiment, the antibody of the invention comprises a light chain variable domain comprising the sequence shown in SEQ ID NO:14 below:

1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser GlyVal Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys107(SEQ ID NO:14)

(HVR residues underlined)

The replacement residues relative to huMAb4D5-8 are indicated in bold/italics above.

The antibodies of the invention may comprise any suitable framework variable domain sequence provided that the binding activity of EphrinB2 is substantially retained. For example, in some embodiments, an antibody of the invention comprises a human subgroup III heavy chain framework consensus sequence. In one embodiment of these antibodies, the framework consensus sequence is at 71, 73 and/or 78The bit contains a substitution. In some embodiments of these antibodies, A is at position 71, T is at position 73, and/or A is at position 78. In one embodiment, the antibodies comprise huMAb4D5-8 (h) Genentech, Inc., South San Francisco, Calif., USA) (see also U.S. Pat. No.6,407,213&5,821,337 and Lee et al, J.mol.biol. (2004),340(5): 1073-93). In one embodiment, these antibodies further comprise a human kappa I light chain framework consensus sequence. In one embodiment, these antibodies comprise the light chain HVR sequence of huMAb4D5-8 (U.S. Pat. No.6,407,213)&5,821,337). In one embodiment, the antibodies comprise huMAb4D5-8 (h)Genentech, Inc., South San Francisco, Calif., USA) (see also U.S. Pat. No.6,407,213&5,821,337 and Lee et al, J.mol.biol. (2004),340(5): 1073-93).

In one embodiment, the antibody of the invention comprises a heavy chain variable domain, wherein the framework 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 the HVR H1, H2, and H3 sequences are SEQ ID NOs 2, 4, and/or 5, respectively. In one embodiment, the antibody of the invention comprises a light chain variable domain, wherein the framework sequence comprises the sequence of SEQ ID NOs 38, 39, 40 and/or 41, and the HVRL1, L2 and L3 sequences are SEQ ID NOs 6, 8 and/or 12, respectively.

In one embodiment, the antibody of the invention comprises a heavy chain variable domain, wherein the framework sequence comprises the sequence of SEQ ID NOs 42, 43, 44, and/or 45, and the HVR H1, H2, and H3 sequences are SEQ ID NOs 1, 3, and/or 5, respectively. In one embodiment, the antibody of the invention comprises a light chain variable domain, wherein the framework sequence comprises the sequence of SEQ ID NOs 15, 16, 17, and/or 18, and the HVR L1, L2, and L3 sequences are SEQ ID NOs 6, 8, and/or 10, respectively.

In one embodiment, the antibody of the invention comprises a heavy chain variable domain, wherein the framework sequence comprises the sequence of SEQ ID NOs 42, 43, 47, and/or 45, and the HVR H1, H2, and H3 sequences are SEQ ID NOs 1, 3, and/or 5, respectively. In one embodiment, the antibody of the invention comprises a light chain variable domain, wherein the framework sequence comprises the sequence of SEQ ID NOs 15, 16, 47, and/or 18, and the HVR L1, L2, and L3 sequences are SEQ ID NOs 7, 9, and/or 11, respectively.

In one embodiment, the antibodies of the invention are affinity matured to obtain the desired target binding affinity. In one embodiment, the affinity matured antibody of the invention comprises a substitution at one or more of amino acids H29, H30, H31, H32, H52, H52a, H54, H56, L29, L30, L31, L32, L33, L50, L53, L55, L89, L91, L92, L93, L94, and L96. In one embodiment, the affinity matured antibody of the invention comprises one or more of the following substitutions: (a) V29I, S30T, S31G, G32S, F52Y, H52aP, K54N, and G56A in the heavy chain, or (b) S30D, T31S, a32S, V33L, S50A, F53N, Y55E, E89Q, T91S, D92Y, S93D or T, T94N, and P96F in the light chain.

In one embodiment, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO. 49. In one embodiment, the antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO 48. In one embodiment, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO. 49 and a light chain variable domain comprising the sequence of SEQ ID NO. 48.

In one embodiment, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO 51. In one embodiment, the antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO. 50. In one embodiment, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO. 51 and a light chain variable domain comprising the sequence of SEQ ID NO. 50.

In one embodiment, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO 53. In one embodiment, the antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO 52. In one embodiment, the 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 52.

In one aspect, the invention provides an antibody that competes with any of the above antibodies for binding to EphrinB 2. In one aspect, the invention provides antibodies that bind to the same or similar epitope on EphrinB2 as the antibodies described above.

As is known in the art and described in more detail below, the amino acid positions/boundaries that define the hypervariable regions of an antibody can vary according to the context and various definitions known in the art (as described below). Some positions within a variable domain may be considered hybrid hypervariable positions in that these positions are considered to be within a hypervariable region under one set of criteria, but are considered to be outside of a hypervariable region under a different set of criteria. One or more of these positions may also be found within an extended hypervariable region (as defined further below).

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 embodiments, the antibody is Fab, Fab '-SH, F (ab') ₂Or scFv.

In one embodiment, the antibody is a chimeric antibody, e.g., an antibody that grafts antigen-binding sequences from a non-human donor to heterologous non-human, or humanized sequences (e.g., framework and/or constant domain sequences). In one embodiment, the non-human donor is a mouse. In one embodiment, the antigen binding sequence is synthetic, such as by mutagenesis (e.g., phage display screening, etc.). In one embodiment, the chimeric antibody of the invention has a murine V region and a 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 human IgG1C region.

Humanized antibodies of the invention include those having amino acid substitutions in the FRs and variants with altered affinity maturation in the grafted CDRs. The substituted amino acids in the CDR or FR are not limited to those present in the donor or acceptor antibody. In other embodiments, the antibodies of the invention further comprise alterations at amino acid residues in the Fc region that result in improved effector function, including enhanced CDC and/or ADCC and B-cell killing functions. Other antibodies of the invention include those with specific modifications that improve stability. In other embodiments, the antibodies of the invention comprise alterations at amino acid residues in the Fc region that result in reduced effector function (e.g., reduced CDC and/or ADCC function and/or reduced B-cell killing). In some embodiments, the antibodies of the invention are characterized by reduced binding (such as loss of binding) to human complement factor C1q and/or a human Fc receptor on Natural Killer (NK) cells. In some embodiments, the antibodies of the invention are characterized by reduced binding (such as loss of binding) to human Fc γ RI, Fc γ RIIA, and/or Fc γ RIIIA. In some embodiments, the antibodies of the invention are of the IgG class (e.g., IgG1 or IgG 4) and comprise at least one mutation (numbering according to the EU index) in E233, L234, G236, D265, D270, N297, E318, K320, K322, a327, a330, P331, and/or P329. In some embodiments, the antibody comprises the mutations L234A/L235A or D265A/N297A.

In one aspect, the invention provides an anti-EphrinB 2 polypeptide comprising any of the antigen binding sequences provided herein, wherein the anti-EphrinB 2 polypeptide specifically binds to EphrinB 2.

Antibodies of the invention bind (such as specifically bind) to EphrinB2, and, in some embodiments, modulate one or more aspects of an EphrinB 2-associated effect, including, but not limited to, EphrinB2 activation, EphrinB2 downstream molecular signaling, EphrinB 2-binding Eph receptor (e.g., EphB1, EphB2, and/or EphB 3) activation, EphrinB 2-binding Eph receptor (e.g., EphB1, EphB2, and/or EphB 3) downstream molecular signaling, EphrinB 2-binding ephh receptor (e.g., EphB1, EphB2, and/or EphrinB 3) binding to EphrinB 3 disruption of EphrinB 3 phosphorylation and/or EphrinB 3, and/or EphrinB 3 binding, EphrinB 3 phosphorylation, EphrinB 3 binding, EphrinB 3, or cell proliferation, or cell 3, or cell growth, And/or treatment or prevention of a condition associated with EphrinB2 expression and/or activity (such as increased EphrinB2 expression and/or activity). In some embodiments, the antibodies of the invention specifically bind to EphrinB 2. In some embodiments, the antibody specifically binds to the extracellular domain (ECD) of EphrinB 2. In some embodiments, the antibody specifically binds to a polypeptide consisting of or consisting essentially of the extracellular domain of EphrinB 2. In some embodiments, the antibody specifically binds EphrinB2 with a Kd of 30pM or stronger. In some embodiments, the antibodies of the invention reduce, inhibit, and/or block EphrinB2 activity in vivo and/or in vitro. In some embodiments, the antibody reduces, inhibits, and/or blocks EphrinB2 autophosphorylation. In some embodiments, the antibody competes (reduces and/or blocks) for binding to an EphrinB 2-binding Eph receptor (e.g., EphB1, EphB2, and/or EphB 3).

In one aspect, the invention provides the use of an antibody of the invention in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder. In some embodiments, the disorder is a neuropathy or neurodegenerative disease.

In one aspect, the invention provides a composition 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 the anti-EphrinB 2 antibodies of the invention.

In one aspect, the invention provides a vector comprising a nucleic acid of the invention.

In one aspect, the invention provides a composition comprising one or more nucleic acids of the invention and a vector. In one embodiment, the carrier is pharmaceutically acceptable.

In one aspect, the invention provides a host cell comprising a nucleic acid or vector of the invention. The vector may be of any type, e.g., a recombinant vector, such as an expression vector. Any of a variety of host cells may be used. In one embodiment, the host cell is a prokaryotic cell, such as E.coli. In one embodiment, the host cell is a eukaryotic cell, for example a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell.

In one aspect, the invention provides methods of making the antibodies of the invention. For example, the invention provides a method of making an anti-EphrinB 2 antibody (as defined herein, including full length and fragments thereof) or immunoconjugate, the method comprising expressing a recombinant vector of the invention encoding the antibody (or fragment thereof) in a suitable host cell, and recovering the antibody.

In one aspect, the invention provides an article of manufacture comprising a container and a composition contained within the container, wherein the composition comprises one or more anti-EphrinB 2 antibodies of the invention. In one embodiment, the composition comprises a nucleic acid of the invention. In one embodiment, the composition comprising the antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment, the article of manufacture of the invention further comprises instructions for administering the composition (e.g., 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 holding a composition comprising one or more anti-EphrinB 2 antibodies of the invention and a second container holding a buffer. In one embodiment, the buffer is pharmaceutically acceptable. In one embodiment, the composition comprising the antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment, the 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-EphrinB 2 antibody of the invention in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder. In some embodiments, the disorder is a neurological disease or neurodegenerative disease. In some embodiments, the disorder is a pathological condition associated with angiogenesis.

In one aspect, the invention provides the use of an antibody of the invention in the manufacture of a medicament for inhibiting 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 a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder. In some embodiments, the disorder is a neurological disease or neurodegenerative disease. In some embodiments, the disorder is a pathological condition associated with angiogenesis.

In one aspect, the invention provides the use of a vector of the invention in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder. In some embodiments, the disorder is a neurological disease or neurodegenerative disease. In some embodiments, the disorder is a pathological condition associated with angiogenesis.

In one aspect, the invention provides the use of a host cell of the invention in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder. In some embodiments, the disorder is a neurological disease or neurodegenerative disease. In some embodiments, the disorder 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 manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder. In some embodiments, the disorder is a neurological disease or neurodegenerative disease.

In one aspect, the invention provides the use of a kit of the invention in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder. In some embodiments, the disorder is a neurological disease or neurodegenerative disease. In some embodiments, the disorder is a pathological condition associated with angiogenesis.

The present invention provides methods or compositions useful for modulating disease states associated with EphrinB2 expression and/or activity (such as increased or decreased expression and/or activity, or unwanted expression and/or activity).

In one aspect, the invention provides methods for treating or preventing a tumor, cancer, and/or cell proliferative disorder associated with increased expression and/or activity of EphrinB2, comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody.

In one aspect, the invention provides methods for killing cells (such as cancer or tumor cells) comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody.

In one aspect, the invention provides methods for reducing, inhibiting, blocking, or preventing tumor or cancer growth, comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody.

In one aspect, the invention provides methods for treating or preventing a neurological or neurodegenerative disease, or repairing damaged nerve cells, comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody.

In one aspect, the invention provides methods for promoting neuronal development, proliferation, maintenance or regeneration, comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody.

In one aspect, the invention provides methods for inhibiting angiogenesis, comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody. In some embodiments, the site of angiogenesis is a tumor or cancer.

In one aspect, the invention provides methods for treating pathological conditions associated with angiogenesis, comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody. In some embodiments, the pathological condition associated with angiogenesis is a tumor, cancer, and/or cell proliferative disorder. In some embodiments, the pathological condition associated with angiogenesis is an intraocular neovascular disease (intraocular neovasular disease).

The methods of the invention may be used to affect any suitable pathological condition. Described herein are exemplary conditions, including cancers selected from the group consisting of: small cell lung cancer, neuroblastoma (neuroblastoma), melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), hepatocellular carcinoma.

In one embodiment, the cells targeted in the methods of the invention are cancer cells. For example, the cancer cell can be selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, a papillary cancer cell, a colon cancer cell, a pancreatic cancer cell, an ovarian cancer cell, a cervical cancer cell, a central nervous system cancer cell, an osteogenic sarcoma cell, a renal cancer cell, a hepatocellular carcinoma cell, a bladder cancer cell, a gastric cancer cell, a head and neck squamous carcinoma cell, a melanoma cell, a leukemia cell, and a colon adenoma cell. In one embodiment, the cells targeted in the methods of the invention are hyperproliferative and/or hyperproliferative cells. In one embodiment, the cells targeted in the methods of the invention are dysplastic (dysplastic) cells. In yet another embodiment, the cells targeted in the methods of the invention are metastatic cells.

The method of the present invention may further comprise additional processing steps. For example, in one embodiment, the method further comprises a step wherein the target cells and/or tissues (e.g., cancer cells) are exposed to a radiation treatment or chemotherapeutic agent.

In one aspect, the invention provides methods comprising administering an effective amount of an anti-EphrinB 2 antibody in combination with an effective amount of another therapeutic agent, such as an anti-angiogenic agent. For example, anti-EphrinB 2 antibodies are used in combination with anti-cancer or anti-angiogenic agents for the treatment of various neoplastic or non-neoplastic conditions. In one embodiment, the neoplastic (neoplastic) or non-neoplastic (non-neoplastic) disorder is a pathological condition associated with angiogenesis. In some embodiments, the other therapeutic agent is an anti-angiogenic agent, an anti-neoplastic agent (anti-neoplastic agent), and/or a chemotherapeutic agent.

The anti-EphrinB 2 antibody can be administered continuously or in combination with another therapeutic agent effective for those purposes, either in the same composition or as a separate composition. The administration of the anti-EphrinB 2 antibody and another therapeutic agent (e.g., an anti-cancer agent, an anti-angiogenic agent) can be performed simultaneously, e.g., as a single composition, or as two or more different compositions, using the same or different routes of administration. Alternatively, or in addition, administration may be sequential in any order. Alternatively, or in addition, the steps may be performed in any order, in any combination of order and simultaneously. In certain embodiments, the interval between administration of two or more compositions may have a time ranging from minutes to days, to weeks, to months. For example, the anti-cancer agent may be administered first, followed by administration of the anti-EphrinB 2 antibody. However, simultaneous or prior administration of anti-EphrinB 2 antibodies is also contemplated. Thus, in one aspect, the invention provides methods comprising administering an anti-EphrinB 2 antibody, followed by administration of an anti-angiogenic agent, such as an anti-VEGF antibody, such as bevacizumab. In certain embodiments, the interval between administration of two or more compositions may have a time ranging from minutes to days, to weeks, to months.

In certain aspects, the invention provides methods of treating disorders (such as tumors, cancers, and/or cell proliferative disorders) by administering an effective amount of an anti-EphrinB 2 antibody and/or angiogenesis inhibitor and one or more chemotherapeutic agents. A variety of chemotherapeutic agents may be used in the combination treatment methods of the present invention. An illustrative and non-limiting list of contemplated chemotherapeutic agents is provided herein in the definitions section. Administration of the anti-EphrinB 2 antibody and the cross-linking agent can be performed simultaneously, e.g., as a single composition, or as two or more different compositions, using the same or different routes of administration. Alternatively, or in addition, administration may be sequential in any order. Alternatively, or in addition, the steps may be performed in any order, in any combination of order and simultaneously. In certain embodiments, the interval between administration of two or more compositions may have a time ranging from minutes to days, to weeks, to months. For example, the chemotherapeutic agent may be administered first, followed by the anti-EphrinB 2 antibody. However, simultaneous or prior administration of anti-EphrinB 2 antibodies is also contemplated. Thus, in one aspect, the invention provides methods comprising administering an anti-EphrinB 2 antibody, followed by administration of a chemotherapeutic agent. In certain embodiments, the interval between administration of two or more compositions may have a time ranging from minutes to days, to weeks, to months.

In one aspect, the invention provides methods for enhancing the efficacy of an anti-angiogenic agent in a subject having a pathological condition associated with angiogenesis, the method comprising administering to the subject an effective amount of an anti-EphrinB 2 antibody in combination with an anti-angiogenic agent, thereby enhancing the inhibitory activity of the anti-angiogenic agent.

In one aspect, the invention provides methods and compositions for inhibiting or preventing recurrent tumor growth (relapse tumorgrowth) or recurrent cancer cell growth. Recurrent tumor growth or recurrent cancer cell growth is used to describe conditions in which patients undergoing or treated with one or more currently available therapies (e.g., cancer therapies such as chemotherapy, radiation therapy, surgery, hormonal therapy and/or biological therapy/immunotherapy, anti-VEGF antibody therapy, particularly standard treatment regimens for particular cancers) are clinically inadequate to treat the patient or are no longer treated by the therapy to achieve any beneficial effect such that the patients require an otherwise effective therapy.

In another aspect, the invention provides a method for detecting EphrinB2, the method comprising detecting EphrinB 2-anti-EphrinB 2 antibody complex in a sample. The term "detecting" as used herein includes qualitative and/or quantitative detection (measuring levels), with or without a reference control.

In another aspect, the invention provides a method for diagnosing a disorder associated with EphrinB2 expression and/or activity, the method comprising detecting EphrinB 2-anti-EphrinB 2 antibody complex in a biological sample from a patient having or suspected of having the disorder. In some embodiments, the EphrinB2 expression is elevated expression or aberrant expression. In some embodiments, the disorder is a tumor, cancer, and/or cell proliferative disorder.

In another aspect, the invention provides any of the anti-EphrinB 2 antibodies described herein, wherein the anti-EphrinB 2 antibody comprises a detectable label.

In another aspect, the invention provides a complex of any of the anti-EphrinB 2 antibodies described herein with EphrinB 2. In some embodiments, the complex is in vivo or in vitro. In some embodiments, the complex comprises a cancer cell. In some embodiments, the anti-EphrinB 2 antibody is detectably labeled.

The present invention relates to the following items.

1. An isolated anti-EphrinB 2 antibody comprising:

2. The antibody of item 1, wherein a HVR-L1 variant comprises 1-4 (1, 2, 3, or 4) substitutions in any combination of the following positions: a7 (S or D), A8 (T or S), A9 (A or S), and A10 (V or L).

3. The antibody of item 1, wherein a HVR-L2 variant comprises 1-3 (1, 2, or 3) substitutions in any combination of the following positions: b1 (S or a), B4 (F or N), and B6 (Y or E).

4. The antibody of item 1, wherein a HVR-L3 variant comprises 1-6 (1, 2, 3, 4, 5, or 6) substitutions in any combination of the following positions: c1 (Q or E), C3 (S or T), C4 (Y or D), C5 (T, D or S), C6 (T or N), and C8 (P or F).

5. The antibody of item 1, wherein a HVR-H1 variant comprises 1-4 (1, 2, 3, or 4) substitutions in any combination of the following positions: d4 (I or V); d5 (T or S), D6 (G or S), and D7 (S or G).

6. The antibody of item 1, wherein a HVR-H2 variant comprises 1-4 (1, 2, 3, or 4) substitutions in any combination of the following positions: e4 (Y or F), E5 (P or H), E7 (N or K), and E9 (A or G).

7. The antibody of item 1, wherein the HVR-H3 variant comprises 1-14 substitutions in the following positions: f1 (a), F2 (R), F3 (T), F4 (S), F5 (a), F6 (W), F7 (a), F8 (Q), F9 (L), F10 (G), F11 (a), F12 (M), F13 (D), and F14 (Y).

8. An isolated anti-EphrinB 2 antibody comprising, consisting of, or consisting essentially of one, two, three, four, five, or six HVRs, wherein each HVR comprises, consists of, or consists of a sequence selected from SEQ ID NOs 1-12, and wherein SEQ ID NOs 6 or 7 corresponds to HVR-L1, SEQ ID NOs 8 or 9 corresponds to HVR-L2, SEQ ID NOs 10, 11, or 12 corresponds to HVR-L3, SEQ ID NOs 1 or 2 corresponds to HVR-H1, SEQ ID NOs 3 or 4 corresponds to HVR-H2, and SEQ ID NO 5 corresponds to HVR-H3.

9. The antibody of item 8, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein HVR-L1 comprises SEQ ID NO:6, HVR-L2 comprises SEQ ID NO:8, HVR-L3 comprises SEQ ID NO:10, HVR-H1 comprises SEQ ID NO:1, HVR-H2 comprises SEQ ID NO:3, and HVR-H3 comprises SEQ ID NO: 5.

10. The antibody of item 8, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein HVR-L1 comprises SEQ ID NO. 7, HVR-L2 comprises SEQ ID NO. 9, HVR-L3 comprises SEQ ID NO. 11, HVR-H1 comprises SEQ ID NO. 1, HVR-H2 comprises SEQ ID NO. 3, and HVR-H3 comprises SEQ ID NO. 5.

