TWI391402B - Anti-fphb4 antibodies and methods using same - Google Patents

Description

Anti-EPHB4 antibody and method of using the same

The invention relates generally to the field of molecular biology. More specifically, the invention relates to anti-EphB4 antibodies and the use of such antibodies.

For many physiological and pathological processes, vascular supply development is a basic need. Actively growing tissues such as embryos and tumors require an adequate blood supply. It fulfils this need by creating pro-angiogenic factors that promote the formation of new blood vessels via a process known as angiogenesis. Angiogenesis is a complex but ordered biological event involving all or more of the following steps: a) endothelial cell (EC) self-existing EC proliferation or differentiation from progenitor cells; b) EC migration and coalescence to form a cord-like structure c) the vascular cord then undergoes tubulogenesis to form a blood vessel with the central lumen; d) the existing cord or blood vessel grows to sprout to form a secondary blood vessel; e) the initial vascular plexus undergoes further remodeling and shaping; and f) The peripheral endothelial cells are recruited to package the endothelial tubes, thereby providing maintenance and regulation functions to the blood vessels; these cells include cells outside the small capillaries, smooth muscle cells of larger blood vessels, and cardiomyocytes in the heart. Hanahan, Science 277: 48-50 (1997); Hogan and Kolodziej, Nat. Rev. Genet . 3: 513-23 (2002); Lubarsky and Krasnow, Cell 112: 19-28 (2003).

It is now fully established: angiogenesis is involved in the pathogenesis of various disorders. Such conditions include solid tumors and metastases, atherosclerosis, post-lens fibrous tissue hyperplasia, hemangioma, chronic inflammation, intraocular neovascular diseases such as proliferative retinopathy (eg, diabetic retinopathy), age-related macula Department of degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis and psoriasis. Folkman et al, J. Biol. Chem. 267: 10931-34 (1992); Klagsbrun et al, Annu. Rev. Physiol . 53: 217-39 (1991); and Garner A., "Vascular diseases" in: Pathobiology A Dynamic Approach, Garner A., Klintworth GK, eds. 2 (Marcel Dekker, NY, 1994), pp. 1625-1710.

In tumor growth, angiogenesis appears to be critical for the transition from hyperplasia to tumor formation and to nourishment to tumor growth and metastasis. Folkman et al, Nature 339: 58 (1989). Compared to normal cells, neovascularization allows tumor cells to gain growth and autonomy. Tumors usually start as a single abnormal cell that can proliferate to a few cubic millimeters in size due to the distance from the available capillary bed, and it can remain "sleep" without further growth and spread over a long period of time. Next, some tumor cells are converted to an angiogenic phenotype to activate endothelial cells that proliferate and mature into new capillaries. These newly formed blood vessels not only allow the primary tumor to continue to grow, but also allow metastatic tumor cells to spread and re-colonize. Therefore, in breast cancer and several other tumors, the correlation between microvessel density and patient survival in tumor sections has been observed. Weidner et al, N. Engl. J. Med. 324: 1-6 (1991); Horak et al, Lancet 340: 1120-24 (1992); Macchiarini et al, Lancet 340: 145-46 (1992). Although the precise mechanism for controlling angiogenesis switching is not fully understood, it is believed that neovascularization of tumor material is produced by the net balance of numerous angiogenic stimulators and inhibitors (Folkman, Nat . Med.1 (1):27-31 (1995)).

The process of vascular development is closely regulated. To date, a large number of molecules (mostly secreted factors produced by surrounding 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 stimulating angiogenesis and inducing vascular permeability. Ferrara et al., Endocr. Rev. 18: 4-25 (1997). It was found that even a single VEGF allele deletion resulted in embryonic death, indicating an irreplaceable role for this factor in vascular system development and differentiation. In addition, VEGF has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders. The aforementioned Ferrara et al., Endocr. Rev. Most human tumors examined overexpressed VEGF mRNA. 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. Cancer 73: 931-34 (1996); Dvorak et al, Am. J. Pathol. 146: 1029-39 (1995).

Similarly, for diabetic and other ischemic-related retinopathy, the level of VEGF in the ocular fluid is highly correlated with the presence of vasoactive hyperplasia in the patient. Aiello et al., N. Engl. J. Med. 331:1480-87 (1994). In addition, studies have shown that the location of VEGF in the choroidal neovascular membrane in patients is affected by AMD. Lopez et al, Invest. Ophthalmol . Vis . Sci. 37: 855-68 (1996).