11. The antibody of item 8, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein HVR-L1 comprises SEQ ID NO:6, HVR-L2 comprises SEQ ID NO:8, HVR-L3 comprises SEQ ID NO:12, HVR-H1 comprises SEQ ID NO:2, HVR-H2 comprises SEQ ID NO:4, and HVR-H3 comprises SEQ ID NO: 5.

12. The antibody of any one of items 1-11, wherein at least a portion of the framework sequence is a human consensus framework sequence.

13. The antibody of item 1, wherein the modification is a substitution, insertion, or deletion.

14. The antibody of any one of items 1-13, wherein the antibody comprises human k subgroup consensus framework sequence.

15. The antibody of any one of items 1-13, wherein the antibody comprises heavy chain human subgroup III consensus framework sequence.

16. The antibody of item 15, wherein the antibody comprises a substitution at one or more of positions 71, 73, or 78.

17. The antibody of item 16, wherein the substitution is one or more of R71A, N73T, or N78A.

18. A polynucleotide encoding the antibody of any one of claims 1-17.

19. A vector comprising the polynucleotide of item 18.

20. The vector of item 19, wherein the vector is an expression vector.

21. A host cell comprising the vector of claim 19 or 20.

22. The host cell of item 21, wherein the host cell is prokaryotic.

23. The host cell of item 21, wherein the host cell is eukaryotic.

24. The host cell of item 23, wherein the host cell is mammalian.

25. A method of making an anti-EphrinB 2 antibody, the method comprising: (a) expressing the vector of item 20 in a suitable host cell, and (b) recovering the antibody.

26. A method of making an anti-EphrinB 2 immunoconjugate, the method comprising: (a) expressing the vector of item 20 in a suitable host cell, and (b) recovering the antibody.

27. The method of clause 25 or 26, wherein the host cell is prokaryotic.

28. The method of item 25 or 26, wherein the host cell is eukaryotic.

29. A method of detecting EphrinB2, the method comprising detecting EphrinB 2-anti-EphrinB 2 antibody complex in a biological sample.

30. A method of diagnosing a disorder associated with EphrinB2 expression, the method comprising detecting an EphrinB 2-anti-EphrinB 2 antibody complex in a biological sample from a patient having or suspected of having the disorder.

31. The method of clause 29 or 30, wherein the anti-EphrinB 2 antibody is detectably labeled.

32. A composition comprising an anti-EphrinB 2 antibody of any of items 1-17.

33. A composition comprising the polynucleotide of any one of claims 18-20.

34. The composition of item 32 or 33, wherein the composition further comprises a carrier.

35. A method of inhibiting angiogenesis, the method comprising administering to a subject in need of such treatment an anti-EphrinB 2 antibody of any one of items 1-17.

36. The method of item 35, further comprising administering to the subject an effective amount of an anti-angiogenic agent.

37. The method of clause 36, wherein the anti-angiogenic agent is administered prior to or after the anti-EphrinB 2 antibody.

38. The method of clause 36, wherein the anti-angiogenic agent is administered concurrently with the anti-EphrinB 2 antibody.

39. The method of any one of claims 36-38, wherein the anti-angiogenic agent is an antagonist of vascular endothelial cell growth factor (VEGF).

40. The method of item 39, wherein said VEGF antagonist is an anti-VEGF antibody.

41. The method of clause 40, wherein the anti-VEGF antibody is bevacizumab.

42. The method of any one of items 35-41, further comprising administering an effective amount of a chemotherapeutic agent.

43. Use of an anti-EphrinB 2 antibody of any of items 1-17 in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disorder.

44. The use of item 43, wherein said disorder is a cancer, a tumor, and/or a cell proliferative disorder.

45. The use of item 43, wherein the disorder is a neuropathy or neurodegenerative disease.

46. The use of item 43, wherein said disorder is a pathological condition associated with angiogenesis.

47. The use of item 46, wherein said pathological condition associated with angiogenesis is a tumor, cancer, and/or cell proliferative disorder.

48. The use of item 46, wherein said pathological condition associated with angiogenesis is an intraocular neovascular disease.

Brief Description of Drawings

FIG. 1: heavy and light chain HVR loop sequences of anti-EphrinB 2 antibodies. The figure shows the heavy chain HVR sequences, i.e., H1, H2, and H3, and the light chain HVR sequences, i.e., L1, L2, and L3. The sequence numbers are as follows: clone 31.19 (HVR-H1 is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 3; HVR-H3 is SEQ ID NO: 5; HVR-L1 is SEQ ID NO: 6; HVR-L2 is SEQ ID NO: 8; HVR-L3 is SEQ ID NO: 10); clone 31.19.1D8 (HVR-H1 is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 3; HVR-H3 is SEQ ID NO: 5; HVR-L1 is SEQ ID NO: 7; HVR-L2 is SEQ ID NO: 9; HVR-L3 is SEQ ID NO: 11); clone 31.19.2D3 (HVR-H1 is SEQ ID NO: 2; HVR-H2 is SEQ ID NO: 4; HVR-H3 is SEQ ID NO: 5; HVR-L1 is SEQ ID NO: 6; HVR-L2 is SEQ ID NO: 8; HVR-L3 is SEQ ID NO: 12).

Amino acid sequence positions are numbered according to the Kabat numbering system described below.

FIGS. 2A, 2B and 3 depict exemplary acceptor human consensus framework sequences for practicing the present invention, with the sequence identifiers as follows:

heavy chain Variable (VH) consensus framework (FIGS. 2A, 2B)

Human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NO:19)

Human VH subgroup I consensus framework minus extended hypervariable regions (SEQ ID NOS: 20-22)

Human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID NO:23)

Human VH subgroup II consensus framework minus extended hypervariable regions (SEQ ID NOS: 24-26)

Human VH subgroup II consensus framework minus extension

Human subgroup III consensus framework minus Kabat CDR (SEQ ID NO:27)

Human VH subgroup III consensus framework minus extended hypervariable regions (SEQ ID NOS: 28-30)

Human VH acceptor framework minus Kabat CDRs (SEQ ID NO:31)

Human VH acceptor framework minus extended hypervariable regions (SEQ ID NOS: 32-33)

Human VH receptor 2 framework minus Kabat CDRs (SEQ ID NO:34)

Human VH receptor 2 framework minus extended hypervariable region (SEQ ID NO:35-37)

Light chain Variable (VL) consensus framework (FIG. 3)

Human VL kappa subgroup I consensus framework (SEQ ID NO:38)

Human VL kappa subgroup II consensus framework (SEQ ID NO:39)

Human VL kappa subgroup III consensus framework (SEQ ID NO:40)

Human VL kappa subgroup IV consensus framework (SEQ ID NO:41)

FIG. 4 depicts the framework region sequences of the light and heavy chains of huMAb4D 5-8. Superscript/bold numerical values indicate amino acid positions according to Kabat.

FIG. 5 depicts the light and heavy chains of huMAb4D5-8Modified/variantFramework region sequences. Superscript/bold numerical values indicate amino acid positions according to Kabat.

FIG. 6 depicts the light chain variable region (SEQ ID NO:48) and the heavy chain variable region (SEQ ID NO:49) of anti-EphrinB 2 monoclonal antibody clone 31.19, the light chain variable region (SEQ ID NO:50) and the heavy chain variable region (SEQ ID NO:51) of anti-EphrinB 2 monoclonal antibody clone 31.19.1D8, and the light chain variable region (SEQ ID NO:52) and the heavy chain variable region (SEQ ID NO:53) of anti-EphrinB 2 monoclonal antibody clone 31.19.2D.

Figure 7 depicts treatment with an anti-ephrinB 2 monoclonal antibody blocking EphB4 receptor-ephrinB 2 ligand signaling in a cell-based assay.

Figure 8 depicts treatment with an anti-ephrinB 2 monoclonal antibody reduces angiogenesis in the rat corneal pocket assay.

Figure 9 depicts the inhibition of tumor growth in vivo by treatment with an anti-ephrinB 2 monoclonal antibody.

Detailed Description

The invention provides anti-EphrinB 2 antibodies that are useful, for example, in the treatment or prevention of disease states associated with EphrinB2 expression and/or activity (such as elevated expression and/or activity or unwanted expression and/or activity). In some embodiments, the antibodies of the invention are used to treat tumors, cancers, and/or cell proliferative disorders.

In another aspect, the anti-EphrinB 2 antibodies of the invention can be used as reagents for detecting and/or isolating EphrinB2, such as detecting EphrinB2 in various tissues and cell types.

The invention further provides methods of making anti-EphrinB 2 antibodies, and polynucleotides encoding anti-EphrinB 2 antibodies.

General technique

The techniques and protocols described or referenced herein are generally well understood by those skilled in the art and are generally employed using conventional methods, such as, for example, the widely used methods described in the following documents: a Laboratory Manual3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y; CURRENT promoters IN MOLECULAR BIOLOGY (f.m. ausubel, et al. eds., (2003)); book of protocols IN enzymolygy (Academic Press, Inc.: PCR2: A PRACTICAL APPROACH (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)); harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL; and ANIMAL CELL CULTURE (r.i. freshney, ed. (1987)).

Definition of

An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified (1) to more than 95% by weight, most preferably more than 99% by weight of the antibody as determined by the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a rotor sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions and staining with Coomassie blue or preferably silver. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, an isolated antibody will typically be prepared by at least one purification step.

An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the natural source of antibody nucleic acid. An isolated nucleic acid molecule is distinguished from the form or context in which it is found in nature. An isolated nucleic acid molecule is thus distinguished from a nucleic acid molecule when present in a natural cell. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that normally expresses the antibody, for example, when the chromosomal location of the nucleic acid molecule in the cell is different from its chromosomal location in a native cell.

The term "variable domain residue numbering according to Kabat" or "amino acid position numbering according to Kabat" and variants thereof refers to the numbering system for heavy or light chain variable domains by antibody editing as in Kabat et al, Sequences of Proteins of Immunological Interest,5th ed. Using this numbering system, the actual linear amino acid sequence may comprise fewer or additional amino acids, corresponding to a shortening or insertion of the variable domain FR or CDR. For example, the heavy chain variable domain may comprise a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat, etc.) after heavy chain FR residue 82. The Kabat residue numbering of a given antibody can be determined by aligning the antibody sequences to the regions of homology with "standard" Kabat numbered sequences.

The phrase "substantially similar" or "substantially the same" as used herein means a sufficiently high degree of similarity between two numerical values (typically one relating to an antibody of the invention and the other relating to a reference/comparison antibody) such that one of skill in the art would consider the difference between the two numerical values to be of little or no biological and/or statistical significance within the context of the biological property measured by the numerical values (e.g., Kd values). The difference between the 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 of the reference/comparison antibody.

"binding affinity" generally refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigen slowly and tend to dissociate readily, while high affinity antibodies generally bind antigen more rapidly and tend to remain bound for a longer period of time. 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 exemplary embodiments are described below.

In one embodiment, the "Kd" or "Kd value" according to the present invention is measured by a radiolabelled antigen binding assay (RIA) carried out using Fab-format antibodies of interest and their antigens as described in the following assays: by using the minimum concentration of the antigen in the presence of a titration series of unlabelled antigen¹²⁵I labelling of the antigen equilibrated Fab, and then capturing the bound antigen with anti-Fab antibody coated plates to measure the solution binding affinity of the Fab for the antigen (Chen, et al, J Mol Biol293:865-881 (1999)). To determine assay conditions, microtiter plates (Dynex) were coated with anti-Fab antibodies (Cappellabs) for capture at 5. mu.g/ml in 50mM sodium carbonate (pH9.6) overnight, followed by blocking with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). In a non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]Antigen with serial dilutions of Fab of interestMixing (e.g., consistent with the evaluation of anti-VEGF antibodies, Fab-12, by Presta et al, Cancer Res.57: 4593-. Then keeping the temperature of the target Fab overnight; however, the incubation may continue for a longer period of time (e.g., 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for room temperature incubation (e.g., 1 hour). The solution was then removed and the plate washed 8 times with PBS containing 0.1% Tween-20. After the plates were dried, 150. mu.l/well scintillation fluid (MicroScint-20; Packard) was added and the plates were counted for 10 minutes on a Topcount Gamma counter (Packard). The concentration at which each Fab gives less than or equal to 20% of the maximum binding is selected for use in competitive binding assays. According to another embodiment, the Kd or Kd value is determined by surface plasmon resonance assay using BIAcore ^TM-2000 or BIAcore^TM-3000(BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor 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 antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% Tween-20 were injected at 25 ℃ at a flow rate of about 25. mu.l/min. The binding rate (k) was calculated by simultaneous fitting of the binding and dissociation sensorgrams using a simple one-to-one Langmuir (Langmuir) binding model (BIAcoreevaluation Software version3.2)_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen, Y., et al, J Mol Biol293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay ⁶M^-1S^-1The rate of binding can then be determined using fluorescence quenching techniques, i.e.according to a spectrometer such as a spectrophotometer equipped with a flow-breaking device (Aviv Inst)Fragments) or 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) in the presence of increasing concentrations of antigen, measuring the fluorescence emission intensity of PBS, 20nM anti-antigen antibody (Fab form) in ph7.2 at 25 ℃ (excitation =295 nM; emission =340nm, 16nm bandpass).

An "association rate" or "k" according to the present invention_on"BIAcore can also be used by the same surface plasmon resonance technique as described above^TM-2000 or BIAcore^TM-3000(BIAcore, inc., Piscataway, NJ) was determined at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor 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 antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% Tween-20 were injected at 25 ℃ at a flow rate of about 25. mu.l/min. The binding rate (k) was calculated by simultaneous fitting of the binding and dissociation sensorgrams using a simple one-to-one Langmuir (Langmuir) binding model (BIAcore Evaluation Software version3.2) _on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen, Y., et al, J Mol Biol293:865-881 (1999). However, if according to the above surface plasmon resonance determination method, the binding rate exceeds 10⁶M^-1S^-1The rate of binding is then preferably determined using fluorescence quenching techniques, i.e.measuring PBS in the presence of increasing concentrations of antigen, pH7.2, according to a measurement in a spectrometer such as a spectrophotometer equipped with a flow cut-off device (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer (Thermospectronic) with a stirred cuvetteOf 20nM anti-antigen antibody (Fab form) at 25 ℃ fluorescence emission intensity (excitation =295 nM; emission =340nM, 16nM band pass) was increased or decreased.

The term "vector" as used herein means a nucleic acid molecule capable of transporting other nucleic acids to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., 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 upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably 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 often in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably, as plasmids are the most commonly used form of vector.

"Polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and/or analogs thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. Modifications to the nucleotide structure, if any, may be made before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, with an uncharged linkage (e.g., methyl phosphonate)Esters, phosphotriesters, phosphoramidates (phosphoramidates), carbamates, etc.) and modifications with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), modifications containing pendant moieties (e.g., such as, for example, modifications of proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications with intercalators (e.g., acridine, psoralen, etc.), modifications containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), modifications containing alkylators, modifications with modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of the polynucleotide. In addition, any hydroxyl groups typically present in sugars may be replaced with, for example, phosphonic acid (phosphonate) groups, phosphoric acid (phosphonate) groups, protected with standard protecting groups, or activated to make additional linkages to additional nucleotides, or may be coupled to solid and semi-solid supports. The 5 'and 3' terminal OH groups may be phosphorylated or substituted with amines or organic capping group moieties of 1-20 carbon atoms. Other hydroxyl groups can also be derivatized to standard protecting groups. Polynucleotides may also contain analog forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2 '-O-methyl, 2' -O-allyl, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methylribonucleosides. One or more phosphodiester linkages may be replaced with alternative linking groups. Such alternative linking groups include, but are not limited to, embodiments wherein the phosphate ester is substituted with P (O) S ("thioester"), P (S) S ("dithioate"), (O) NR ₂("amide ester"), P (O) R, P (O) OR', CO OR CH₂(formacetal)) wherein R or R' are each independently H or substituted or unsubstituted alkyl (1-20C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl (araldyl). Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

"oligonucleotide" as used herein generally refers to short polynucleotides, generally single-stranded, generally synthetic, generally but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides is equally and fully applicable to oligonucleotides.

"percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared. However, for purposes of the present invention,% amino acid sequence identity values are obtained using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table A below. The ALIGN-2 sequence comparison computer program was written by Genentech corporation and the source code shown in table a below has been submitted to the US copyright office (US CopyrightOffice, Washington d.c.,20559) along with the user's text file and registered with US copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech corporation (South San Francisco, Calif.) or may be compiled from source code provided in the following table. The ALIGN2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

TABLE A

In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a with respect to, with, or against a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the A and B alignments of this program, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not be equal to the% amino acid sequence identity of B relative to a.

Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "EphrinB 2" (interchangeably referred to as "EphrinB 2 ligand"), as used herein, refers to any natural or variant (whether natural or synthetic) EphrinB2 polypeptide, unless otherwise specifically indicated or the context indicates otherwise. The term "native sequence" specifically encompasses naturally occurring truncated or secreted forms (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants. The term "wild-type EphrinB 2" generally refers to a polypeptide comprising the amino acid sequence of a naturally occurring EphrinB2 protein. The term "wild-type EphrinB2 sequence" generally refers to the amino acid sequence found in naturally occurring EphrinB 2.

The term "Eph receptor" (such as an EphB receptor, such as EphB1, EphB2, and/or EpgB 3), as used herein, refers to any natural or variant (whether natural or synthetic) Eph receptor polypeptide, unless otherwise expressly stated or the context indicates otherwise. The term "native sequence" specifically encompasses naturally occurring truncated or secreted forms (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants. The term "wild-type Eph receptor" generally refers to a polypeptide comprising the amino acid sequence of a naturally occurring Eph receptor protein. The term "wild-type Eph receptor sequence" generally refers to an amino acid sequence found in a naturally occurring Eph receptor.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), and may also include certain antibody fragments (as described in more detail herein). The antibody may be human, humanized and/or affinity matured.

The term "variable" refers to the fact that certain portions of the variable domains differ widely between antibody sequences and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of the antibodies. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light chain and heavy chain variable domains. The more highly conserved portions of the variable domains are called the Framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet conformation, connected by three CDRs which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR region and, together with the CDRs of the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of proteins of Immunological Interest, 5 th edition, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as antibody participation in cells of antibody-dependent cells.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having an antigen-binding site, and a remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produced an F (ab') ₂A fragment which has two antigen binding sites and is still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. In the two-chain Fv species, this region consists of a dimer of one heavy and one light variable domain in tight, non-covalent association. In single-chain Fv species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker, such that the light and heavy chains associate in a "dimeric" structure analogous to that of a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. The six CDRs collectively confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, with only a lower affinity than the entire binding site.

The Fab fragment also comprises the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments inA few residues have been added to the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domain carry a free thiol group. F (ab') ₂Antibody fragments were originally generated as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), depending on the amino acid sequence of their constant domains.

Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The constant domains of the heavy chains corresponding to different classes of immunoglobulins are referred to as α, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

An "antibody fragment" comprises only a portion of an intact antibody, wherein said portion preferably retains at least one, preferably most or all of the functions normally associated with the portion when present in an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment comprises the antigen binding site of an intact antibody, such that the ability to bind antigen is retained. In another embodiment, an antibody fragment, e.g., an antibody fragment comprising an Fc region, retains at least one biological function normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to that of an intact antibody. For example, such an antibody fragment may comprise an antigen binding arm And which is 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 an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Typically, antibodies comprise six hypervariable regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). A description of many hypervariable regions is used and encompassed herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the position of the structural loops (Chothia and Lesk, J.mol.biol.196:901-917 (1987)). The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of the available complex crystal structure. The residues for each of these hypervariable regions are noted below.

Hypervariable regions can comprise "extended hypervariable regions" as follows: 24-36 or 24-34(L1), 46-56 or 50-56(L2) and 89-97(L3) in VL and 26-35(H1), 50-65 or 49-65(H2) and 93-102, 94-102 or 95-102(H3) in VH. For each of these definitions, the variable region residues are numbered according to Kabat et al, supra.

"framework" or "FR" residues refer to those residues in the variable domain other than the hypervariable region residues defined herein.

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are those in which residues from a hypervariable region of a human immunoglobulin (recipient antibody) are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see Jones et al, Nature321:522-525(1986); Riechmann et al, Nature332:323-329(1988); and Presta, curr Op, struct, biol.2:593-596 (1992). See also the following reviews and references cited therein: vaswani and Hamilton, Ann. allergy, Asthma & Immunol.1: 105-.

A portion of the heavy and/or light chain in a "chimeric" antibody (immunoglobulin) is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl.Acad.Sci.81: 6851. 6855 (1984)). As used herein, humanized antibodies are a subset of chimeric antibodies.

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein the domains are present on a single polypeptide chain. In general, scFv polypeptides are at V_HAnd V_LAlso included between the domains is a polypeptide linker enabling scFv formationThe structure required for binding the antigen. For an overview of scFv see Pluckthun, Springer-Verlag, New York, pp.269-315(1994), edited by Rosenburg and Moore, volume 113 of The Pharmacology of monoclonal antibodies.

"antigen" refers to a predetermined antigen to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. Preferably, the target antigen is a polypeptide.

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments are in the same polypeptide chain (V)_H-V_L) Comprising a linked heavy chain variable domain (V)_H) And a light chain variable domain (V)_L). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain, thereby creating two antigen binding sites. Diabodies are described more fully in, for example, EP404,097, WO93/11161, Hollinger et al, Proc. Natl. Acad. Sci. USA90:6444-6448 (1993).

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more CDRs of the antibody that result in an improvement in the affinity of the antibody for an antigen as compared to a parent antibody without the alterations. Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies can be generated by procedures known in the art. Marks et al, Bio/Technology10:779-783(1992) describe affinity maturation by VH and VL domain shuffling. The following documents describe random mutagenesis of CDR and/or framework residues: barbas et al, Proc.Nat.Acad.Sci.USA91: 3809-.

Antibody "effector functions" refer to those biological activities that can be attributed to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region) and vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted Ig bound to Fc receptors (fcrs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. The antibody "arms" (arm) cytotoxic cells and is absolutely required for such killing. The main cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravechand Kinet, annu.rev.immunol.9:457-92(1991) page 464 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. Pat. No.5,500,362 or 5,821,337 or Presta U.S. Pat. No.6,737,056. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models such as those disclosed in Clynes et al, PNAS (USA)95: 652-.

"human effector cells" refer to leukocytes which express one or more fcrs and which exert effector function. Preferably, the cell expresses at least Fc γ RIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMC), Natural Killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, preferably PBMC and NK cells. The effector cells may be isolated from their natural source, e.g., blood.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds an IgG antibody (gamma receptor), including receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see for review) Annu.Rev.Immunol.15:203-234 (1997)). For an overview of FcRs see ravech and Kinet, Annu.Rev.Immunol.9:457-492(1991); Capel et al, Immunomethods4:25-34(1994); de Haas et al, J.Lab.Clin.Med.126:330-41 (1995). The term "FcR" encompasses other fcrs herein, including those that will be identified in the future. 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 for the regulation of immunoglobulin homeostasis. WO00/42072(Presta) describes antibody variants with increased or decreased binding to FcR. The contents of this patent publication are expressly incorporated herein by reference. See also, Shields et al, J.biol.chem.9(2):6591-6604 (2001).

Methods for measuring binding to FcRn are known (see e.g. Ghetie1997, Hinton 2004). The in vivo binding and serum half-life of human FcRn high affinity binding polypeptides to human FcRn can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with the Fc variant polypeptides.

"complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Initiation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to the antibody (of the appropriate subclass) to which its cognate antigen binds. To assess complement initiation, CDC assays may be performed, for example as described in Gazzano-Santoro et al, j.immunol.methods202:163 (1996).

Polypeptide variants with altered Fc region amino acid sequence and increased or decreased C1q binding ability are described in U.S. patent nos. 6,194,551B1 and WO 99/51642. The contents of those patent publications are expressly incorporated herein by reference. See also Idusogene et al, J.Immunol.164: 4178-.

An "Fc region-containing polypeptide" refers to a polypeptide comprising an Fc region, such as an antibody or immunoadhesin (see definition below). The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be eliminated, for example, during purification of the polypeptide or by recombinant engineering of the nucleic acid encoding the polypeptide. Thus, a composition comprising a polypeptide having an Fc region according to the invention may comprise a polypeptide having K447, a polypeptide that eliminates all K447, or a mixture of polypeptides having and without the K447 residue.

A "blocking" antibody or an "antagonist" antibody refers to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonistic antibodies substantially (substentially) or completely inhibit the biological activity of the antigen.

"agonistic antibodies" as used herein refers to antibodies that mimic at least one functional activity of a polypeptide of interest.

For purposes herein, an "acceptor human framework" is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence as it, or may comprise pre-existing amino acid sequence variations. When there are pre-existing amino acid changes, preferably there are no more than 5, preferably 4 or fewer, or 3 or fewer pre-existing amino acid changes. When pre-existing amino acid changes are present in the VH, preferably those changes are located only at three, two or one of positions 71H, 73H and 78H; for example, the amino acid residues at those positions may be 71A, 73T and/or 78A. In one embodiment, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"human consensus framework" refers to a framework representing the most common amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable region sequences. Typically, the sequence subtypes are subtypes such as Kabat et al. In one embodiment, for VL, the subtype is subtype kappa I as in Kabat et al. In one embodiment, for the VH, the subtype is subtype III as in Kabat et al.

The "VH subgroup III consensus framework" comprises a consensus sequence obtained from the amino acid sequences in variable heavy chain subgroup III of Kabat et al. In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a portion of, or all of, the following sequences:

EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:42)-H1-WVRQAPGKGLEWV(SEQ ID NO:43)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:44)-H3-WGQGTLVTVSS(SEQ ID NO:45)。

the "VL subgroup I consensus framework" comprises the consensus sequence obtained from the amino acid sequences in variable light chain kappa subgroup I of Kabat et al. In one embodiment, the VL subtype I consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences:

DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:15)-L1-WYQQKPGKAPKLLIY(SEQ ID NO:16)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQID NO:17)-L3-FGQGTKVEIK(SEQ ID NO:18)。

"disorder" or "disease" refers to any condition that would benefit from treatment with the agents/molecules or methods of the invention. This includes chronic and acute conditions or diseases, including those pathological conditions that predispose a mammal to the condition in question. Non-limiting examples of conditions to be treated herein include malignant and benign tumors; carcinomas, blastomas, and sarcomas.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

"tumor" as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancerous) and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma (hepatic carcinosoma), gastric cancer, melanoma, and various types of head and neck cancer.

Deregulation of angiogenesis can result in a number of conditions that can be treated by the compositions and methods of the present invention. These disorders include non-neoplastic and neoplastic disorders. Neoplastic disorders include, but are not limited to, those described above. Non-neoplastic conditions include, but are not limited to, unwanted or abnormal hypertrophy, arthritis, Rheumatoid Arthritis (RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft rejection, retinal/choroidal neovascularization, neovascularization of the corners of the eye (rubeosis), ocular diseases, vascular restenosis, arteriovenous malformations (AVM), meningiomas, hemangiomas, angiofibromas, thyroid hyperplasia (including Graves' disease), corneal and other tissue transplants, chronic inflammation, pneumonia, acute lung injury/ARDS, Sepsis, primary pulmonary hypertension, malignant pulmonary effusion (malignant pulmony effusions), cerebral edema (e.g., associated with acute stroke/closed head trauma/trauma), synovial inflammation, pannus formation in RA, myositis ossificans, hypertrophic bone formation, Osteoarthritis (OA), refractory ascites, polycystic ovarian disease, endometriosis, third space fluid disease (3rd spacing of fluid diseases) (pancreatitis, compartment syndrome, burns, enteropathy), uterine fibroids (uterine fibroids), premature labor, chronic inflammation such as IBD (Crohn's disease and ulcerative colitis), renal allograft rejection, inflammatory bowel disease, nephrotic syndrome, unwanted or abnormal tissue mass growth (noncancerous), hemophilic joints, thick scars of hair, inhibition of hair growth, oly-Weber syndrome, Osler-Weber syndrome, Retrolental fibroplasia of pyogenic granulomas, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion.

The terms "neurodegenerative (degenerative) disease" and "neurodegenerative (degenerative) disorder" are used in the broadest sense, including all disorders whose pathology involves neuronal degeneration and/or dysfunction, including but not limited to peripheral neuropathy; motor neuron disorders such as amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Bell's (Bell) paralysis, and various disorders 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.

"peripheral neuropathy" refers to a neurodegenerative disorder affecting peripheral nerves, most commonly manifested as one or a combination of motor, sensory, sensorimotor, or autonomic dysfunction. Peripheral neuropathy may be, for example, genetically acquired, may originate from systemic diseases, or may be induced by toxic agents such as neurotoxic drugs, e.g., antineoplastic agents, or industrial or environmental pollutants. "peripheral sensory neuropathy" is characterized by degeneration of peripheral sensory neurons, which may be idiopathic, and may be used as, for example, a cytostatic drug therapy (e.g., treatment with a chemotherapeutic agent, such as vincristine, cisplatin, methotrexate, 3 '-azido-3' -deoxythymidine, or a taxane, such as paclitaxel (paclitaxel), "for example Bristol-Myers Squibb Oncology,Princeton,N.J.]And docetaxel (doxetaxel), [ solution of doxetaxel and a pharmaceutically acceptable salt thereofRhne-Poulenc Rorer,Antony,France]) Alcohol intoxication, Acquired Immune Deficiency Syndrome (AIDS), or genetic predisposition. Peripheral neuropathies obtained by genetics include, for example, Refsum's disease, Krabbe's disease, metachromatic leukodystrophy, Fabry's (Fabry) disease, Dejerine-Sottas syndrome, betalipoproteinemia, and Charcot-mary-Tooth (CMT) disease (also known as protein muscular atrophy or Hereditary Motor Sensory Neuropathy (HMSN)). Most types of peripheral neuropathy develop/develop slowly with a course of months or years. At presentIn bed practice, such neuropathy is called chronic. Peripheral neuropathy sometimes occurs/develops rapidly, with a course of days, called acute. Peripheral neuropathies often affect both sensory and motor nerves, resulting in a mixed type of sensory and motor neuropathy, but both purely sensory and purely motor neuropathies are also known.

As used herein, "treatment" or "treating" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, which may be for the purpose of prevention or in the course of clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and remission or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the onset/progression of a disease or disorder.

"individual" refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, livestock (such as cattle), sport animals, pets (such as cats, dogs, and horses), primates, mice, and rats.

For the purpose of treatment, "mammal" refers to any animal classified as a mammal, including humans, domestic and livestock animals, and zoo, sports or pet animals, such as dogs, horses, cats, cattle, etc. Preferably, the mammal is a human.

An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic effect.

The "therapeutically effective amount" of a substance/molecule, antagonist or agonist of the invention may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount also refers to an amount of the substance/molecule, agonist or antagonist that outweighs any toxic or detrimental effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic effect. Typically, but not necessarily, since the prophylactic dose is administered to the subject prior to the onset of the disease or at an early stage of the disease, the prophylactically effective amount will be lower than the therapeutically effective amount.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of a cell and/or causes destruction of a cell. The term is intended to include: radioisotopes, e.g. At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²And radioactive isotopes of Lu; chemotherapeutic agents, such as methotrexate (methotrexate), doxorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antineoplastic or anticancer agents disclosed hereinafter. Other cytotoxic agents are described below. Tumoricidal agents cause destruction of tumor cells.

"chemotherapeutic agent" refers to a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and thiotepaCyclophosphamide (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa), and uretepa (uredepa); ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide amide), triethylenethiophosphoramide (triethylenethiophosphoramide), and trimethylolmelamine (trimethlomelamine); annonaceous acetogenins (especially bullatacin and bullatacin); -9-tetrahydrocannabinol (dronabinol),beta-lapachone (lapachone); lapachol (lapachol); colchicines (colchicines); betulinic acid (betulinic acid); camptothecin (camptothecin) (including the synthetic analogue topotecan)CPT-11 (irinotecan),acetyl camptothecin, scopoletin (scopoletin), and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); podophyllotoxin (podophylotoxin); podophyllinic acid (podophyllic acid); teniposide (teniposide); cryptophycins (especially cryptophycins 1 and 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin (spongistatin); nitrogen mustards (nitrosgen mustards), such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (l) omustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics such as enediynes antibiotics (enediynes) (e.g., calicheamicins, especially calicheamicin γ 1I and calicheamicin ω I1 (see, e.g., Agnew, chem. Intl. Ed. Engl.33:183-186(1994)), anthracyclines (dynemicin), including dynemicin A; epothilones (esperamicin), and neocarzinostatin (neocarzinostatin) and related chromoproteenediynes chromophores), aclacins (aclacinomycin), actinomycins (actinomycin), anidamycin (antrhramycin), azaserine (azacine), bleomycin (bleomycin), actinomycins C (cacodymycin), carbamycin, caroicins (carminomycin), daunomycins (carmycin), daunomycins (daunomycin D-5), daunomycins (daunomycin-D-6-D), daunomycins (daunomycinin, daunomycin (daunomycin, daunomycin,Doxorubicin (doxorubicin) (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcellomomycin), mitomycins (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (pelomycin), pelomycin (polyplomycin), potfiromycin, puromycin (puromycin), triiron doxorubicin (quelamycin), rodobicin (rodorubicin), streptonigrin (streptagrin), streptozocin (streptazocin), tubercidin (tunicin), ubenidin (metrizax), zocin (zostatin), zorubicin (zorubicin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as, for example, ancitabine (ancitabine), Azacitidine (azacitidine), 6-azauridine (azauridine), carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; epothilone (epothilone); etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mogradol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine); Polysaccharide complex (JHS natural products, Eugene, OR); razoxane (rizoxane); rhizomycin (rhizoxin); sizofuran (sizofiran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2',2' ' -trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin A, rorodin A and snake-fish (anguidin);

urethane (urethan); changchun wineDixin (vindesine) ((B))) Dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); piperobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); thiotepa (thiotepa); taxoids, e.g. taxolPaclitaxel (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE^TMWithout Cremophor, albumin engineered nanoparticle dosage forms of paclitaxel (American pharmaceutical Partners, Schaumberg, Illinois) anddocetaxel (doxetaxel) ((doxetaxel))Rorer, antonyy, France); chlorambucil (chlorambucil); gemcitabine (gemcitabine)6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs such as cisplatin (cissplatin) and carboplatin (carboplatin); vinblastine (vinblastine) Platinum (platinum); etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); vincristine (vincristine)Oxaliplatin (oxaliplatin); leucovorin (leucovorin); vinorelbine (vinorelbine)Oncostatin (novantrone); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); ibandronate (ibandronate); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids (retinoids), such as retinoic acid (retinoic acid); capecitabine (capecitabine)A pharmaceutically acceptable salt, acid or derivative of any of the foregoing; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy) and FOLFOX (oxaliplatin)^TM) Abbreviation for treatment regimen combining 5-FU and folinic acid).

The definition also includes anti-hormonal agents that act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic treatment. They may themselves be hormones. Examples include antiestrogens and Selective Estrogen Receptor Modulators (SERMs), including for example tamoxifen (tamoxifen) (including Tamoxifen) and,Raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxyttamoxifen, trioxifene (trioxifene), naloxifene (keoxifene), LY117018, onapristone (onapristone), andtoremifene (toremifene); anti-pregnenones; estrogen receptor down-regulators (ERD); agents acting to inhibit or shut down the ovary, e.g. Luteinizing Hormone Releasing Hormone (LHRH) agonists, such asAndleuprolide acetate, goserelin acetate, buserelin acetate and triptorelin acetate; anti-androgens such as flutamide (flutamide), nilutamide (nilutamide), and bicalutamide (bicalutamide); other anti-androgens, such as flutamide (flutamide), nilutamide (nilutamide), and bicalutamide (bicalutamide); and aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal gland, such as, for example, 4(5) -imidazole, aminoglutethimide,Megestrol acetate (megestrol acetate),Exemestane (exemestane), formestane (formestane), fadrozole (fadrozole),Vorozole (vorozole), Letrozole (letrozole) andanastrozole (anastrozole). In addition, this definition of chemotherapeutic agents includes diphosphonates such as clodronate (e.g., clodronate)Or）、Etidronate (etidronate), NE-58095, and,Zoledronic acid/zoledronate,Alendronate (alendronate),Pamidronate (pamidronate),Tiludronate (tiludronate) orRisedronate (risedronate); and troxacitabine (a 1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in adherent cell proliferation, such as, for example, PKC- α, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines, e.g.Vaccines and gene therapy vaccines, e.g.A vaccine,A vaccine anda vaccine;a topoisomerase 1 inhibitor;rmRH, lapatinib ditosylate (ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor, also known as GW 572016); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

"growth inhibitory agent" as used herein refers to a compound or composition that inhibits the growth of a cell (such as a cell expressing EphrinB 2) in vitro or in vivo. Thus, the growth inhibitory agent may be one that significantly reduces the percentage of cells in S phase (such as cells expressing EphrinB 2). Examples of growth inhibitory agents include agents that block cell cycle progression (at a position outside the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes (taxanes), and topoisomerase II inhibitors such as doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide), and bleomycin (bleomycin). Those agents that block G1 also spill over into S phase arrest, for example, DNA alkylating agents such as tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), mechloroethylmethylamine (mechloroethylamine), cisplatin (cissplatin), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil), and ara-C. For more information see The "Molecular Basis of Cancer", eds. Mendelsohn and Israel, Chapter 1 entitled "cell regulation, oncogenes, and antisense drugs", Murakani et al, WB saunders, Philadelphia, 1995, especially page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel derived from taxus baccata (c) Rhone-Poulenc Rorer) is paclitaxel (paclitaxel)Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerizationAnd, in turn, results in the inhibition of mitosis in the cell.

"Doxorubicin (Doxorubicin)" is an anthracycline antibiotic. The full chemical name of doxorubicin is (8S-cis) -10- [ (3-amino-2, 3, 6-trideoxy- α -L-lysu-hexopyranosyl) oxy ] -7,8,9, 10-tetrahydro-6, 8, 11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5, 12-naphthalenedione.

(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione

The term "anti-tumor composition" refers to a composition useful for treating cancer, which comprises at least one active therapeutic agent, such as an "anti-cancer agent. Examples of therapeutic agents (anti-cancer agents, also referred to herein as "anti-neoplastic agents") include, but are not limited to, agents such as chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, toxins, and other agents used to treat cancer such as anti-VEGF neutralizing antibodies, VEGF antagonists, anti-HER-2, anti-CD 20, epidermal Growth Factor Receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1/EGFR inhibitors, erlotinib, COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more targets of ErbB2, ErbB3, ErbB4, or VEGF receptors, platelet-derived growth factor (PDGF), and/or inhibitors of Stem Cell Factor (SCF) receptor tyrosine kinases (e.g., imatinib mesylate). ) TRAIL/Apo2, and other biologically active and organic chemical agents, and the like.

The term "prodrug" as used herein refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is capable of being enzymatically activated or converted to a more active parent drug form. See, e.g., 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, eds., pp. 247-267, Humana Press (1985). Prodrugs of the present 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, β -lactam-containing prodrugs, prodrugs containing optionally substituted phenoxyacetamide or prodrugs containing optionally substituted phenylacetamide, 5-fluorocytosine and other 5-fluorouridine prodrugs convertible to more active, non-cytotoxic drugs. Examples of cytotoxic drugs that may be derivatized into prodrug forms for use in the present invention include, but are not limited to, those chemotherapeutic agents described above.

An "anti-angiogenic agent" or "angiogenesis inhibitor" refers to a small molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody, or conjugate or fusion protein thereof that inhibits, either directly or indirectly, angiogenesis (vasculogenesis), or unwanted vascular permeability. For example, the anti-angiogenic agent is an antibody or other antagonist of an angiogenic agent as defined above, e.g., an antibody to VEGF receptor, a small molecule that blocks VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, SUTENT/SU11248 (sunitinib), AMG 706). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin (angiostatin), endostatin (endostatin), and the like. See, e.g., Klagsbrun and D' Amore Annu. Rev. physiol.53: 217-39 (1991); Streit and Detmar Oncogene 22:3172-3179(2003) (e.g., Table 3 listing anti-angiogenic therapies in malignant melanoma), Ferrara & AlitaloNature Medicine 5(12):1359-1364 (1999); Tonini et al Oncogene 22:6549-6556(2003) (e.g., Table 2 listing anti-angiogenic factors), Sato int. J. Clin. oncol.8: 200-206(2003) (e.g., Table 1 listing anti-angiogenic agents used in clinical trials).

Compositions of the invention and methods of making the same

The invention encompasses compositions, including pharmaceutical compositions, comprising anti-EphrinB 2 antibodies; and polynucleotides comprising the coding sequence of an anti-EphrinB 2 antibody. As used herein, a composition comprises one or more antibodies that bind EphrinB2, and/or one or more polynucleotides comprising sequences encoding one or more antibodies that bind EphrinB 2. These compositions may further comprise suitable carriers such as pharmaceutically acceptable excipients, including buffers, which are well known in the art.

The invention also encompasses embodiments of the isolated antibodies and polynucleotides. The invention also encompasses embodiments of substantially pure antibodies and polynucleotides.

The anti-EphrinB 2 antibodies of the invention are preferably monoclonal. The scope of the invention also encompasses Fab, Fab '-SH and F (ab')₂. These antibody fragments may be created by conventional means, such as enzymatic digestion, or may be generated by recombinant techniques. Such antibody fragments may be chimeric or humanized. These fragments are useful for diagnostic and therapeutic purposes as set forth below.

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of different or polyclonal antibodies.

The anti-EphrinB 2 monoclonal antibodies of the invention can be prepared using the hybridoma method originally described by Kohler et al, Nature256:495(1975), or can be prepared by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies to EphrinB2 are typically generated by multiple subcutaneous (sc) or intraperitoneal (ip) injections of EphrinB2 and an adjuvant in animals. EphrinB2 can be prepared using methods well known in the art, some of which are described herein. For example, recombinant production of EphrinB2 is described below. In one embodiment, the animal is immunized with an EphrinB2 derivative comprising EphrinB2 extracellular domain (ECD) fused to the Fc portion of an immunoglobulin heavy chain. In a preferred embodiment, the animal is immunized with the EphrinB2-IgG1 fusion protein. Animals are typically immunized against an immunogenic conjugate or derivative of EphrinB2 with monophosphoryl lipid a (MPL)/Trehalos Dicrynomycolate (TDM) (ribi immunochem. research, inc., Hamilton, MT) and solutions injected intradermally at multiple sites. After 2 weeks, animals were boosted. After 7-14 days, the animals were bled and anti-EphrinB 2 titers were determined. Animals were boosted until the titer reached a plateau (plateaus).

Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, monoclonal antibodies: Principles and Practice, pp.59-103, Academic Press, 1986).

The hybridoma cells so prepared are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent HGPRT-deficient cells from growing.

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these cells, preferred myeloma Cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center (San Diego, California, USA) and SP-2 or X63-Ag8-653 cells available from the American Type culture Collection (American Type culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human heteromyeloma cell lines 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., NewYork, 1987).

The culture broth in which the hybridoma cells are growing can be assayed for production of monoclonal antibodies directed against EphrinB 2. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal. biochem.107:220 (1980).

After identification of hybridoma cells producing Antibodies with the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, academic Press, 1986). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo in animals as ascites tumors.