Anti-VEGF neutralizing antibodies inhibit the growth of various human tumor cell lines in nude mice (Kim et al, Nature 362: 841-44 (1993); Warren et al, J. Clin. Invest. 95: 1789-97 (1995) ;Borgstr M et al, Cancer Res. 56:4032-39 (1996); Melnyk et al, Cancer Res. 56:921-24 (1996)) and also inhibits intraocular angiogenesis in a model of ischemic retinopathy (Adamis et al) Man, 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 various intraocular neovascular disorders. Such antibodies are described in, for example, EP 817,648, issued Jan. 14, 1998; the disclosure of which is incorporated herein by reference. An anti-VEGF antibody, bevacizumab, has been approved by the FDA for use in combination with chemotherapy to treat metastatic colorectal cancer (CRC). Bevacizumab was studied in a number of ongoing clinical trials for the treatment of various cancer indications.

The EphB4 receptor ("EphB4" or "EphB4R") is a member of the eph receptor family, which constitutes the largest family of tyrosine kinase receptors in the human genome (reviewed in Dodelet, Oncogene, 19: 5614-5619, 2000) . Human eph receptor tyrosine kinases are classified into class A and class B according to sequence identity, wherein the corresponding A-type and B-type ligands are called ephrin. Signal transduction can occur in a manner in which the receptor tyrosine kinase is activated by a ligand; and can occur in a reversible manner, wherein the transmembrane ephrinB ligand is activated by interaction with the receptor. A variety of biological functions have been implicated in Eph receptor ligand interactions, including axon guidance, tissue boundary formation, angiogenesis, and cellular activity (Kullander et al., Nat. Rev. Mol. Cell. Biol. , 3: 475-486, 2002; Cheng et al, Cytokine Growth Factor Rev., 13: 75-85, 2002; Coulthard et al, Int. J. Dev. Biol., 46: 375-384, 2002). EphB4 binds to ligands such as ephrin-B1, ephrin-B2, and ephrin-B3. The EphB4 receptor has an extracellular region in which a cysteine-rich motif extends beyond its amino terminus half, followed by two type II fibronectin motifs. There is an intracellular domain characterized by a conserved kinase domain and a transmembrane domain.

Clearly, there remains a need for agents that have the best clinical properties for developing therapeutic agents. The invention described herein satisfies this need and provides other advantages.

All references cited herein are hereby incorporated by reference in their entireties in their entireties in the the the the the the the the the

The invention is based, in part, on the recognition of various EphB4 binding agents (e.g., immunoconjugates, antibodies, and fragments thereof). EphB4 represents an important and advantageous therapeutic goal, and the present invention provides compositions and methods based on the combination of EphB4. The EphB4 binding agents of the invention as described herein provide important therapeutic and diagnostic agents for the targeted use of pathological conditions associated with EphB4-EphB4 ligand pathway expression and/or activity. Accordingly, the present invention provides methods, compositions, kits, and articles of manufacture associated with the incorporation of EphB4.

The invention provides antibodies that bind (eg, specifically bind) to EphB4.

In one aspect, the invention provides an isolated anti-EphB4 antibody, wherein the full length IgG form of the antibody specifically binds to human EphB4, wherein the binding affinity is about 50 pM or better. As is well established in the art: the binding affinity of a ligand to its receptor can be determined using various assays and can be expressed in various quantitative values. Thus, in one embodiment, binding affinity is expressed as a Kd value and reflects intrinsic binding affinity (eg, with minimal affinity effects). In general and preferably, binding affinity is measured in vitro, whether in a cell-free setting or in a cell-associated setting. Any of a number of assays known in the art, including those assays described herein, can be used to obtain binding affinity measurements, including, for example, Biacore, Radioimmunoassay (RIA), and ELISA.

In one aspect, the invention provides an isolated antibody that binds to a ligand binding region of EphB4. In some embodiments, the isolated antibody binds to a polypeptide comprising or consisting of an EphB4 extracellular domain or consisting essentially of an EphB4 extracellular domain.

In one aspect, the invention provides an isolated anti-EphB4 antibody that competes for binding to EphB4 with an EphB4 ligand.

In one aspect, the invention provides an isolated anti-EPhB4 antibody that inhibits, reduces and/or blocks EphB4 activity. In some embodiments, EphB4 autophosphorylation is inhibited, reduced, and/or blocked.