Monoclonal antibodies secreted by the subclones can be suitably separated from the culture fluid, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The anti-EphrinB 2 antibodies of the invention can be created by screening synthetic antibody clones for the desired activity using combinatorial libraries. In principle, synthetic antibody clones are selected by screening phage libraries containing phage displaying various antibody variable region (Fv) fragments fused to phage coat proteins. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and are thus separated from those not bound in the library. The bound clones are then eluted from the antigen and can be further enriched by additional antigen adsorption/elution cycles. Any anti-EphrinB 2 antibody of the invention can be obtained by designing an appropriate antigen screening protocol to select phage clones of Interest, followed by construction of a full-length anti-EphrinB 2 antibody clone using the Fv sequence from the phage clone of Interest and an appropriate constant region (Fc) sequence as described in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication91-3242, Bethesda MD (1991), vols.1-3.

The antigen-binding domain of an antibody is formed by two variable (V) regions of about 110 amino acids, from the heavy (VL) and light (VH) chains, respectively, that both exhibit three hypervariable loops or Complementarity Determining Regions (CDRs). The variable domains can be functionally displayed on phage, either as single chain Fv (scFv) fragments, in which VH and VL are covalently linked by a short, flexible linker, or as Fab fragments, in which they are each 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, scFv-encoding phage clones and Fab-encoding phage clones are collectively referred to as "Fv phage clones" or "Fv clones".

The repertoire of VH and VL genes can be cloned separately by Polymerase Chain Reaction (PCR) and recombined randomly in a phage library, and then can be searched for antigen binding clones as described in Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Libraries from immunized sources can provide antibodies with high affinity for the immunogen without the need to construct hybridomas. Alternatively, the non-immunized repertoire can be cloned to provide a single human antibody source for a wide range of non-self and self antigens without any immunization, as described in Griffiths et al, EMBO J,12:725-734 (1993). Finally, unimmunized libraries can also be constructed synthetically, i.e., by cloning unrearranged V genes from stem cells and using PCR primers comprising random sequences to encode the highly variable CDR3 regions and to effect rearrangement in vitro, as described in Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992).

Filamentous phage was used to display antibody fragments by fusion with the minor coat protein pIII. Antibody fragments may be displayed as single chain Fv fragments in which the VH and VL domains are linked by a flexible polypeptide spacer on the same polypeptide chain, for example as described in Marks et al, J.mol.biol.,222:581-597(1991), or as Fab fragments in which one chain is fused to pIII and the other chain is secreted into the periplasm of the bacterial host cell, where a Fab-coat protein structure is assembled which is displayed on the phage surface by substitution of some wild-type coat protein, for example as described in Hoogenboom et al, Nucl.acids Res.,19:4133-4137 (1991).

Generally, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If it is desired to bias the library toward anti-EphrinB 2 clones, the subject can be immunized with EphrinB2 to generate an antibody response, and spleen cells and/or circulating B cells or other Peripheral Blood Lymphocytes (PBLs) recovered for library construction. In a preferred embodiment, a library of human antibody gene fragments biased toward anti-EphrinB 2 clones is obtained by generating an anti-EphrinB 2 antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that EphrinB2 immunization generates B cells that produce human antibodies against EphrinB 2. The generation of human antibody-producing transgenic mice is described below.

Further enrichment of the anti-EphrinB 2-reactive cell population can be obtained by isolating B-cells expressing EphrinB 2-specific membrane-bound antibodies using a suitable screening protocol, e.g. by cell separation using EphrinB2 affinity chromatography or adsorption of cells to fluorescently labeled EphrinB2 followed by Fluorescence Activated Cell Sorting (FACS).

Alternatively, the use of splenocytes and/or B cells or other PBLs from non-immunized donors provides a better representation of the possible repertoire of antibodies, and also allows the construction of antibody libraries using animal (human or non-human) species in which EphrinB2 is not antigenic. To construct a library of antibody genes incorporated in vitro, stem cells are harvested from a subject to provide nucleic acids encoding unrearranged antibody gene segments. Immune cells of interest can be obtained from a variety of animal species (such as human, mouse, rat, rabbit, luprine, dog, cat, pig, cow, horse, and avian species, among others).

Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. For the rearranged VH and VL gene libraries, the desired DNA may be obtained by isolating genomic DNA or mRNA from lymphocytes followed by Polymerase Chain Reaction (PCR) using primers matched to the 5 'and 3' ends of the rearranged VH and VL genes, as described in Orlandi et al, Proc.Natl.Acad.Sci. (USA),86:3833-3837(1989), thereby constructing a diverse V gene repertoire for expression. The V gene can be amplified from cDNA and genomic DNA, with a reverse primer located at the 5' end of the exon encoding the mature V domain, and a forward primer based within the J segment, as described by Orlandi et al (1989) and Ward et al, Nature,341:544-546 (1989). However, for amplification from cDNA, the reverse primer may also be based within the leader exon, as described by Jones et al, Biotechnol.,9:88-89(1991), and the forward primer within the constant region, as described by Sasty et al, Proc.Natl.Acad.Sci. (USA),86:5728-5732 (1989). To maximize complementarity, degeneracy can be incorporated into the primers, as described by Orlandi et al (1989) or Sastry et al (1989). Preferably, library diversity is maximized by amplifying all available VH and VL rearrangements present in immune cell Nucleic acid samples using PCR primers targeting each V gene family, for example as described in Marks et al, J.mol.biol.,222:581-597(1991) or Orum et al, Nucleic Acids Res.,21:4491-4498 (1993). For cloning the amplified DNA into an expression vector, rare restriction sites can be introduced as tags at one end of the PCR primers, as described by Orlandi et al (1989), or further PCR amplification can be performed using tagged primers, as described by Clackson et al, Nature,352:624-628 (1991).

The synthetic recombinant V gene repertoire can be derived in vitro from V gene segments. Most human VH gene segments have been cloned and sequenced (Tomlinson et al, J.mol.biol.,227:776-798(1992)), and located (Matsuda et al, Nature Genet.,3:88-94 (1993)); segments of these clones (including all major constructs of the H1 and H2 loops) can be used to generate a diverse VH gene repertoire using PCR primers for the H3 loop with coding sequence and length diversity, as described in Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). The VH repertoire can also be generated by concentrating all sequence diversity in a single length long H3 loop, as described in Barbas et al, Proc.Natl.Acad.Sci.USA,89:4457-4461 (1992). The human V.kappa.and V.lambda.segments have been cloned and sequenced (Williams and Winter, Eur.J.Immunol.,23:1456-1461(1993)) and can be used to generate a repertoire of synthetic light chains. A repertoire of synthetic V genes based on a range of VH and VL fold structures and lengths of L3 and H3 will encode antibodies with considerable structural diversity. After amplification of the DNA encoding the V gene, germline V gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992).

The repertoire of antibody fragments can be constructed by combining VH and VL genes in several ways. Each repertoire can be created in a different vector and the vector recombined in vitro, e.g., as described in Hogrefe et al, Gene,128:119-126(1993), or in vitro by combinatorial infection, e.g., the loxP system described in Waterhouse et al, Nucl. acids Res.,21:2265-2266 (1993). The in vivo recombination method exploits the double-stranded nature of the Fab fragment to overcome the library volume limitation imposed by e. The non-immunized VH and VL repertoires were cloned separately, one into the phagemid and the other into the phage vector. The two libraries were then combined by infecting phage-containing bacteria with phage so that each cell contained a different combination, the library capacity being limited only by the number of cells present (about 10) ¹²Individual clones). Both vectors contain in vivo recombination messagesNo. VH and VL genes were recombined onto a single replicon and co-packaged into phage virions. These giant libraries provide large numbers of libraries with good affinity (Kd)^-1Is about 10^-8M) of a diverse antibody.

Alternatively, the repertoires can be cloned sequentially into the same vector, e.g., as described in Barbas et al, Proc.Natl.Acad.Sci.USA,88:7978-7982(1991), or assembled together by PCR and then cloned as described in Clackson et al, Nature,352:624-628 (1991). PCR assembly can also be used to link VH and VL DNA with DNA encoding flexible peptide spacers to form single chain Fv (scFv) repertoires. In another technique, "intracellular PCR assembly" is used to combine VH and VL genes in lymphocytes by PCR, and then clone the complete set of linked genes, as described in Embleton et al, Nucl. acids Res.,20:3831-3837 (1992).

Antibodies generated by the unimmunized library (natural or synthetic) may have moderate affinity (Kd)^-1Is about 10⁶-10⁷M^-1) However, affinity maturation can also be simulated in vitro by constructing and reselecting secondary libraries as described in Winter et al (1994), supra. For example, in the method of Hawkins et al, J.mol.biol.,226:889-896(1992) or the method of Gram et al, Proc.Natl.Acad.Sci USA,89:3576-3580(1992), mutations are introduced randomly in vitro using an error-prone polymerase (Leung et al, Technique,1:11-15 (1989)). In addition, affinity maturation can be performed by randomly mutating one or more CDRs, e.g., PCR using primers that carry random sequences across the CDRs of interest in selected individual Fv clones and screening for higher affinity clones. WO9607754 (published on 3/14/1996) describes a method for inducing mutations in complementarity determining regions of immunoglobulin light chains to create a light chain gene library. Another highly efficient method is to combine VH or VL domains selected by phage display with naturally occurring V domain variants from non-immune donors and screen for higher affinity in rounds of chain shuffling, as described in Marks et al, Biotechnol.,10:779-783 (1992). This technique allows the generation of affinities at 10 ^-9M range antibodies andan antibody fragment.

EphrinB2 nucleic acid and amino acid sequences are known in the art. The nucleic acid sequence encoding EphrinB2 can be designed using the amino acid sequence of the desired EphrinB2 region. Alternatively, a cDNA sequence (or fragment thereof) of GenBank accession No. NM _004093 may be used. The nucleic acid encoding EphrinB2 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. int. ed. engl.,28:716-734(1989), such as the triester, phosphite, phosphoramidite, and H-phosphonate methods. In one embodiment, the DNA encoding EphrinB2 is designed using codons preferred by the expression host cell. Alternatively, the DNA encoding EphrinB2 can be isolated from genomic or cDNA libraries.

Following construction of the DNA encoding EphrinB2, the DNA molecule is operably linked to expression control sequences in an expression vector, such as a plasmid, which control sequences are recognized by a host cell transformed with the vector. Generally, plasmid vectors contain replication and control sequences that are derived from a species compatible with the host cell. Vectors typically carry a site of replication, as well as sequences encoding proteins 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 encoding EphrinB2 is operably linked to a secretion leader sequence, resulting in secretion of the expression product into the culture medium by the host cell. Examples of secretory leaders include stII, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and α -factor. Also suitable for use herein is the 36 amino acid leader sequence of protein A (Abrahmsen et al, EMBO J.,4:3901 (1985)).

Host cells are transfected, preferably transformed, with the expression or cloning vectors of the invention described above and cultured in conventional nutrient media, which may be modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Transfection refers to the uptake of an expression vector by a host cell, whether or not the coding sequence is actually expressed. One of ordinary skill in the art is well versed in a variety of transfection methods, e.g., CaPO⁴Precipitation and electroporation. Transfection is considered successful if there is any indication of the operation of this vector within the host cell. Methods for transfection are well known in the art and some are described herein.

Transformation refers to the introduction of DNA into an organism such that the DNA may be replicated, either as an extrachromosomal element, or by chromosomal integration. Depending on the host cell used, transformation is carried out using standard techniques appropriate for the cell. Methods for transformation are well known in the art, and some are described herein.

Prokaryotic host cells used to produce EphrinB2 can be cultured as generally described in Sambrook et al, supra.

The mammalian host cells used to produce EphrinB2 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 cultured in vitro as well as cells within a host animal.

Purification of EphrinB2 can be accomplished using art-recognized methods, some of which are described herein.

Purified EphrinB2 can be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic acid copolymers, hydroxy methacrylate gels, polyacrylic and polymethacrylic acid copolymers, nylon, neutral and ionic carriers, and the like, for affinity chromatographic separation of phage display clones. Attachment of EphrinB2 protein to the substrate can be achieved by techniques described in Methods in enzymology, vol.44 (1976). A common technique for attaching protein ligands to polysaccharide matrices, such as agarose, dextran or cellulose, involves activation of the support with cyanogen halides followed by coupling of the primary aliphatic or aromatic amines of the peptide ligands to the activated matrix.

Alternatively, EphrinB2 can be used to coat the wells of an adsorption plate, expressed on host cells attached to an adsorption plate, or used for cell sorting, or coupled to biotin for capture with streptavidin-coated beads, or used in any other method known in the art for panning phage display libraries.

The phage library sample is contacted with immobilized EphrinB2 under conditions suitable for binding of at least a portion of the phage particles to the adsorbent. Normally, conditions including pH, ionic strength, temperature, and the like are selected to mimic physiological conditions. Phage bound to the solid phase are washed and then eluted with acid, e.g. as described in Barbas et al, Proc.Natl.Acad.Sci USA,88:7978-7982(1991), or with base, e.g. as described in Marks et al, J.mol.biol.,222:581-597(1991), or by Ephrin B2 antigen competition, e.g. in a protocol similar to that of Clackson et al, Nature,352:624-628 (1991). Phages can be enriched 20-1,000-fold in a single round of selection. In addition, the enriched phage can be cultured in bacterial culture and subjected to more rounds of selection.

The efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage are capable of binding antigen simultaneously. Antibodies with faster dissociation kinetics (and weak binding affinity) can be retained by using short washes, multivalent phage display, and high antigen coating density in the solid phase. The high density not only stabilizes the phage through multivalent interactions, but also facilitates the recombination of dissociated phage. Selection of antibodies with slower dissociation kinetics (and strong binding affinity) can be facilitated by the use of prolonged washing and monovalent phage display (as described in Bass et al, Proteins,8:309-314(1990) and WO 92/09690) and low antigen coating density (as described in Marks et al, Biotechnol.,10:779-783 (1992)).

It is possible to select between phage antibodies with different affinities for EphrinB2, even with slightly different affinities. However, random mutagenesis of selected antibodies (e.g., as performed by some of the affinity maturation techniques described above) has the potential to produce many mutants, most of which bind antigen, and a few of which have higher affinity. By limiting EphrinB2, rare high affinity phages were able to compete out. To retain all higher affinity mutants, the phage can be incubated with an excess of biotinylated EphrinB2, but the molar concentration of biotinylated EphrinB2 is lower than the target molar affinity constant of EphrinB 2. High affinity binding phage were then captured with streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows selection of antibodies according to binding affinity, with sensitivity that allows isolation of mutant clones with only 2-fold higher affinity from a large excess of low affinity phage. Conditions for washing phage bound to the solid phase can also be manipulated to perform dissociation kinetic-based differentiation.

anti-EphrinB 2 clones can be actively selected. In one embodiment, the invention provides anti-EphrinB 2 antibodies that block binding between EphB receptors (such as EphB1, EphB2, and/or EphB3) and EphrinB2, but do not block binding of EphB receptors to second proteins (such as EphrinB1 and/or EphrinB 3). Fv clones corresponding to such anti-EphrinB 2 antibodies can be selected as follows: (1) isolating anti-EphrinB 2 clones from the phage library as described above, and optionally amplifying the population of isolated phage clones by culturing in a suitable bacterial host; (2) selecting EphrinB2 and a second protein for which blocking and non-blocking of activity, respectively, is desired; (3) adsorbing an anti-EphrinB 2 phage clone to immobilized EphrinB 2; (4) using an excess of the second protein to elute any unwanted clones that recognize the EphrinB2 binding determinant (which overlaps or shares with the binding determinant of the second protein); and (5) eluting the clones still adsorbed after step (4). Optionally, clones with the desired blocking/non-blocking properties can be further enriched by repeating the selection procedure described herein one or more times.

DNA encoding the hybridoma-derived monoclonal antibodies or phage display Fv clones of the invention are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from a hybridoma or phage DNA template). Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell that does not otherwise produce immunoglobulin protein, such as an escherichia coli cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell, to obtain synthesis of the desired monoclonal antibody in the recombinant host cell. A review of the recombinant expression of DNA encoding antibodies in bacteria includes 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 may be combined with known DNA sequences encoding the constant regions of the heavy and/or light chains (e.g., suitable DNA sequences are available from Kabat et al, supra) to form clones encoding full-length or partial heavy and/or light chains. It will be appreciated that constant regions of any isotype may be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and that such constant regions may be derived from any human or animal species. Fv clones derived from variable domain DNA of one animal species (such as human) and then fused with constant region DNA of another animal species to form coding sequences for "hybrid" full-length heavy and/or light chains are included in the definition of "chimeric" and "hybrid" antibodies as used herein. In a preferred embodiment, Fv clones derived from human variable DNA are fused to human constant region DNA to form fully human, full-length or partial heavy and/or light chain coding sequences.

The DNA encoding the anti-EphrinB 2 antibody derived from the hybridoma of the invention may also be modified, for example by replacing, with the coding sequences for the human heavy and light chain constant regions, the homologous murine sequences derived from the hybridoma antibody (e.g. as in Morrison et al, proc.natl.acad.sci.usa,81:6851-6855 (1984)). The DNA encoding the hybridoma or Fv clone-derived antibody or fragment may be further modified by covalently joining an immunoglobulin-encoding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide. "chimeric" or "hybrid" antibodies having the binding specificity of the Fc-or hybridoma clone-derived antibodies of the invention can be prepared in this manner.

Antibody fragments

The invention encompasses antibody fragments. In some cases, it may be advantageous to use antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and may result in easier access to solid tumors.

Various techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical Methods24:107-117(1992); Brennan et al, Science229:81 (1985)). However, these fragments can now be produced directly from recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted by E.coli, thus allowing easy production of large quantities of these fragments. Antibody fragments can be isolated from the phage antibody libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab')2 fragments (Carter et al, Bio/Technology10:163-167 (1992)). According to another approach, the F (ab')2 fragment can be isolated directly from the recombinant host cell culture. Fab and F (ab') with extended in vivo half-life comprising salvage receptor binding epitope residues ₂Fragments are described in U.S. Pat. No.5,869,046. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO93/16185, U.S. Pat. No.5,571,894, and U.S. Pat. No.5,587,458. Fv and sFv are the only types with intact binding sites, lacking constant regions; as such, they are suitable for reducing non-specific binding when used in vivo. sFv fusion proteins can be constructed to generate fusions of effector proteins at the amino or carboxy terminus of an sFv. See, for example, Antibody Engineering, edited by Borebaeck, supra. Antibody fragments may also be "linearAntibodies ", for example, as described in U.S. Pat. No.5,641,870. 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 may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable region. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature321:522-525(1986); Riechmann et al, Nature332:323-327(1988); Verhoeyen et al, Science239:1534-1536 (1988)), using non-human hypervariable region sequences in place of the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which significantly less than the entire human variable region is replaced with the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

The choice of human light and heavy chain variable regions for making humanized antibodies is very important for reducing antigenicity. The entire library of known human variable region sequences was screened using the variable region sequences of rodent antibodies according to the so-called "best-fit" method. The closest human sequence to rodents is then selected as the human framework for the humanized antibody (Sims et al, J.Immunol.151:2296(1993); Chothia et al, J.mol.biol.196:901 (1987)). Another approach uses a specific framework derived from the consensus sequence of all human antibodies of a specific subclass of light or heavy chains (subgroups). The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA89:4285(1992); Presta et al, J. Immunol.151:2623 (1993)).

More importantly, the antibodies retain high affinity for the antigen and other favorable biological properties after humanization. To achieve this, according to one method, humanized antibodies are prepared by a process of analyzing the parent sequence and various conceptual humanized products using three-dimensional models of the parent sequence and the humanized sequence. Three-dimensional models of immunoglobulins are generally available, as will be familiar to those skilled in the art. Computer programs are also available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. By examining these display images, one can analyze the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the acceptor and import sequences and combined to obtain a desired antibody characteristic, such as increased affinity for the target antigen. In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Human antibodies

The human anti-EphrinB 2 antibodies of the invention can be constructed by combining Fv clone variable domain sequences selected from human-derived phage display libraries with known human constant domain sequences as described above. Alternatively, the human monoclonal anti-EphrinB 2 antibodies of the invention can be produced by hybridoma methods. Human myeloma and mouse-human heteromyeloma cell lines used to generate human Monoclonal antibodies have been described, for example, in Kozbor J.Immunol.,133:3001(1984); Brodeur et al, Monoclonal Antibody production techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York,1987); and Borner et al, J.Immunol.,147:86(1991).

For example, it is now possible to generate transgenic animals (e.g., mice) that are capable of generating a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of large numbers of human germline immunoglobulin genes in such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc.Natl.Acad.Sci.USA90:2551(1993), Jakobovits et al, Nature362:255-258(1993), Bruggemann et al, Yeast in Immunol.7:33 (1993).

Gene shuffling can also be used to derive human antibodies from non-human (e.g., rodent) antibodies in vitro, where the human antibodies have similar affinity and specificity as the starting non-human antibody. According to this method, which is also known as "epitope imprinting", the variable regions of the heavy or light chains of the non-human antibody fragments obtained by phage display techniques as described above are replaced with a repertoire of human V domain genes, resulting in a population of non-human chain scFv or Fab chimeras. Selection with antigen results in the isolation of a non-human chain/human chain chimeric scFv or Fab, wherein the human chain restores the antigen binding site after removal of the corresponding non-human chain in the primary phage display clone, i.e. the epitope determines (imprints) the selection of the human chain partner. When this process is repeated to replace the remaining non-human chains, human antibodies are obtained (see PCT WO93/06213, published at 1/4/1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides fully human antibodies that do not contain FR or CDR residues of non-human origin.

Human antibodies can also be produced by in vitro activated B cells (see U.S. Pat. nos. 5,567,610 and 5,229,275).

Bispecific antibodies

Bispecific antibodies refer to monoclonal antibodies, preferably human or humanized antibodies, having binding specificity for at least two different antigens. In this case, one of the binding specificities is for EphrinB2 and the other one of the binding specificities is for any other antigen. Exemplary bispecific antibodies can bind to two different epitopes of the EphrinB2 protein. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing EphrinB 2. These antibodies possess an EphrinB2 binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-alpha, vinca alkaloids, ricin a chain, methotrexate, or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies).

Methods for constructing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature305:537 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is rather cumbersome and the product yield is low. A similar procedure is disclosed in WO93/08829, 5.13.1993 and Trunecker et al, EMBO J.10:3655 (1991).

According to a different and more preferred method, antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. Preferably, at least part of the hinge, C_H2 and C_HRegion 3 immunoglobulin heavy chain constant domain fusion. Preferably, a first heavy chain constant region (C) comprising the site necessary for light chain binding is present in at least one of the fusions_H1). The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used for construction provide optimal yields, this provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when expression of at least two polypeptide chains in the same ratio leads to high yields or when the ratio is of no particular significance.

In a preferred embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. Since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient separation route, it was found that this asymmetric structure facilitates the separation of the desired bispecific complex from the unwanted immunoglobulin chain combinations. This method is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology121:210 (1986).

According to another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise at least part of an antibody constant domain C_H3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size to the large side chains are created at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody of the heterologous conjugate may be conjugated to avidin and the other antibody to biotin. For example, such antibodies have been suggested for targeting immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treating HIV infection (WO91/00360, WO92/00373, and EP 03089). Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No.4,676,980, along with a number of crosslinking techniques.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science229:81(1985) describes proteolytic cleavage of intact antibodies to F (ab')₂Protocol for fragmentation. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The Fab' fragments produced are then converted intoA Thionitrobenzoate (TNB) derivative. One of the Fab ' -TNB derivatives is then reverted back to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immobilization reagents for enzymes.

Recent advances have facilitated the direct recovery of Fab' -SH fragments from E.coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, J.Exp.Med.175:217-225(1992) describe a fully humanized bispecific antibody F (ab')₂And (4) generation of molecules. Each Fab' fragment was secreted separately from E.coli and subjected to directed chemical coupling in vitro to form bispecific antibodies. The bispecific antibody so formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for the direct production and isolation of bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies have been generated using leucine zippers. Kostelny et al, J.Immunol.148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The "diabody" technique described in Hollinger et al, Proc. Natl. Acad. Sci. USA90:6444-6448(1993) provides an alternative mechanism for the construction of bispecific antibody fragments. The fragment comprises heavy chain constant domains (V) connected by a linker_H) And a light chain constant domain (V)_L) The linker is too short to allow pairing between the two domains on the same strand. Thus, V on a segment is forced_HAnd V_LDomain and complementary V on another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for constructing bispecific antibody fragments by using single chain Fv (sFv) dimers has also been reported. See Gruber et al, j. 152:5368(1994)。

Antibodies with more than two titers are contemplated. For example, trispecific antibodies can be prepared. Tutt et al, J.Immunol.147:60 (1991).

Multivalent antibodies

Multivalent antibodies can be internalized (and/or catabolized) by a cell expressing an antigen to which the antibody binds more rapidly than bivalent antibodies. The antibodies of the invention can be multivalent antibodies (other than the IgM class) having three or more antigen binding sites (e.g., tetravalent antibodies) that can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. A multivalent antibody may comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. Preferred multivalent antibodies herein comprise (or consist of) three to about eight, but preferably four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chains comprise two or more variable domains. For example, a polypeptide chain can comprise VD1- (X1)_n-VD2-(X2)_n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, a polypeptide chain can comprise: VH-CH 1-flexible linker-VH-CH 1-Fc region chain; or VH-CH1-VH-CH1-Fc domain chain. Preferably, the multivalent antibody herein further comprises at least two (and preferably four) light chain variable domain polypeptides. A multivalent antibody herein may comprise, for example, about two to about eight light chain variable domain polypeptides. Light chain variable domain polypeptides contemplated herein comprise a light chain variable domain, and optionally further comprise a CL domain.

Antibody variants

In some embodiments, amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics. Amino acid changes can be introduced into the subject antibody amino acid sequences at the time the sequences are prepared.

One method that can be used to identify certain residues or regions of an antibody that are preferred mutagenesis positions is "alanine scanning mutagenesis" as described in Cunningham and Wells, Science244:1081-1085 (1989). Here, a residue or group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine and glutamic acid) are identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Amino acid positions that exhibit functional sensitivity to substitution are then refined by introducing more or other variants at or for the substitution site. Thus, while the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the consequences of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region and the expressed immunoglobulin is screened for the desired activity.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion of the N-or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment 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 recognition sequences for enzymatic attachment of a carbohydrate module to an asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates potential glycosylation sites. 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, but 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence to include one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding or replacing one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

If the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, U.S. patent application 2003/0157108(Presta, L.) describes antibodies having a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody. See also US2004/0093621(Kyowa Hakko kogyo co., Ltd.). Antibodies having an aliquot of N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody are mentioned in WO2003/011878(Jean-Mairet et al) and U.S. Pat. No. 6,602,684 (Umana et al). Antibodies having at least one galactose residue in an oligosaccharide attached to the Fc region of an antibody are reported in WO1997/30087(Patel et al). See also WO1998/58964(Raju, S.) and WO1999/22764(Raju, S.) for antibodies with altered carbohydrate attachment to their Fc regions. For antigen binding molecules with improved glycosylation see also US2005/0123546(Umana et al).

Preferred glycosylation variants herein comprise an Fc region, wherein the carbohydrate structure attached to the Fc region lacks fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions that further improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (Eu residue numbering). Examples of publications relating to "defucose" or "fucose-deficient" antibodies include: US2003/0157108, WO2000/61739, WO2001/29246, US2003/0115614, US2002/0164328, US2004/0093621, US2004/0132140, US2004/0110704, US2004/0110282, US2004/0109865, WO2003/085119, WO2003/084570, WO2005/035586, WO2005/035778, WO2005/053742, Okazakiet al J.mol.biol.336:1239-1249(2004), Yamane-Ohnuki et al Biotech.Bioeng.87:614 (2004). Examples of cell lines producing defucosylated antibodies include protein fucosylated deficient Lec13CHO cells (Ripka et al Arch. biochem. Biophys.249:533-545(1986); U.S. patent application Ser. No. US2003/0157108A1, Presta, L; and WO2004/056312A1, Adams et al, especially example 11) and knock-out cell lines such as the alpha-1, 6-fucosyltransferase gene, FUT8, knock-out CHO cells (Yamane-Ohnuki et al Biotech. Bioeng.87:614 (2004)).

Another class of variants are amino acid substitution variants. These variants have at least one amino acid residue in the antibody molecule replaced with a different residue. Sites of greatest interest for substitutional mutagenesis include hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in table 1 in the "preferred substitutions" column. If such substitutions result in a change in biological activity, more substantial changes, referred to as "exemplary substitutions" in Table 1, or as further described below with reference to amino acid classifications, can be introduced and the products screened.

TABLE 1

Substantial modification of antibody biological properties can be achieved by selecting substitutions that differ significantly in their effectiveness in maintaining: (a) the structure of the polypeptide backbone in the replacement region, e.g., (folded) sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain.

Naturally occurring residues may be grouped as follows, according to common side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp and Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions entail replacing one of these classes with a member of the other class.

One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were produced. One convenient method for generating such surrogate variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) were mutated to generate all possible amino acid substitutions at each site. The antibodies so generated are displayed on filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues which contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. The contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

Nucleic acid molecules encoding antibody amino acid sequence variants can be 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 naturally occurring amino acid sequence variants), or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications in the Fc region of an immunoglobulin polypeptide of the invention, thereby generating an Fc region variant. Fc region variants may include human Fc region sequences (such as human IgG1, IgG2, IgG3, or IgG4Fc regions) comprising amino acid modifications (such as substitutions) at one or more amino acid positions, including the hinge cysteine.

In accordance with this description and the teachings of the art, it is contemplated that in some embodiments, the antibodies used in the methods of the invention may comprise one or more alterations in, for example, the Fc region, as compared to the wild-type counterpart antibody. These antibodies will still retain essentially the same properties required for therapeutic efficacy compared to their wild type counterparts. For example, it is believed that certain changes in the Fc region may be made which will result in altered (i.e. either enhanced or attenuated) C1q binding and/or Complement Dependent Cytotoxicity (CDC), for example as described in WO 99/51642. See also Duncan and Winter, Nature322:738-40(1988), U.S. Pat. No. 5,648,260, U.S. Pat. No. 5,624,821, and WO94/29351, which are directed to other examples of Fc region variants.

WO00/42072(Presta) and WO2004/056312(Lowman) describe antibody variants with increased or decreased binding to FcR. The contents of these patent publications are expressly incorporated herein by reference. See also the fields et al J biol chem.9(2):6591-6604 (2001). Antibodies with increased half-life 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 having one or more substitutions that improve binding of the Fc region to FcRn. Polypeptide variants having altered amino acid sequences of the Fc region and increased or decreased binding of C1q are described in U.S. Pat. No.6,194,551B1, WO 99/51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogene et al.J.Immunol.164:4178-4184 (2000).

Antibody derivatives

The antibodies of the invention can be further modified to include additional non-proteinaceous moieties known in the art and readily available. Preferably, the moiety suitable for derivatization of the antibody is a water-soluble polymer. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached 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 may be determined based on considerations including, but not limited to, the specific properties or function of the antibody to be improved, whether the antibody derivative is to be used in a treatment under specified conditions, and the like.

Screening for antibodies with desired Properties

Antibodies of the invention can be characterized for their physical/chemical properties and biological functions by a variety of assays known in the art. In some embodiments, the antibody is characterized by one or more of: reducing or blocking EphrinB2 activation, reducing or blocking EphrinB2 downstream molecular signaling, reducing or blocking EphrinB 2-binding Eph receptor (such as EphB1, EphB2, and/or EphB 3) activation, reducing or blocking EphrinB 2-binding ephh receptor (such as EphB1, EphB2, and/or EphB 3) downstream molecular signaling, disrupting or blocking EphrinB 2-binding Eph receptor (such as EphB1, EphB2, and/or EphB 3) binding to EphrinB2, EphrinB2 phosphorylation and/or EphrinB 5 multimerization, and/or EphrinB 2-binding ephh receptor (such as EphB1, EphB2, and/or EphrinB 6342), and/or treating and/or preventing tumor cell proliferation, or preventing and/or inhibiting EphrinB 57323-binding to EphrinB activity, such as inhibiting or preventing or increasing or treating and/or inhibiting angiogenesis, such as cancer, such as EphrinB 1, EphrinB 6324, and/or tumor cell proliferation, and/or cancer development and/or preventing or cancer.

Purified antibodies can be further identified 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 generated herein are analyzed for their biological activity. In some embodiments, antibodies of the invention are tested for their antigen binding activity. Antigen binding assays known in the art and useful herein include, but are not limited to, any direct or competitive binding assay using techniques such as Western blotting, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, and protein a immunoassays. Exemplary antigen binding assays are provided below in the examples section.

In yet another embodiment, the invention provides an anti-EphrinB 2 monoclonal antibody that competes for binding to EphrinB2 with the 31.19, 31.19.1D8, and/or 31.19.2D3 antibody. Such competitive antibodies include antibodies that recognize the same or overlapping epitope of EphrinB2 as the EphrinB2 epitope recognized by the 31.19, 31.19.1D8, and/or 31.19.2D3 antibodies. Such competitive antibodies can be obtained by screening anti-EphrinB 2 hybridoma supernatants for binding that competes with labeled 31.19, 31.19.1D8, and/or 31.19.2D3 antibody for immobilized EphrinB 2. Hybridoma supernatants containing the competitive antibody will reduce the amount of bound, labeled antibody detected in the test competition binding mixture compared to the amount of bound, labeled antibody detected in the control binding mixture containing the irrelevant antibody or no antibody. Any of the competitive binding assays described herein are suitable for use in the foregoing procedures.

In another aspect, the invention provides an anti-EphrinB 2 monoclonal antibody comprising one or more (such as 2, 3, 4, 5, and/or 6) HVRs of the 31.19 antibody, the 31.19.1D8 antibody, or the 31.19.2D3 antibody. An anti-EphrinB 2 monoclonal antibody comprising one or more HVRs of the 31.19 antibody, 31.19.1D8 antibody, and/or 31.19.2D3 antibody can be constructed by grafting one or more HVRs of the 31.19 antibody, 31.19.1D8 antibody, and/or 31.19.2D3 antibody onto a template antibody sequence, e.g., a human antibody sequence closest to the corresponding murine sequence of the parent antibody or a consensus sequence of all human antibodies of a particular light or heavy chain subgroup of the parent antibody, as described above, and expressing the resulting chimeric light and/or heavy chain variable region sequences, with or without accompanying constant region sequences, in a recombinant host cell.

The anti-EphrinB 2 antibodies of the invention having the unique properties described herein can be obtained by screening anti-EphrinB 2 hybridoma clones for the desired properties by any convenient method. For example, if it is desired to block or not block EphrinB2 ligand binding to an anti-EphrinB 2 monoclonal antibody of EphrinB2, candidate antibodies can be tested in a binding competition assay, such as a competitive binding ELISA, wherein a well of the plate is coated with EphrinB2, an antibody solution in excess of the EphrinB2 binding partner of interest is plated onto the coated plate, and the bound antibodies are detected enzymatically, e.g., by contacting the bound antibodies with an anti-Ig antibody coupled with HRP or a biotinylated anti-Ig antibody and developing an HRP color reaction, e.g., by developing the plate with streptavidin-HRP and/or hydrogen peroxide and detecting the HRP color reaction at 490nm with an ELISA reader by spectrophotometry.

If an anti-EphrinB 2 antibody that inhibits or activates EphrinB2 activation is desired, candidate antibodies can be tested in an EphrinB2 phosphorylation assay. Such assays are known in the art and are described in the examples section.

If an anti-EphrinB 2 antibody that inhibits cell growth is desired, the candidate antibody can be tested in an in vitro and/or in vivo assay that measures inhibition of cell growth. Such assays are known in the art and are further described and exemplified herein.

In one embodiment, the invention contemplates an improved antibody with some, but not all, effector functions, which makes it a desirable candidate in many applications where the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In certain embodiments, the Fc activity of the resulting immunoglobulin is measured to ensure that only the desired properties are retained. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to confirm that the antibody lacks fcyr binding (and therefore potentially lacks ADCC activity) but retains FcRn binding ability. The main cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravech and Kinet, annu.rev.immunol.9:457-92(1991) page 464 summarizes FcR expression on hematopoietic cells. Examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No.5,500,362 or 5,821,337. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of the molecule of interest may be assessed in vivo, for example in animal models such as disclosed in Clynes et al, PNAS (USA)95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. To assess complement activation, CDC assays may be performed, for example as described in Gazzano-Santoro et al, j.immunol.methods202:163 (1996). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art, such as described in the examples section.

Vectors, host cells and recombinant methods

For recombinant production of the antibody of the invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. DNA encoding the antibody can be readily isolated and sequenced using conventional procedures (e.g., 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 will depend in part on the host cell to be used. Generally, preferred host cells are of prokaryotic or eukaryotic (typically mammalian) origin. It will be appreciated that constant regions of any isotype may be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and that such constant regions may be obtained from any human or animal species.

a. Production of antibodies using prokaryotic host cells:

i. vector construction

The polynucleotide sequences encoding the polypeptide building blocks of the antibodies of the invention can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated from antibody producing cells such as hybridoma cells and sequenced. Alternatively, polynucleotides may be synthesized using nucleotide synthesizers or PCR techniques. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replication and expression of the heterologous polynucleotide in a prokaryotic host. For the purposes of the present invention, a wide variety of vectors available and known in the art may be used. The choice of an appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains a variety of components, depending on its function (either to amplify or express the heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. Vector components generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a Ribosome Binding Site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

In general, plasmid vectors for use with host cells contain replicon and control sequences that are derived from species compatible with these hosts. Vectors typically carry a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells. For example, E.coli is typically transformed with plasmid pBR322 derived from the E.coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thereby providing easy means for identifying transformed cells. pBR322, derivatives thereof, or other microbial plasmids or phages may also contain or be modified to contain promoters that can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives useful for the expression of particular antibodies are described in detail in Carter et al, U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transformation vectors for these hosts. For example, phage such as λ gem.tm. -11 can be used to construct recombinant vectors that can be used to transform susceptible host cells such as e.coli LE 392.

The expression vectors of the invention may comprise two or more promoter-cistron pairs which encode each polypeptide building block. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that regulates its expression. Prokaryotic promoters are generally divided into two classes, inducible and constitutive. An inducible promoter is a promoter that initiates an elevated level of transcription of a cistron under its control in response to a change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.

Numerous promoters recognized by a variety of potential host cells are well known. The selected promoter may be operably linked to cistron DNA encoding the light or heavy chain by excising the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Both native promoter sequences and many heterologous promoters can be used to direct amplification and/or expression of a target gene. In some embodiments, heterologous promoters are used because they generally allow for higher transcription and higher yields of expressed target genes as compared to the native target polypeptide promoter.

Promoters suitable for use in prokaryotic hosts include the PhoA promoter, the β -galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoters. However, other promoters functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published whereby the skilled worker is able to operably link them to cistrons encoding the target light and heavy chains using linkers or adaptors which provide any desired restriction sites (Siebenlist et al, Cell20:269 (1980)).

In one aspect of the invention, each cistron within the recombinant vector contains a secretory signal sequence component that directs the transmembrane transport of the expressed polypeptide. 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 purposes of the present 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 native signal sequence of the heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected, for example, from the group consisting of: alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the invention, the signal sequence used in both cistrons of the expression system is a STII signal sequence or a variant thereof.

In another aspect, the production of immunoglobulins according to the present invention may occur in the cytoplasm of the host cell, and thus does not require the presence of a secretion signal sequence within each cistron. At that point, the immunoglobulin light and heavy chains are expressed, folded and assembled within the cytoplasm to form functional immunoglobulins. Certain host strains (e.g., E.coli trxB-strains) provide cytoplasmic conditions favorable for disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pluckthun, Gene159:203 (1995)).

Prokaryotic host cells suitable for expression of the antibodies of the invention include Archaebacteria (Archaebaceria) and Eubacteria (Eubacterium), such as gram-negative or gram-positive organisms. Examples of useful bacteria include Escherichia (e.g. Escherichia coli), Bacillus (e.g. Bacillus subtilis), enterobacter (e.g. enterobacter), Pseudomonas (e.g. Pseudomonas aeruginosa) species, Salmonella typhimurium (Salmonella typhimurium), Serratia marcescens (Serratia marcoceans), Klebsiella (Klebsiella), Proteus (Proteus), Shigella (Shigella), Rhizobium (Rhizobium), Vitreoscilla (Vitreoscilla), or Paracoccus (Paracoccus). In one embodiment, gram-negative cells are used. In one embodiment, E.coli cells are used as hosts in the present invention. Examples of E.coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol.2, Washington, D.C., society of American microbiology, 1987, pp.1190-1219; ATCC accession No. 27,325) and derivatives thereof, including strain 33D3 (U.S. Pat. No. 5,639,635) having the genotype W3110. delta. fhuA (Δ tonA) ptr3lac Iq lacL 8. delta. ompT. delta. mpc-fepE) degP41 kanR. Other strains and derivatives thereof, such as E.coli 294 (ATCC 31,446), E.coli B, E.coli lambda 1776 (ATCC 31,537) and E.coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative only and not limiting. Methods for constructing any of the above bacterial derivatives having a specified genotype are known in the art, see, e.g., Bass et al, Proteins8:309-314 (1990). It is generally necessary to select an appropriate bacterium in consideration of replicability of the replicon in bacterial cells. For example, E.coli, Serratia, or Salmonella species may be suitable for use as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to provide the replicon. In general, the host cell should secrete minimal amounts of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors in the cell culture.

Antibody production

Host cells are transformed with the above expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Transformation is the introduction of DNA into a prokaryotic host so that the DNA can be replicated, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is carried out using standard techniques appropriate for these cells. Calcium treatment with calcium chloride is commonly used for bacterial cells with a strong cell wall barrier. Another transformation method used polyethylene glycol/DMSO. Yet another technique used is electroporation.

Prokaryotic cells for producing the polypeptides of the invention are cultured in media known in the art and suitable for culturing the selected host cells. Examples of suitable media include LB media (Luria broth) supplemented with essential nutrient supplements. In some embodiments, the medium further contains a selection agent selected based on the construction of the expression vector to selectively permit growth of the prokaryotic cell comprising the expression vector. For example, ampicillin is added to a medium for culturing cells expressing an ampicillin resistance gene.

In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may be present at appropriate concentrations, either alone or as a mixture with another supplement 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.

Prokaryotic host cells are cultured at a suitable temperature. For example, for culturing E.coli, preferred temperatures range from about 20 ℃ to about 39 ℃, more preferably from about 25 ℃ to about 37 ℃, and even more preferably about 30 ℃. The pH of the medium may be any pH ranging from about 5 to about 9, depending primarily on the host organism. For E.coli, the pH is preferably from about 6.8 to about 7.4, more preferably about 7.0.

If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for activating the promoter. In one aspect of the invention, the PhoA promoter is used to control transcription of the polypeptide. Thus, for induction, the transformed host cells are cultured in phosphate-limited medium. Preferably, the phosphate-limited medium is C.R.A.P medium (see, e.g., Simmons et al, J.Immunol. methods263:133-147 (2002)). Depending on the vector construct employed, a variety of other inducers may be employed, as is known in the art.

In one embodiment, the expressed polypeptide of the invention is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery typically involves destruction of the microorganism, usually by means such as osmotic shock (osmoticshock), sonication or lysis. Once the cells are disrupted, the cell debris or whole cells can be removed by centrifugation or filtration. The protein may be further purified by, for example, affinity resin chromatography. Alternatively, the protein may be transported into the culture broth and isolated therefrom. The cells can be removed from the culture broth and the culture supernatant filtered and concentrated for further purification of the produced protein. The expressed protein 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 quantities by a fermentation process. A variety of large-scale fed-batch fermentation processes are available for the production of recombinant proteins. Large scale fermentations have a capacity of at least 1000 liters, preferably about 1,000 to 100,000 liters. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source). Small-scale fermentation generally refers to fermentation in a fermentor that is no more than about 100 liters in volumetric capacity, and may range from about 1 liter to about 100 liters.

During fermentation, induction of protein expression is typically initiated after the cells are cultured under appropriate conditions to a desired density (e.g., OD550 of about 180-. Depending on the vector construct employed, a variety of inducers may be used, as is known in the art and described above. Cells can be cultured for shorter periods of time prior to induction. Cells are typically induced for about 12-50 hours, although longer or shorter induction times may be used.

In order to improve the production and quality of the polypeptides of the invention, a variety of fermentation conditions may be modified. For example, to improve proper assembly and folding of the secreted antibody polypeptide, additional vectors that overexpress chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and/or DsbG) or FkpA (a peptidylprolyl-cis, trans-isomerase with chaperone activity) may be used to co-transform the host prokaryotic cell. Chaperonins have been shown to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al, J.biol.chem.274: 19601-.

In order to minimize proteolysis of the expressed heterologous protein, particularly one that is sensitive to proteolysis, certain host strains deficient in proteolytic enzymes may be used in the present invention. For example, a host cell strain may be modified to carry out genetic mutations 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 E.coli protease deficient strains are available, see for example Joly et al, (1998) supra, Georgiou et al, U.S. Pat. No. 5,264,365, Georgiou et al, U.S. Pat. No. 5,508,192, Hara et al, Microbial Drug Resistance2:63-72 (1996).

In one embodiment, an E.coli strain deficient in proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins is used as host cell in the expression system of the invention.

Antibody purification

Standard protein purification methods known in the art can be used. The following scheme is illustrative of a suitable purification scheme: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resins such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, 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 a full-length antibody product of the invention. Protein a is a 41kD cell wall protein from Staphylococcus aureus (Staphylococcus aureus), which binds with high affinity to the antibody Fc region. Lindmark et al, J.Immunol.meth.62:1-13 (1983)). The solid phase on which protein A is immobilized is preferably a column having a glass or quartz surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column is coated with a reagent, such as glycerol, in an attempt to prevent non-specific adhesion of contaminants.

As a first step of purification, a preparation derived from the cell culture as described above is applied to a protein a immobilized solid phase such that the antibody of interest specifically binds to protein a. The solid phase is then washed to remove contaminants that are non-specifically bound to the solid phase. Finally, the desired antibody is recovered from the solid phase by elution.

b. Production of antibodies using eukaryotic host cells:

carrier members typically include, 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

The vectors used in eukaryotic host cells 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. Preferably a heterologous signal sequence that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretion leaders, such as the herpes simplex virus gD signal, can be used.

The DNA of these precursor regions is ligated in-frame to the DNA encoding the antibody.

(ii) Origin of replication

Typically, mammalian expression vectors do not require an origin of replication component. For example, the SV40 origin may typically only be used because it contains the early promoter.

(iii) Selection gene components

Expression and cloning vectors may comprise a selection gene, also referred to as a selectable marker. Typical selection genes encode the following proteins: (a) conferring resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) complementing the corresponding nutritional deficiency; or (c) provide key nutrients not available from complex media.

One example of a selection scheme utilizes drugs to retard the growth of host cells. Those cells successfully transformed with the heterologous gene produce proteins that confer drug resistance and thus survive the selection protocol. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of a suitable selectable marker for mammalian cells is one that is capable of identifying cells competent to take up antibody nucleic acids, such as DHFR, thymidine kinase, metallothionein I and II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is a Chinese Hamster Ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts comprising endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, a wild-type DHFR protein, and another selectable marker such as aminoglycoside 3' -phosphotransferase (APH) can be selected by culturing the cells in a medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter component

Expression and cloning vectors typically comprise a promoter recognized by the host organism and are operably linked to the antibody polypeptide nucleic acid. Promoter sequences for eukaryotic cells are known. Virtually all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is the CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence, which may be a signal to add a poly A tail to the 3' end of the coding sequence. All these sequences are suitably inserted into eukaryotic expression vectors.

Transcription of antibody polypeptides by vectors in mammalian host cells is under the control of promoters from heat shock promoters, e.g., obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis b virus, and simian virus 40(SV40), from heterologous mammalian promoters such as the actin promoter or an immunoglobulin promoter, provided that such promoters are compatible with the host cell system.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using 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. Pat. No. 4,601,978 or, alternatively, the use of the Rous sarcoma virus long terminal repeat as a promoter.

(v) Enhancer element component

Transcription of DNA encoding an antibody polypeptide of the invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. For enhanced elements for activation of eukaryotic promoters see also Yaniv, Nature297:17-18 (1982). Enhancers may be spliced into the vector at positions 5' or 3' to the coding sequence of the antibody polypeptide, but are preferably located at sites 5' to the promoter.

(vi) Transcription termination component

Expression vectors used in eukaryotic host cells also typically contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are typically available from the 5 'and occasionally 3' ends of untranslated regions of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide segments transcribed as polyadenylated fragments in the untranslated region of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and expression vectors disclosed therein.

(vii) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney line (293 cells or 293 cells subcloned for 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, Urlaub et al, Proc.Natl.Acad.Sci.USA77:4216 (1980)), mouse Sertoli (Sertoli) cells (TM 4, Mather, biol.23: 243. 31 (1980)), monkey kidney cells (CV 1, ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC 158L 1587), human cervical cancer cells (HELA, CCL 2), canine kidney cells (ATCC 73 34, ATCC 1444), bovine liver kidney cells (ATCC 1445, ATCC 14435), bovine liver kidney cells (ATCC 14435, ATCC 14465), bovine liver mouse liver kidney cells (ATCC 14435), bovine liver kidney 4665), mouse liver cells (ATCC 14435, ATCC 1442), mouse liver 469, ATCC 1447, mouse liver cells (ATCC 1442), mouse liver 469, mouse liver cells (BRL) transformed by SV 3877), mouse lung cells (TM) and mouse lung tumor cells (MRL), mouse lung tumor), mouse lung tumor cells (MRL) cells (TM 4, mouse lung tumor), mouse lung tumor, annals N.Y.Acad.Sci.383:44-68 (1982)), MRC5 cells, FS4 cells, and the human hepatoma (Hep G2) line.

For antibody production, host cells are transformed with the expression or cloning vectors described above and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

(viii) Culture of host cells

Host cells for producing the antibodies of the invention can be cultured in a variety of media. Commercial media such as Ham's F10 (Sigma), minimal essential medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media described in the following documents can be used as the medium for the host cells: ham et al, meth.Enz.58:44(1979), Barnes et al, anal.biochem.102:255(1980), U.S. Pat. No. 4,767,704, 4,657,866, 4,927,762, 4,560,655, 5,122,469, WO90/03430, WO87/00195, or U.S. patent review 30,985. Any of these media may be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN) ^TMMedicine), trace elementAn element (defined as an inorganic compound typically present in a final concentration in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included at suitable concentrations. Culture conditions such as temperature, pH, etc. are previously used for the host cell selected for expression, as will be apparent to the ordinarily skilled artisan.

(ix) Purification of antibodies

When recombinant techniques are used, the antibodies can be produced intracellularly or secreted directly into the culture medium. If the antibody is produced intracellularly, it is first necessary to remove particulate debris, either the host cells or the lysed fragments, for example by centrifugation or ultrafiltration. If the antibody is secreted into the culture medium, the supernatants from these expression systems are typically first concentrated using a commercial protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (the preferred purification technique is affinity chromatography). The suitability of protein a as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein a can be used to purify human gamma 1, gamma 2, or gamma 4 heavy chain-based antibodies (Lindmark et al, j. immunol. meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ 3 (Guss et al, EMBOJ.5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices may be used. Physically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow faster flow rates and shorter processing times than agarose. If the antibody comprises a CH3 domain, Bakerbond ABX may be used ^TMPurification was performed on resin (j.t. baker, phillips burg, NJ). Depending on the antibody to be recovered, other protein purification techniques such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC may also be usedChromatography on silica, heparin SEPHAROSE^TMChromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification step, the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., about 0-0.25M salt).

Immunoconjugates

The invention also provides immunoconjugates (interchangeably referred to as "antibody-drug conjugates" or "ADCs") comprising any of the anti-EphrinB 2 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 a fragment thereof), or a radioisotope (i.e., a radioconjugate).

Use of Antibody-drug conjugates for the local delivery of Cytotoxic or cytostatic Agents (i.e., drugs for killing or inhibiting tumor cells) In the treatment of Cancer (Syrigos And Epeneros, Anticancer Research19: 605; (1999); Niculescu-Duvaz And Springer, Adv.Drg.Del.Rev.26: 151. 172(1997); U.S. Pat. No. 4,975,278) enables targeted delivery of drug moieties to tumors And intracellular accumulation there, whereas systemic administration of these unconjugated drug Agents may result In unacceptable levels of toxicity to normal cells beyond the tumor cells sought to be eliminated (Baldwin, Lancet603-05 (15.1986); Thorape, "Anticancer of Cytotoxic Agents In Cancer Therapy: A Review," Biological Research, 19884, incorporated by Pictures, et al, Inc.; Biological Research, Pictures, 1985). Thereby attempting to achieve maximum efficacy and minimal toxicity. Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al, Cancer Immunol. Immunother.21:183-87 (1986)). Drugs used in these methods include daunomycin (daunomycin), doxorubicin (doxorubicin), methotrexate (methotrexate) and vindesine (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, journal.of the Nat. Cancer Inst.92(19):1573-1581(2000); Mandler et al, Bioorganic & Med. chem. Letter10: 1025-1028(2000); Mandler et al, Bioconjugate chem.13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al, Proc. Natl. Acad. Sci.93: 8618-8623 (1996)), and calicheamicin (Lode et al, Cancer Res.58:2928(1998); man et al, Cancer Res.3353: 3342 (1993)). Toxins can exert their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when coupled to large antibody or protein receptor ligands.

(ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope conjugate composed of a murine IgG1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes, and either 111In or 90Y radioisotopes conjugated via thiourea linker-chelators (Wiseman et al, Eur. Jour. Nucl. Med.27(7):766-77 (2000); Wiseman et al, Blood99(12):4336-42(2002); Witzig et al, J.Clin. Oncol.20(10):2453-63(2002); Witzig et al, J.Clin. Oncol.20(15):3262-69 (2002)). Although zevanin has activity against B-cell non-Hodgkin's (Hodgkin) lymphoma (NHL), administration results in severe and prolonged cytopenia in most patients. MYLOTARG^TM(gemtuzumab Pharmaceuticals, Wyeth Pharmaceuticals), an antibody-drug conjugate composed of human CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myelogenous leukemia by injection (Drugs of the Future25(7):686(2000); U.S. Pat. No. 4970198; 5079)233, 5585089, 5606040, 5693762, 5739116, 5767285, 5773001). Phase II trials for the treatment of CanAg expressing cancers such as colon, pancreatic, gastric and other cancers are ongoing with Cantuzumab mertansine (Immunogen Inc.), an antibody-drug conjugate consisting of the huC242 antibody linked via a disulfide linker SPP to a maytansinoid drug moiety DM 1. MLN-2704 (Millennium pharm., BZL Biologics, immunolgen Inc.), an antibody-drug conjugate consisting of a monoclonal antibody directed against Prostate Specific Membrane Antigen (PSMA) linked to maytansinoid drug moiety DM1, is under development for potential treatment of prostate tumors. Synthetic analogs of dolastatin (dolastatin), Auristatin E (AE), and monomethyl auristatin (mmae), were conjugated to chimeric monoclonal antibodies cBR96 (specific for Lewis Y on carcinomas) and cAC10 (specific for CD30 on hematological malignancies) (Doronina et al, Nature Biotechnology21(7): 778-one 784 (2003)) and are under therapeutic development.

Chemotherapeutic agents useful for generating immunoconjugates are described herein (e.g., above). Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α -sarcin (sarcocin), Aleutites fordii (Aleuties fordii) toxic protein, dianthus chinensis (dianthin) toxic protein, Phytolacca americana (Phytolacca) toxic protein (PAPI, PAPII, and PAPA-S), Momordica charantia (Momordica chaetia) inhibitor, Jatropha curcin (curcin), crotin (crotin), Saponaria officinalis (saponaria officinalis) inhibitor, gelonin alba (gelonin), mitomycin (gelonin), tricin (resphin), trichothecin (triomycin), and trichothecin (enomycin). See, e.g., WO93/21232, published on month 10 and 28, 1993. A variety of radionuclides are available for use in the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186 Re. Conjugates of the antibody and cytotoxic agent may be prepared using a variety of bifunctional protein coupling agents, such as bifunctional derivatives of N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) hexanediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026.

Also contemplated herein are conjugates of antibodies with one or more small molecule toxins such as calicheamicin (calicheamicin), maytansinoids (maytansinoids), dolastatins (dolastatins), aurostatins, trichothecenes (trichothecene), and CC1065, and fragments of these toxins that have toxin activity.

i. Maytansine and maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (full length or fragment) of the invention conjugated to one or more maytansinoid molecules.

Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was originally isolated from the east African shrub Maytenus serrata (Maytenus serrata) (U.S. Pat. No. 3,896,111). It was subsequently discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). The synthesis of maytansinol and its derivatives and analogues is disclosed, for example, in the following U.S. patents: 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.

Maytansinoid drug moieties are attractive drug moieties in antibody drug conjugates because they: (i) relatively easy to prepare by fermentation or chemical modification, derivatization of the fermentation product; (ii) are readily derivatized with functional groups suitable for coupling through non-disulfide linkers; (iii) is stable in plasma; and (iv) is effective against a variety of tumor cell lines.

Immunoconjugates comprising maytansinoids and their preparation and therapeutic use are disclosed, for example, in the following patents: U.S. Pat. Nos. 5,208,020, 5,416,064, and European patent EP0425235B1, the disclosures of which are expressly incorporated herein by reference. Liu et al, Proc.Natl.Acad.Sci.USA93:8618-8623(1996) describe immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic against cultured colon cancer cells and showed antitumor activity in an in vivo tumor growth assay. Chari et al, Cancer Research52: 127-. Cytotoxicity of TA.1-maytansinoid conjugates was tested in vitro on human breast cancer cell line SK-BR-3, which expresses 3x10 per cell ⁵A HER-2 surface antigen. The drug conjugates achieve a degree of cytotoxicity similar to free maytansinoid drugs, which can be increased by increasing the number of maytansinoid molecules conjugated per antibody molecule. The a 7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates can be prepared by chemically linking an antibody to a maytansinoid molecule without significantly impairing the biological activity of the antibody or the maytansinoid molecule. See, for example, U.S. Pat. No.5,208,020, the disclosure of which is expressly incorporated herein by reference. An average of 3-4 maytansinoid molecules per antibody molecule coupled showed efficacy in enhancing cytotoxicity against target cells without negatively affecting the function or solubility of the antibody, although it is expected that even one molecule of toxin/antibody will enhance cytotoxicity compared to the use of 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. Pat. No.5,208,020 and in the other patents and non-patent publications referred to above. Preferred maytansinoids are maytansinol and maytansinol analogs modified at aromatic rings or other positions of the maytansinol molecule, such as various maytansinol esters.

A number of linking groups are known in the art for use in the preparation of antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or European patent 0425235B1, Chari et al, cancer research52:127-131(1992), U.S. patent application No.10/960,602 filed 10/9/2004, the disclosure of which is expressly incorporated herein by reference. Antibody-maytansinoid conjugates comprising linker component SMCC can be prepared as disclosed in U.S. patent application No.10/960,602 filed on 8/10/2004. The linking group includes a disulfide group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group, or an esterase labile group, as disclosed in the patents mentioned above, disulfide and thioether groups being preferred. Additional linking groups are described and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. Particularly preferred coupling agents include N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al, biochem. J.173:723-737 (1978)) and N-succinimidyl-4- (2-pyridylthio) valerate (SPP), thereby providing a disulfide linkage.

Depending on the type of linkage, linkers can be attached to various positions of the maytansinoid molecule. For example, ester linkages can be formed by reaction with hydroxyl groups using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.

Auristatin and dolastatin

In some embodiments, immunoconjugates comprise an antibody of the invention conjugated to dolastatins (dolastatins) or dolastatin peptide analogs and derivatives, auristatins (U.S. Pat. Nos. 5,635,483;5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemothers.45 (12):3580-3584) and to have anti-cancer (US5,663,149) and anti-fungal activity (Pettit et al (1998) Antimicrob. Agents Chemothers.42: 2961-2965). Dolastatin or auristatin drug moieties can be attached to an antibody via the N (amino) terminus or the C (carboxyl) terminus of a peptide drug moiety (WO 02/088172).

Exemplary auristatin embodiments include N-terminally attached monomethyl auristatin drug moieties DE and DF, as disclosed in "monomer Compounds Cable of Conjugation to lipids", U.S. Pat. No.10/983,340,2004, 11/5, the disclosure of which is expressly incorporated herein by reference in its entirety.

Typically, peptide-based drug moieties can 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 liquid phase synthesis methods well known in the art of peptide chemistry (see E.and K.L u bke, The Peptides, volume1, pp76-136,1965, Academic Press). The auristatin/dolastatin drug module can be prepared according to the methods in the following references: U.S. Pat. No. 5,635,483, U.S. Pat. No. 5,780,588, Pettit et al (1989) J.Am.chem.Soc.111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design13:243-277; Pettit, G.R., et al.Synthesis,1996, 725; Pettit et al (1996) J.chem.Soc.PerkinTrans.15:859-863; and Doronina (2003) Nat Biotechnol21(7):778-784; monomeric Compounds binding of ligation Ligands, U.S. Pat. No.10/983,340,2004, filed 11.5.A., incorporated by reference in its entirety (disclosing, for example, methods for preparing single valine and MMvaline coupling linkers such as linkers to linkers and for coupling).

iii. calicheamicin

In other words, an immunoconjugate comprises an antibody of the invention conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of generating double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates see U.S. Pat. Nos. 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 to the company Cynamid, U.S.A.). Useful calicheamicin structural analogs include, but are not limited to, γ 1I, α 2I, α 3I, N-acetyl- γ 1I, PSAG and θ I1 (Hinman et al, Cancer Research53:3336-3342(1993); Lode et al, Cancer Research58:2925-2928(1998); and the aforementioned U.S. patents to Cyanamid, USA). Another anti-tumor drug that can be conjugated to an antibody is QFA, which is an antifolate drug. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, cellular uptake of these agents via antibody-mediated internalization greatly enhances their cytotoxic effects.

Other cytotoxic agents

Other anti-tumor agents that can be conjugated to the antibodies of the invention include BCNU, streptavidin, vincristine (vincristine), 5-fluorouracil, the family of agents collectively referred to as the LL-E33288 complex described in U.S. Pat. No. 5,053,394, 5,770,710, and epothilones (esperamicins) (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α -sarcin (sarcocin), Aleutites fordii (Aleuties fordii) toxic protein, dianthus chinensis (dianthin) toxic protein, Phytolacca americana (Phytolacca americana) toxic protein (PAPI, PAPII and PAP-S), Momordica charantia (Mordicacharantia) inhibitor, Jatropha curcin (curcin), crotin (crotin), Saponaria officinalis (saponaria officinalis) inhibitor, gelonin alba (gelonin), mitomycin (morin), tricin (restrictocin), trichothecin (triomycin), and trichothecin (enomycin). See, for example, WO93/21232 published on month 10 and 28, 1993.

The invention also contemplates immunoconjugates formed between an antibody and a compound having nucleic acid degrading activity (e.g., a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNase).

For selective destruction of tumors, the antibody may comprise a highly radioactive atom. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include At ²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. Where the conjugate is used for detection, the radioactive atom may be included for scintigraphic studiesE.g. Tc^99mOr I¹²³Or spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

Incorporation of radioactive or other labels into the conjugate can be carried out in known manner. For example, the peptides may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as Tc99m or I123, Re186, Re188 and In111 may be attached via a cysteine residue In the peptide. Yttrium-90 can be attached via lysine residues. The IODOGEN method (Frakeret al, biochem. Biophys. Res. Commun.80:49-57 (1978)) can be used to incorporate iodine-123. Other methods are described in detail in Monoclonal Antibodies in Immunoscintigraphy (Chatal, CRC Press, 1989).

Conjugates of the antibody and cytotoxic agent may be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisothiocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Research52: 127-.

The compounds of the present invention specifically encompass, but are not limited to, ADCs prepared with the following crosslinkers: commercial 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), e.g., available from Pierce Biotechnology Inc., Rockford, IL, U.S.A.). See 2003-2004 application manual and Catalog (Applications handbook and Catalog) pp.467-498.

v. preparation of antibody-drug conjugates

In the antibody-drug conjugates (ADCs) of the invention, the antibody (Ab) is conjugated via a linker (L) to one or more drug moieties (D), for example from about 1 to about 20 drug moieties per antibody. The ADCs of formula I can be prepared by several routes using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) the nucleophilic group of the antibody reacts via a covalent bond with a divalent linker reagent to form Ab-L, which then reacts with the drug moiety D; and (2) reaction of the nucleophilic group of the drug moiety with a bivalent linker reagent via a covalent bond to form D-L, followed by reaction with the nucleophilic group of the antibody. Additional methods for making ADCs are described herein.

Ab-(L-D)p I

The joint may be made up of one or more joint members. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimido 4- (2-pyridylthio) pentanoate ("SPP"), N-succinimido 4- (N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC"), and N-succinimido (4-iodo-acetyl) aminobenzoate ("SIAB"). Other linker components are known in the art, some of which are also described herein. See also "monomer valentineCompunds Cable of Conjugation to Ligands", U.S. Ser. No.10/983,340,2004, filed 11/5/11, the entire contents of which are incorporated herein by reference.

In some embodiments, the linker may comprise amino acid residues. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. 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 (gly-gly-gly). Amino acid residues that make up the amino acid linker moiety include those naturally occurring amino acids, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid linker components can be designed and optimized for their selectivity in enzymatic cleavage by specific enzymes (e.g., tumor associated proteases, cathepsin B, C and D, or plasmin proteases).

Nucleophilic groups of antibodies include, but are not limited to: (i) an N-terminal amino group; (ii) side chain amino groups, such as lysine; (iii) side chain sulfhydryl groups, such as cysteine; and (iv) glycosylating the hydroxyl or amino groups of the sugar in the antibody. The amino, thiol, and hydroxyl groups are nucleophilic and capable of reacting with electrophilic groups on a linker moiety to form a covalent bond, and linker reagents include: (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 groups and maleimide groups. Some antibodies have reducible interchain disulfide bonds, i.e., cysteine bridges. The antibody may be rendered reactive for conjugation to a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody via reaction of lysine with 2-iminothiolane (Traut's reagent), resulting in conversion of the amine to a thiol. Reactive thiol groups can be introduced into an antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing a mutant antibody comprising one or more non-native cysteine amino acid residues).

Antibody-drug conjugates of the invention can also be produced by modifying the antibody, i.e., introducing electrophilic moieties that can react with nucleophilic substituents on the linker reagent or drug. The sugar of the glycosylated antibody can be oxidized with, for example, a periodate oxidizing agent to form an aldehyde or ketone group that can react with the amine group of the linker reagent or drug moiety. The resulting imine Schiff base groups may form stable linkages or may be reduced, for example, with borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate moiety of a glycosylated antibody with galactose oxidase or sodium metaperiodate can generate carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate technologies). In another embodiment, a protein containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate, resulting in the formation of an aldehyde at the first amino acid (Geoghegan & Stroh, Bioconjugate chem.3:138-146(1992); U.S. Pat. No. 5,362,852). Such aldehydes may react with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on the drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting with electrophilic groups on a linker moiety to form a covalent bond, and linker reagents including: (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 groups, and maleimide groups.

Alternatively, fusion proteins comprising an antibody and a cytotoxic agent may be prepared, for example, by recombinant techniques or peptide synthesis. The length of the DNA may comprise regions encoding the two parts of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for tumor pre-targeting, wherein the antibody-receptor conjugate is administered to the patient, followed by clearance of unbound conjugate from the circulation using a clearing agent, followed by administration of a "ligand" (such as avidin) conjugated to a cytotoxic agent (such as a radionucleotide).

Pharmaceutical formulations

Therapeutic formulations comprising The antibodies of The invention may be prepared by mixing an antibody of The desired purity with an optional physiologically acceptable carrier, excipient or stabilizer, and stored as an aqueous solution, lyophilized or other dry dosage form (Remington: The Science and Practice of Pharmacy20 (2000)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; 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 including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as TWEEN ^TM、PLURONICS^TMOr polyethylene glycol (PEG).

The formulations herein may also contain more than one active compound necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. Suitably, such molecules are combined in amounts effective for the intended purpose.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington, The science and Practice of Pharmacy20th edition (2000).

Formulations for in vivo administration must be sterile. This can be easily achieved by filtration using sterile filtration membranes.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ -ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of sustained release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated antibodies are maintained in vivo for extended periods of time, they may denature or aggregate by exposure to a humid environment at 37 ℃, resulting in a loss of biological activity and possible changes in immunogenicity. Rational stabilization strategies can be designed according to the relevant mechanisms. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation via thiol-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling humidity, employing appropriate additives, and developing specific polymer matrix compositions.

Use of

The antibodies of the invention are useful, for example, in vitro, ex vivo and in vivo therapeutic methods.

In one aspect, the invention provides methods of treating or preventing a tumor, cancer, and/or cell proliferative disorder associated with increased expression and/or activity of EphrinB2, comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody.

In one aspect, the invention provides a method of reducing, inhibiting, or preventing tumor or cancer growth, comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody.

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 a method of inhibiting angiogenesis, the method comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody.

In one aspect, the invention provides a method of treating a pathological condition associated with angiogenesis, the method comprising administering to a subject in need of such treatment an effective amount of an anti-EphrinB 2 antibody. In some embodiments, the pathological condition associated with angiogenesis is a tumor, cancer, and/or 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 may bind to antigens from other species. Thus, the antibodies of the invention can be used to bind a particular antigenic activity, for example, in cell cultures containing the antigen, in human subjects, or in other mammals having the antigen with which the antibodies of the invention cross-react (e.g., chimpanzees, baboons, marmosets, rhesus monkeys and rhesus monkeys, pigs or mice). In one embodiment, the antibodies of the invention can be used to inhibit antigenic activity by contacting the antibody with an antigen such that the antigenic activity is inhibited. Preferably, the antigen is a human protein molecule.

In one embodiment, the antibodies of the invention are useful in a method of binding an antigen in a subject having a disorder associated with increased expression and/or activity of the antigen, comprising administering to the subject an antibody of the invention such that the antigen is bound in the subject. Preferably, the antigen is a human protein molecule and the subject is a human subject. Alternatively, the subject may be a mammal expressing an antigen to which an antibody of the invention binds. Still alternatively, the subject can be a mammal into which an antigen has been introduced (e.g., by administration of the antigen or by expression of an antigenic transgene). The antibodies of the invention can be administered to a human subject for therapeutic purposes. In addition, the antibodies of the invention can be administered for veterinary purposes to a non-human mammal (e.g., primate, pig or mouse) expressing an antigen with which an immunoglobulin is cross-reactive, or to an animal model of a human disease. With respect to the latter, such animal models can be used to assess the therapeutic efficacy of the antibodies of the invention (e.g., testing the dose and time course of administration).

The antibodies of the invention are useful for treating, inhibiting, delaying progression of, preventing/delaying relapse of, ameliorating, or preventing a disease, disorder, or condition associated with the expression and/or activity of one or more antigenic molecules.

Exemplary conditions include carcinoma, lymphoma, blastoma (blastoma), sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung), peritoneal cancer, hepatocellular cancer (hepatocellular cancer), gastric cancer (gastric or stomach cancer) (including gastrointestinal cancer), pancreatic cancer, glioblastoma (glioblastomama), cervical cancer, ovarian cancer, liver cancer (liverconer), bladder cancer, urethral cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (kidney or renal cancer), prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma (hepatic carcinoma), anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma, brain, and related metastases. In some embodiments, the cancer is selected from: small cell lung cancer, neuroblastoma (neuroblastoma), melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma.

The antibodies of the invention may also be used in the treatment (including prevention) of disorders whose pathology involves cell decline/degeneration or dysfunction, such as the treatment of various (chronic) neurodegenerative disorders and acute neuronal cell injury. Such neurodegenerative disorders include, but are not limited to, peripheral neuropathy; motor neuron disorders such as amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Bell's (Bell) paralysis, and various disorders 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 injury, such as that resulting from trauma or spinal cord injury.

In certain embodiments, an immunoconjugate comprising an antibody coupled to one or more cytotoxic agents is administered to a patient. In some embodiments, the immunoconjugate and/or the antigen to which it binds is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the target cell to which it binds. In one embodiment, the cytotoxic agent targets or interferes with a nucleic acid in a 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 described herein (such as maytansinoids, auristatins, dolastatins, or calicheamicins), radioisotopes, or ribonucleases or DNA endonucleases.

In therapy, the antibodies of the invention may be used alone or in combination with other compositions. For example, an antibody of the invention can be co-administered with another antibody, a chemotherapeutic agent (including a mixture of chemotherapeutic agents), other cytotoxic agents, anti-angiogenic agents, cytokines, and/or growth inhibitory agents. When the antibodies of the invention inhibit tumor growth, it may be particularly desirable to combine them with one or more other therapeutic agents that also inhibit tumor growth. Alternatively, or in addition, the patient may receive combination radiation therapy (e.g., external beam irradiation or therapy with radiolabeled agents such as antibodies). Such combination therapies described above include co-administration (when the two or more agents are contained in the same or separate formulations) and separate administration, in which case administration of the antibody of the invention may occur before and/or after administration of the adjunctive therapy.

Combination therapy

As noted above, the present invention provides combination therapies in which an anti-EphrinB 2 antibody is administered with another therapy. For example, anti-EphrinB 2 antibodies are used in combination with anti-cancer therapeutics or anti-neovascularization therapies to treat various neoplastic or non-neoplastic disorders. In one embodiment, the neoplastic or non-neoplastic condition is characterized by a pathological condition associated with abnormal or undesired angiogenesis. The anti-EphrinB 2 antibody can be administered sequentially or in combination with another agent effective for those purposes, either in the same composition or as a separate composition. Alternatively, or in addition, multiple inhibitors of EphrinB2 may be administered.

The administration of the anti-EphrinB 2 antibody can be performed simultaneously, e.g., as a single composition or as two or more different compositions, using the same or different routes of administration. Alternatively, or additionally, administration may be performed sequentially in either order. In certain embodiments, the interval between administration of two or more compositions may have a time ranging from minutes to days, to weeks, to months. For example, the anti-cancer agent may be administered first, followed by the EphrinB2 inhibitor. However, simultaneous or prior administration of anti-EphrinB 2 antibodies is also contemplated.

The effective amount of the therapeutic agent administered in combination with the anti-EphrinB 2 antibody will depend on the judgment of the physician or veterinarian. Dose administration and adjustment is done to achieve maximum control over the condition to be treated. The dosage will also depend on factors such as the type of therapeutic agent to be used and the particular patient being treated. Suitable doses of the anti-cancer agent are those currently used and may be reduced by the combined action (synergy) of the anti-cancer agent and the anti-EphrinB 2 antibody. In certain embodiments, the combination of inhibitors enhances the efficacy of a single inhibitor. The term "potentiate" refers to an increase in the efficacy of a therapeutic agent at its usual or approved dosage.

Typically, anti-EphrinB 2 antibodies and anti-cancer agents are suitable for the same or similar diseases to block or reduce pathological conditions such as tumor growth or growth of cancer cells. In one embodiment, the anti-cancer agent is an anti-angiogenic agent.

Anti-angiogenic therapies associated with cancer are cancer treatment strategies aimed at inhibiting the development of tumor blood vessels needed to provide nutrients to support tumor growth. Because angiogenesis is involved in both primary tumor growth and metastasis, the angiogenic therapy provided by the present invention is capable of inhibiting not only the neoplastic growth of tumors at primary sites, but also preventing metastasis of tumors at secondary sites, thereby allowing the tumors to be attacked by other therapeutic agents.

A number of anti-angiogenic agents have been identified and known in the art, including those listed herein, such as those listed in the definitions, see also, e.g., Carmeliet and Jain, Nature407:249-257(2000); Ferrara et al, Nature Reviews: Drug Discovery,3:391-400(2004); Sato int.J.Clin.Oncol.,8:200-206 (2003). See also US patent application US 20030055006. In one embodiment, the anti-EphrinB 2 antibody is used in combination with an anti-VEGF neutralizing antibody (or fragment) and/or another VEGF antagonist or VEGF receptor antagonist (including, but not limited to, for example, a fragment of a soluble VEGF receptor (e.g., VEGFR-1, VEGFR-2, VEGFR-3, neurophilins (e.g., NRP1, NRP 2)), an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, a low molecular weight inhibitor of VEGFR tyrosine kinase (RTK), an antisense strategy for VEGF, a ribozyme directed against VEGF or VEGF receptor, an antagonistic variant of VEGF, and combinations thereof. Alternatively, or in addition, optionally, two or more angiogenesis inhibitors may be co-administered to the patient in addition to the VEGF antagonist and the other agent. In certain embodiments, one or more additional therapeutic agents, such as an anti-cancer agent, may be administered in combination with the anti-EphrinB 2 antibody, VEGF antagonist, and anti-angiogenic agent.

In certain aspects of the invention, other therapeutic agents useful in combination tumor therapy with anti-EphrinB 2 antibodies include other cancer therapies (e.g., surgery, radiological treatments (e.g., involving irradiation or administration of radioactive substances), chemotherapy, treatment with anti-cancer agents listed herein and known in the art, or combinations thereof). Alternatively, or in addition, two or more antibodies that bind to the same antigen disclosed herein or two or more different antigens disclosed herein can be co-administered to a patient. Sometimes, it may be beneficial to also administer one or more cytokines to the patient.

Chemotherapeutic agents

In certain aspects, the invention provides methods of blocking or reducing tumor growth or growth of cancer cells by administering an effective amount of an EphrinB2 antagonist and/or an angiogenesis inhibitor and one or more chemotherapeutic agents to a patient susceptible to or diagnosed with cancer. A variety of chemotherapeutic agents may be used in the combination treatment methods of the present invention. An illustrative and non-limiting list of contemplated chemotherapeutic agents is provided herein in the "definitions".

As will be appreciated by those of ordinary skill in the art, suitable doses of chemotherapeutic agents will generally vary around those doses that are earlier employed in clinical therapy where the chemotherapeutic agent is administered alone or in combination with other chemotherapeutic agents. The dosage may vary depending on the condition being treated. The physician administering the treatment will be able to determine the appropriate dosage for the individual subject.

Recurrent tumor growth

The invention also provides methods and compositions for inhibiting or preventing recurrent tumor growth or recurrent cancer cell growth. Recurrent tumor growth (relapse tumor growth) or recurrent cancer cell growth is used to describe conditions in which patients undergoing or being treated with one or more currently available therapies (e.g., cancer therapies such as chemotherapy, radiation therapy, surgery, hormonal therapy and/or biological therapy/immunotherapy, anti-VEGF antibody therapy, particularly standard treatment regimens for particular cancers) are clinically inadequate to treat the patient or are no longer receiving any beneficial effect from the therapy such that the patients require an otherwise effective therapy. As used herein, the expression also refers to the condition of a "non-responsive/refractory" patient, e.g., describing that the patient is responsive to therapy but suffers from side effects, develops resistance, is not responsive to therapy, is not satisfactorily responsive to therapy, etc. In various embodiments, the cancer is recurrent tumor growth or recurrent cancer cell growth, wherein the number of cancer cells has not been satisfactorily reduced, or increased, or the tumor size has not been significantly reduced, or increased, or the size or number of cancer cells has failed to further reduce or decrease. Determining whether a cancer cell is a recurrent tumor growth or a recurrent cancer cell growth can be performed in vivo or in vitro, using the art-recognized meaning of "recurrent" or "refractory" or "non-responsive" in such context, by any method known in the art for determining the effectiveness of a treatment on cancer cells. Tumors resistant to anti-VEGF therapy are one example of recurrent tumor growth.

The present invention provides methods of blocking or reducing recurrent tumor growth or recurrent cancer cell growth in a subject by administering one or more anti-EphrinB 2 antibodies to block or reduce recurrent tumor growth or recurrent cancer cell growth in the subject. In certain embodiments, the antagonist can be administered after the cancer therapeutic. In certain embodiments, the anti-EphrinB 2 antibody is administered concurrently with cancer therapy. Alternatively, or in addition, the anti-EphrinB 2 antibody therapy is alternated with another cancer therapy, which may be performed in any order. The invention also encompasses the administration of one or more inhibitory antibodies to prevent the onset of cancer in a patient predisposed to cancerOr a method of recurrence. In one embodiment, the cancer therapy is treatment with an anti-angiogenic agent, such as a VEGF antagonist. Anti-angiogenic agents include those known in the art and those listed in the definitions herein. In one embodiment, the anti-angiogenic agent is an anti-VEGF neutralizing antibody or fragment (e.g., humanized a4.6.1, AVASTIN)(Genentech, South San Francisco, Calif.), Y0317, M4, G6, B20, 2C3, etc.). See, for example, U.S. Pat. Nos. 6,582,959,6,884,879,6,703,020, WO98/45332, WO96/30046, WO94/10202, EP0666868B1, U.S. patent application 20030206899,20030190317,20030203409,20050112126, Popkov et al, Journal of Immunological Methods288:149-164(2004), and WO 2005012359. Additional agents may be administered in combination with the VEGF antagonist and the anti-EphrinB 2 antibody to block or reduce recurrent tumor growth or recurrent cancer cell growth, e.g., see section herein entitled "combination therapy".

The antibodies (and adjunctive therapeutic agents) of the invention are administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, as well as intralesional (if local treatment is desired) administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, pulse infusion of the antibody is also suitable, particularly in the form of a gradual reduction in the antibody dose. The medication may be administered by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term.

The antibody compositions of the invention can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site at which the agent is delivered, the method of administration, the schedule of administration, and other factors known to medical practitioners. It is not necessary but optional to formulate the antibody with one or more agents currently used for preventing or treating the disorder in question. The effective amount of such other agents depends on the amount of the antibody of the invention present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and routes of administration as used above, or about 1-99% of the dosages used so far.

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, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1. mu.g/kg to 15mg/kg (e.g., 0.1mg/kg-10 mg/kg) of antibody is an initial candidate dose for administration to a patient, whether, for example, by one or more separate administrations, or by continuous infusion. Typical daily dosages may range from about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is maintained until the desired suppression of disease symptoms occurs. An exemplary dose of the antibody ranges from about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient. The dose may be administered intermittently, e.g., weekly or every three weeks (once) (e.g., such that the patient receives about 2 to about 20 doses, e.g., about 6 doses, of the antibody). One dose of a higher loading dose may be administered followed by one or more doses of a lower dose. An exemplary dosing regimen comprises administering one dose of a loading agent of about 4mg/kg followed by one dose per week of a maintenance agent of about 2mg/kg antibody. However, other dosage regimens may also be useful. The progress of such therapy can be conveniently monitored by conventional techniques and assays.

The anti-EphrinB 2 antibodies of the invention can be used in assays (such as diagnostic or prognostic assays) that detect EphrinB2 expression in specific cells or tissues, wherein the antibodies are labeled and/or immobilized on an insoluble substrate as described below.

In another aspect, the invention provides a method of diagnosing a disorder associated with EphrinB2 expression and/or activity, the method comprising detecting an EphrinB 2-anti-EphrinB 2 antibody complex in a biological sample from a patient having or suspected of having the disorder. In some embodiments, EphrinB2 expression is increased expression or aberrant (undesired) expression. In some embodiments, the disorder is a tumor, cancer, and/or cell proliferative disorder.

The anti-EphrinB 2 antibodies can be used for EphrinB2 detection in any of a number of well-known detection assays. For example, EphrinB2 can be assayed on a biological sample by obtaining the sample from a desired source, mixing the sample with an anti-EphrinB 2 antibody to allow the antibody to form an antibody/EphrinB 2 complex with any EphrinB2 present in the mixture, and detecting any antibody/EphrinB 2 complex present in the mixture. Biological samples can be prepared for assay by methods known in the art appropriate for the particular sample. The method of mixing the sample with the antibody and the method of detecting the antibody/EphrinB 2 complex are selected according to the type of assay used. Such assays include immunohistochemistry, competitive and sandwich/sandwich assays, and steric inhibition assays (steric inhibition assays).

The analytical methods for EphrinB2 all use one or more of the following reagents: labeled EphrinB2 analogs, immobilized EphrinB2 analogs, labeled anti-EphrinB 2 antibodies, immobilized anti-EphrinB 2 antibodies, and steric conjugates. The labelled reagent is also referred to as a "tracer".

The label used is any detectable functional group that does not interfere with the binding of EphrinB2 to the anti-EphrinB 2 antibody. Many labels are known for use in immunoassays, examples include moieties that can be detected directly (moity), such as fluorescent dyes, chemiluminescent and radioactive labels, and moieties that must be reacted or derivatized for detection, such as enzymes. Examples of such markers include:

The label used is any detectable functional group (functional) that does not interfere with the binding of EphrinB2 to the anti-EphrinB 2 antibody. Many labels are known for use in immunoassays, examples include moieties that can be detected directly, such as fluorescent dyes, chemiluminescent and radioactive labels, and moieties that must be reacted or derivatized for detection, such as enzymes. Examples of such labels include radioisotopes³²P、¹⁴C、¹²⁵I、³H and¹³¹i, fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No.4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with enzymes that oxidize dye precursors using hydrogen peroxide such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

Conventional methods for covalently binding these labels to proteins or polypeptides are available. For example, coupling agents such as dialdehydes, carbodiimides, bismaleimides, bisimidates, bisdiazotized benzidine, and the like may be used to label the antibodies with the above-described fluorescent, chemiluminescent, and enzymatic labels. See, e.g., U.S. Pat. Nos. 3,940,475 (fluorometry) and 3,645,090 (enzymes), Hunter et al, Nature,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 labels herein are enzymes such as horseradish peroxidase and alkaline phosphatase. Coupling such labels, including enzymes, to antibodies is a standard procedure for those of ordinary skill in the immunoassay art. See, for example, O' Sullivan et al, "Methods for the preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," Methods in Enzymology, ed.J.J.Langlone and H.Van Vunakis, Vol.73(Academic Press, New York, New York,1981), pp.147-166.

Some assays require immobilization of reagents. Immobilization enables separation of the anti-EphrinB 2 antibody from any EphrinB2 still free in solution. For this purpose, either the anti-EphrinB 2 antibody or EphrinB2 analogue is insolubilized before the assay protocol by adsorption to a water-insoluble substrate or surface (Bennich et al, u.s.3,720,760), by covalent coupling (e.g. cross-linking with glutaraldehyde), or the anti-EphrinB 2 antibody or EphrinB2 analogue is insolubilized after the assay protocol, e.g. by immunoassay precipitation.

Immunohistochemistry and staining protocols can be used to examine protein expression in a sample. Immunohistochemical staining of tissue sections has proven to be a reliable method of assessing or detecting the presence of proteins in a sample. Immunohistochemistry ("IHC") techniques utilize antibodies to probe and visualize cellular antigens in situ, usually by chromogenic or fluorescent methods. For sample preparation, a tissue or cell sample from a mammal (typically a human patient) may be used. Examples of samples include, but are not limited to, cancer cells such as colon, breast, prostate, ovarian, lung, stomach, pancreatic, lymphoma, and leukemia cancer cells. The sample may be obtained by a variety of procedures known in the art, including but not limited to surgical resection, aspiration, or biopsy. The tissue may be fresh or frozen. In one embodiment, the sample is fixed and embedded in paraffin or the like. The tissue sample may be fixed (i.e., preserved) by conventional methods. One of ordinary skill in the art will appreciate that the choice of fixative is determined by whether the sample is to be used for histological staining or other analytical purposes. One of ordinary skill in the art will also appreciate that the length of fixation will depend on the size of the tissue sample and the fixative used.

IHC may be performed with other techniques such as morphological staining and/or fluorescence in situ hybridization. There are two commonly used IHC methods: direct and indirect assays. According to the first assay, the binding of an antibody to a target antigen (e.g., EphrinB 2) is determined directly. This direct assay uses a labeled reagent, such as a fluorescent label or an enzyme-labeled primary antibody, which is visualized without further antibody interaction. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. If the second antibody is conjugated to an enzyme 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 antibodies used for immunohistochemistry are typically labeled with a detectable moiety. There are a number of markers which can be generally divided into the following types:

in addition to the sample preparation protocol discussed above, further processing of the tissue slices before, during, or after IHC may be required. For example, epitope retrieval methods can be performed, such as heating tissue samples in citrate buffer (see, e.g., Leong et al. appl. Immunohistochem.4(3):201 (1996)).

Following the optional blocking step, the tissue section is exposed to the first antibody under suitable conditions for a sufficient time such that the first antibody binds to the target protein antigen in the tissue sample. Suitable conditions for achieving this can be determined by routine experimentation. The extent of binding of the antibody to the sample is determined by using any of the detectable labels discussed above. Preferably, the label is an enzymatic label (e.g., HRPO) that catalyzes a chemical change in a chromogenic substrate such as 3,3' -diaminobenzidine chromogen. Preferably, the enzyme label is conjugated to an antibody that specifically binds to the first antibody (e.g., the first antibody is a rabbit polyclonal antibody and the second antibody is a goat anti-rabbit antibody).

The specimen thus prepared can be placed and covered with a cover slip. Slide evaluation is then performed, for example, using a microscope, and staining intensity criteria routinely used in the art can be employed. The staining intensity criteria can be evaluated as follows:

TABLE 2

Dyeing pattern	Score of
		No staining was observed in the cells.	0

Faint/barely detectable staining was detected in more than 10% of the cells.	1+
		Weak to moderate staining was observed in more than 10% of the cells.	2+
Moderate to strong staining was observed in more than 10% of the cells.	3+

Typically, staining patterns that score about 2+ or higher in IHC assays are diagnostic and/or prognostic. In some embodiments, a staining pattern with a score of about 1+ or greater is diagnostic and/or prognostic. In other embodiments, a staining pattern with a score of about 3 or higher is diagnostic and/or prognostic. It will be appreciated that in examining cells and/or tissue from a tumor or colon adenoma using IHC, staining in tumor cells and/or tissue (rather than in the stroma or surrounding tissue that may be present in the sample) is typically measured or assessed.

Other assays, known as competitive or sandwich assays, have been well established and widely used in the commercial diagnostic industry.

Competitive assays rely on the ability of the tracer EphrinB2 analog to compete with the test customer sample EphrinB2 for a limited number of antigen binding sites of anti-EphrinB 2 antibodies. anti-EphrinB 2 antibody is typically insolubilized before or after competition, and the tracer and EphrinB2 bound to the anti-EphrinB 2 antibody are then separated from the unbound tracer and EphrinB 2. This separation is achieved by pouring (where the binding partners are insolubilized beforehand) or by centrifugation (where the binding partners are precipitated after the competitive reaction). The amount of the test sample EphrinB2 is inversely proportional to the amount of bound tracer, which is measured by the amount of the labeling substance. A dose-response curve is plotted for a known amount of EphrinB2, which is compared to the test results to quantitatively determine the amount of EphrinB2 present in the test sample. When enzymes are used as detectable labels, these assays are referred to as ELISA systems.

Another competitive assay, known as the "homogeneous" assay, does not require phase separation. Here, conjugates of enzymes and EphrinB2 were prepared and used such that the presence of anti-EphrinB 2 antibodies altered the enzyme activity when anti-EphrinB 2 antibodies bound EphrinB 2. In this case, EphrinB2 or an immunologically active fragment thereof is coupled to an enzyme such as a peroxidase with a bifunctional organic bridge. The conjugate is selected for use with an anti-EphrinB 2 antibody such that binding of the anti-EphrinB 2 antibody inhibits or enhances the enzymatic activity of the marker. This method is widely practiced per se, and is called EMIT.

Steric conjugates (steric conjugates) are used in steric hindrance methods of homogeneous assays. These conjugates are synthesized by covalently linking a low molecular weight hapten to a small EphrinB2 fragment such that antibodies directed against the hapten are substantially unable to bind the conjugate at the same time as the anti-EphrinB 2 antibody. Under this assay protocol, EphrinB2 present in the test sample will bind to the anti-EphrinB 2 antibody, thereby allowing the anti-hapten to bind to the conjugate, resulting in a change in the hapten properties of the conjugate, such as a change in fluorescence when the hapten is a fluorophore.

The sandwich assay is particularly useful for the determination of EphrinB2 or anti-EphrinB 2 antibodies. In a sequential sandwich assay (sequential sandwich assay), an immobilized anti-EphrinB 2 antibody is used to adsorb the test sample EphrinB2, the test sample is removed by washing, the bound EphrinB2 is used to adsorb a labeled second anti-EphrinB 2 antibody, and the bound species is then separated from the residual tracer. The amount of bound tracer was directly proportional to the test sample EphrinB 2. In the "simultaneous" sandwich assay (simultaneous sandwich assay), test samples were not isolated prior to addition of labeled anti-EphrinB 2. A sequential sandwich assay using an anti-EphrinB 2 monoclonal antibody as one antibody and a polyclonal anti-EphrinB 2 antibody as the other antibody can be used to test the sample for EphrinB 2.

The above is merely an exemplary detection assay for EphrinB 2. Other methods now or hereafter developed for determining EphrinB2 using anti-EphrinB 2 antibodies are included within the scope of the invention, including the biological assays described herein.

Article of manufacture

In another aspect of the invention, there is provided an article of manufacture comprising a substance useful in the treatment, prevention and/or diagnosis of the disorders described above. An article of manufacture comprises a container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be made of various materials, such as glass or plastic. The container contains a composition that is effective, by itself or in combination with other compositions, in the treatment, prevention and/or diagnosis of the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable 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 for use in treating a selected condition, such as cancer. In addition, an article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition therein, wherein the composition comprises an additional cytotoxic agent. The article of manufacture of this embodiment of the invention may further comprise a package insert indicating that the first and second antibody compositions are useful for treating a particular condition, such as cancer. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be implemented in accordance with the general description provided above.

Examples

Example 1: generation of anti-EphrinB 2 antibodies

A variety of methods are known in the art for generating phage display libraries from which antibodies of interest can be obtained. Synthetic phage antibody libraries (Lee, C.V.et al.J. Mol Biol340,1073-93(2004); Liang, W.C.et al.J. Biol Chem281,951-61(2006)) were constructed on a single framework (humanized anti-ErbB 2 antibody, 4D 5) by introducing diversity within the Complementarity Determining Regions (CDRs) of the heavy and light chains. The naive library was plate panned against His-tagged human EphrinB2 immobilized on a maxisorp immune plate. After four rounds of enrichment, clones were randomly picked and specific binders identified using phage ELISA. The resulting heprinb 2-binding clones were further screened with His-tagged murine EphrinB2 protein to identify cross-species clones. Clone 19 performed well in these assays and was selected for further characterization. For each positive phage clone, the variable regions of the heavy and light chains were subcloned into a pRK expression vector engineered to express full-length IgG chains. The heavy and light chain constructs were co-transfected into 293 or CHO cells and the expressed antibodies were purified from serum-free culture using a protein a affinity column. The purified antibodies were tested for blocking the interaction between EphrinB2 and EphB receptor by ELISA and for binding to stable cell lines expressing full-length human EphrinB2 or murine EphrinB2 by FACS. For affinity maturation, phage libraries with different combinations of three CDR loops (CDR-L3, -H1, and-H2) derived from the original clone of interest were constructed by a soft randomization strategy (soft randomization strategy) such that each selected position was mutated to a non-wild-type residue or maintained wild-type at a frequency of about 50:50 (Liang et al, 2006, supra). High affinity clones were then identified by four rounds of increasing stringency solution phase panning against human and murine His-tagged EphrinB2 protein. Selected clones were screened by phage ELISA, then expressed as Fab proteins and their affinities determined using Biacore. The sequences of the HVR regions of the parental clone 19 and the affinity matured clone are shown in FIG. 1.

Example 2: characterization of anti-EphrinB 2 antibodies

To determine the binding affinity of mouse anti-EphrinB 2 mAb, BIAcore was used^TMSurface plasmon resonance (SRP) measurements were performed at-3000 (BIAcore, inc., Piscataway, NJ). Briefly, carboxymethylated dextran biosensor 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-EphrinB 2 antibody was diluted to 5. mu.g/ml with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain approximately 500 Response Units (RU) of conjugated antibody. Next, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, human or murine EphrinB2-His molecules, diluted twice in serial in PBS with 0.05% Tween-20, were injected at 25 ℃ at a flow rate of about 25. mu.l/min. The binding rate (k) was calculated using a simple one-to-one Langmuir binding model (BIAcore EvaluationSoftware version3.2)_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. The results of this experiment are shown in table 3. "NA" means no measurement was made.

Table 3: binding affinity and kinetics of anti-EphrinB 2 antibodies binding to human and mouse EphrinB2

Example 3: anti-EphrinB 2 antibody treatment blocks EphB4 signaling in cell-based assays

To demonstrate the ability of anti-EphrinB 2 antibodies to block the interaction of membrane-bound EphrinB2 and EphB4, we performed cell-based assays in which EphB4 and EphrinB2 are presented by different cell types. 3T3 cells overexpressing human EphrinB2 were used to stimulate HUVEC cells expressing high levels of EphB4 but low levels of EphrinB2, and the ability of anti-EphrinB 2 antibodies to inhibit EphB4 activation was tested.

3T3 cells overexpressing human EphrinB2 were prepared as follows: human full-length EphrinB2 was cloned into pcDNA5/FRT vector (Invitrogen) and subsequently used to generate stable cell lines together with 3t3.flp cells (Invitrogen) according to the manufacturer's manual.

3T3 cells overexpressing human EphrinB2 were plated on HUVEC cells for 15 or 30 minutes with or without anti-EphrinB 2 antibody. Activation of the EphB4 receptor was assessed by immunoprecipitating EphB4 protein and then detecting the presence or absence of tyrosine phosphorylation of EphB4 receptor by western blotting using an anti-phospho-tyrosine antibody (antibody 4G10; Upstate). Briefly, cells were lysed with RIPA buffer. Cell lysates were clarified by centrifugation and anti-EphrinB 2 antibody 19.2D3 was added at 5 μ g/sample. After 2 hours of incubation at 4 ℃, the immunocomplexes were electrophoresed using protein a agarose. EphB4 phosphorylation was analyzed by Western blotting using the anti-phosphotyrosine antibody 4G10(Upstate) at a concentration of 1. mu.g/ml.

The results of this experiment are shown in figure 7. Plating of 3T3 cells on HUVEC cells caused significant EphB4 tyrosine phosphorylation (lane 2). Pre-incubation of HUVEC with anti-EphrinB 2 antibody (clone 19.2D3, 5. mu.g/ml) for 30 minutes effectively abolished the 3T3-EphrinB2 cell coverage induced tyrosine phosphorylation of EphB4 (lane 3). In contrast, untreated HUVEC cells did not exhibit EphB4 activation (lane 1), while untreated 3T3-ephrinB2 cells did not exhibit EphB4 activation (lane 4). These results establish that anti-EphrinB 2 antibody treatment blocks EphrinB2 ligand-EphB 4 receptor interaction in the context of direct cell-cell contact.

Example 4: anti-EphrinB 2 antibody treatment inhibited angiogenesis in rat corneal pocket assay

The anti-activity (anti-activity) of the monoclonal anti-EphrinB 2 antibody was tested in the rat corneal pocket assay (rat corneal pocket assay). Briefly, Harlan Sprague-Dawley rats were anesthetized with isoflurane and a low dose injectable anesthetic (low dose injectable anesthesia). The eyeball was gently protruded and fixed in place with atraumatic forceps (seminal in place). Using a No. 15 blade, a 1.5mm incision was made near the center of the cornea. Using a microscraper (microspatula), a blunt-disconnect (blunt-disconnect) incision is carefully made, passed through the stroma (stroma), and passed to the external canthus. Hydron-coated pellets containing growth factor (200ng VEGF), methylcellulose, and aluminum sulfate (100 μ g) were inserted into the bottom of the bag. The anti-EphrinB 2 antibody clone 19.2D3 (10 μ g/pill), if added, was included in the pill. After surgery, the eyes were coated with gentamicin ointment. On day 6, animals were injected with high molecular weight fluorescein isothiocyanate-dextran and euthanized to allow visualization of the vascular structure. Corneal total specimen embedding was performed using the removed eyeball, and the new blood vessel area was measured using computer-aided Image analysis (Image-Pro Plus).

The results of this example are shown in FIG. 8. anti-EphrinB 2 antibody treatment significantly reduced VEGF-induced neovascularization, demonstrating that anti-EphrinB 2 antibody has anti-angiogenic activity in this model. Control treatment (lacking VEGF) showed limited neovascularization, while VEGF-treated positive control showed significant neovascularization.

Example 5: anti-EphrinB 2 antibody treatment inhibits angiogenesis in mouse dorsal cavity assays

Monoclonal anti-EphrinB 2 antibodies were tested for anti-angiogenic activity in a mouse dorsalwindow chamber assay (mouse dorsal window chamber assay). The protocols and techniques used in the back-window assay were essentially performed as described in paperfuss, d.microviscs.res., 18:311-318,1979 and Shan, s.clinical cancer Research,7:2590-2596, 2001. Briefly, the anatomical midline (anatomical line) is marked along the back and the C clip is sutured into place with a 4-0 wire. A template corresponding to the outer diameter of the collared (collar) was used to delineate the incision with a sterile marker. A circular cut is made along the contour perimeter, followed by an effort to make a cross cut following the subcutaneous tissue above the fascia. The area is then trimmed with a pair of fine tweezers and iris scissors. All two fascia layers were removed from one side of the skin fold: these layers maintain the intact vascular structure (vasculatures). The C clip was then removed. The groove with the window frame in the center was inserted into the skin fold and secured in place with 4-0 silk thread. Tumor cells were injected into the fascia in the window. The window was sealed with a cover slip. A thin layer of neospora antibiotic ointment was applied to the suture and incision wound to prevent infection. Animals were observed under a stereomicroscope for dissection to confirm circulation within the cavity. Animals were allowed to recover on a circulating heating blanket until they were completely recovered from anesthesia and finally returned to their room in their mini-isolation cages. The antibodies (anti-EphrinB 2 antibody clone 19.2D3 and anti-mouse VEGF antibody G6) were administered twice at a dose of 10mg/kg, one dose was i.v. administered at the time of cell injection, and the second dose was administered by direct injection into the lumen on day 3. Tumor area and tumor vasculature were calculated using Image-Pro.

anti-EphrinB 2 antibodies significantly reduced neovascularization, demonstrating anti-EphrinB 2 anti-angiogenic activity in this model. Treatment with the positive control anti-mouse VEGF antibody showed significantly reduced neovascularization, as expected. In contrast, the untreated control showed extensive neovascularization.

Example 6: anti-EphrinB 2 antibody treatment inhibits tumor growth in vivo

To determine the ability of anti-EphrinB 2 antibodies to inhibit tumor growth in vivo, anti-EphrinB 2 antibodies were tested in a tumor xenograft model as described below. Briefly, light brown nude mice (Charles river laboratories, Hollister, Calif.) were treated and used according to the experimental animal care and use guidelinesSouth (the guide for the car and use of laboratory animals). A673 human rhabdomyosarcoma cells were suspended in serum-free medium and mixed with an equal volume of matrigel (matrigel). To establish subcutaneous tumor xenografts, 5x10⁶Individual cells were injected into the right side of 6-8 week old female mice. 24 hours after cell inoculation, animals were dosed i.p. with 0.2ml of antibody at a dose of 10mg/kg body weight, twice a week. Tumor growth was quantified by caliper measurements. Tumor volume (mm) was determined by measuring length (l) and width (w) and calculating volume (V = lw2/2) ³). 10 animals per group received PBS, 10mg/kg body weight anti-EphrinB 2 antibody clone 19.2D3, or 10mg/kg body weight anti-mouse VEGF (B6), twice weekly (indicated by arrows in FIG. 9).

The results of this example are shown in FIG. 9. anti-EphrinB 2 antibody treatment reduced mean tumor volume, demonstrating that anti-EphrinB 2 antibody treatment inhibited tumor growth in vivo. Mean tumor volume with SE is indicated in the figure.

Although the foregoing aspects have been described in some detail by way of illustration for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention.

Claims

1. Use of an anti-EphrinB 2 antibody for the manufacture of a medicament for reducing, inhibiting and/or blocking EphB2 activity, wherein the antibody is selected from the group consisting of:

(a) antibodies comprising HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3 in the order SEQ ID NOS 6, 8, 10, 1, 3, 5;

(b) antibodies comprising HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3 in the order SEQ ID NOS 7, 9, 11, 1, 3, 5; and

(c) antibodies comprising HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3 in the order SEQ ID NO 6, 8, 12, 2, 4, 5.

2. The use of claim 1, wherein the medicament reduces, inhibits and/or blocks EphB2 autophosphorylation.

3. The use of claim 1, wherein said antibody in said medicament competes for binding to an EphB2 ligand.

4. The use of claim 1, wherein said antibody in said medicament binds EphrinB2 with a dissociation constant (Kd) of about 30pM or stronger.

5. The use of claim 1, wherein said antibody in said medicament disrupts or blocks ligand binding to EphB 2.

6. The use of claim 1, wherein said antibody in said medicament reduces EphB2 tyrosine phosphorylation.

7. An antibody fragment of an anti-EphrinB 2 antibody, wherein the anti-EphrinB 2 antibody is selected from the group consisting of:

(c) antibodies comprising HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3 in the order SEQ ID NOS 6, 8, 12, 2, 4, 5; and wherein the antibody fragment is a Fab, Fab ', F (ab')2 or Fv fragment.

8. A polynucleotide encoding the antibody fragment of claim 7.

9. A vector comprising the polynucleotide of claim 8.

10. The vector of claim 9, wherein the vector is an expression vector.

11. An isolated host cell comprising the vector of claim 9 or 10.

12. The host cell of claim 11, wherein the host cell is prokaryotic.

13. The host cell of claim 11, wherein the host cell is eukaryotic.

14. The host cell of claim 13, wherein the host cell is mammalian.

15. A method of making an anti-EphrinB 2 antibody fragment, the method comprising: (a) expressing the vector of claim 9 or 10 in a suitable isolated host cell, and (b) producing said antibody fragment.

16. The method of claim 15, further comprising: (c) recovering the antibody fragment.

17. Use of an effective amount of an antibody fragment of claim 7 in the manufacture of a medicament for inhibiting angiogenesis in a subject in need of such treatment, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3 in the order SEQ ID NOs 6, 8, 12, 2, 4, 5, and wherein the antibody fragment is Fab, Fab ', F (ab')₂Or an Fv fragment.

18. The use of claim 17, wherein the medicament is a medicament for use in combination with an anti-angiogenic agent.

19. The use of claim 18, wherein the anti-angiogenic agent is administered prior to or after the anti-EphrinB 2 antibody fragment.

20. The use of claim 18, wherein the anti-angiogenic agent is administered concurrently with the anti-EphrinB 2 antibody fragment.

21. The use of claim 18, wherein the anti-angiogenic agent is an antagonist of vascular endothelial cell growth factor (VEGF).

22. The use of claim 21, wherein the VEGF antagonist is an anti-VEGF antibody.

23. The use of claim 22, wherein the anti-VEGF antibody is bevacizumab.

24. The use of claim 17, wherein the medicament is a medicament in combination with an effective amount of a chemotherapeutic agent.

25. The use of any one of claims 17-24, wherein said anti-EphrinB 2 antibody fragment is conjugated to a detectable label or a cytotoxic agent.

26. The use of any one of claims 17-24, wherein the subject is diagnosed with a cancer, a tumor, and/or a cell proliferative disorder.

27. The use of any one of claims 17-24, wherein the anti-EphrinB 2 antibody fragment is coupled to a detectable label or a cytotoxic agent, and wherein the subject is diagnosed with a cancer, a tumor, and/or a cell proliferative disorder.

28. Use of an anti-EphrinB 2 antibody fragment of claim 7 in the manufacture of a product or kit for detecting EphrinB2, said detection comprising detecting EphrinB 2-anti-EphrinB 2 antibody fragment complex in a biological sample.

29. The use of claim 28, wherein said anti-EphrinB 2 antibody fragment is detectably labeled.

30. The use of claim 28 or 29, wherein the biological sample is from a subject having a cancer, a tumor, and/or a cell proliferative disorder.