In one aspect, an anti-EphB4 antibody of the invention comprises: (a) at least one, two, three, four or five hypervariable region (HVR) sequences selected from the group consisting of: (i) HVR-L1 comprising the sequence A1-A11, wherein A1-A11 is RASQDVSTAVA (SEQ ID NO: 9), (ii) HVR-L2 comprising the sequence B1-B7, wherein B1-B7 is SASFLYS (SEQ ID NO: 11) (iii) HVR-L3 comprising the sequence C1-C9, wherein C1-C9 is QESTTTPPT (SEQ ID NO: 15), (iv) HVR-H1 comprising the sequence D1-D10, wherein D1-D10 is GFSISNYYLH (SEQ ID NO: 2), (v) HVR-H2 comprising the sequence E1-E18, wherein E1-E18 is GGIYLYGSSSEYADSVKG (SEQ ID NO: 4) and (vi) HVR-H3 comprising the sequence F1-F17, wherein F1-F17 is ARGSGLRLGGLDYAMDY (SEQ ID NO: 7); and (b) at least one variant HVR, wherein the variant HVR sequence comprises a change in at least one residue of the sequence set forth in SEQ ID NOs: 1-17.

In one aspect, the invention provides an antibody comprising one, two, three, four, five or six HVRs, wherein each HVR comprises or consists of a sequence consisting of SEQ ID NOs: 1-17 The sequence consists of or consists essentially of the sequence, and wherein SEQ ID NO: 9 or 10 corresponds to HVR-L1, SEQ ID NO: 11 or 12 corresponds to HVR-L2, SEQ ID NO: 13, 14, 15, 16 or 17 corresponds to HVR-L3, SEQ ID NO: 1 or 2 corresponds to HVR-H1, SEQ ID NO: 3, 4, 5 or 6 corresponds to HVR-H2 and SEQ ID NO: 7 or 8 corresponds to HVR-H3.

In one embodiment, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each of which in turn comprises SEQ ID NO: 9, 11, 13, 1 3, 7.

In one embodiment, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each of them comprises SEQ ID NO: 10, 12, 14, 1 in sequence. 3, 8.

In one embodiment, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each of which in turn comprises SEQ ID NO: 9, 11, 15, 2 4, 7.

In one embodiment, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each of which in turn comprises SEQ ID NO: 9, 11, 16, 1 , 5, 7.

In one embodiment, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each of which in turn comprises SEQ ID NOs: 9, 11, 17, 1 6,6.

A variant HVR in an antibody of the invention may have one or more (eg, two, three, four, five or more) residues within the HVR.

In one embodiment, the HVR-L1 variant comprises 1-5 (1, 2, 3, 4 or 5) substitutions at any combination of the following positions: A6 (V or S), A7 (S or E) , A8 (T or I), A9 (A or F) and A10 (V or L).

In one embodiment, the HVR-L2 variant comprises 1-2 (1 or 2) substitutions at any combination of: B4 (F or N) and B6 (Y or E).

In one embodiment, the HVR-L3 variant comprises 1-7 (1, 2, 3, 4, 5, 6 or 7) substitutions at any combination of: C2 (Q, E or K), C3 (S or T), C4 (Y, N, T, E or A), C5 (T, A or Q), C6 (T, V or I), C8 (P, L or E) and C9 (T) Or S).

In one embodiment, the HVR-H1 variant comprises 1-3 (1, 2 or 3) substitutions at any combination of: D3 (T or S), D6 (G or N), and D9 (I) Or L).

In one embodiment, the HVR-H2 variant comprises 1-5 (1, 2, 3, 4 or 5) substitutions at any combination of the following positions: E5 (P or L), E7 (S or G) , E8 (G or S), E10 (T, S or R) and E11 (D, E or G).

In one embodiment, the HVR-H3 variant comprises one substitution at the following position: F3 (G or S). The letters in parentheses after each position indicate an illustrative substitution (i.e., replacement) of the amino acid; it will be apparent to those skilled in the art that conventional amines can be routinely analyzed using techniques known in the art and/or described herein. The base acid serves as an adaptation of the substituted amino acid in the context described herein.

In one aspect, the invention provides an antibody comprising an HVR-H1 region comprising the sequence of SEQ ID NO: 1 or 2. In one aspect, the invention provides an antibody comprising an HVR-H2 region comprising the sequence of SEQ ID NO: 3, 4, 5 or 6. In one aspect, the invention provides an antibody comprising an HVR-H3 region comprising the sequence of SEQ ID NO: 7 or 8. In one embodiment, the invention provides an antibody comprising an HVR-L1 region comprising the sequence of SEQ ID NO: 9 or 10. In one embodiment, the invention provides an antibody comprising an HVR-L2 region comprising the sequence of SEQ ID NO: 11 or 12. In one embodiment, the invention provides an antibody comprising an HVR-L3 region comprising the sequence of SEQ ID NO: 13, 14, 15, 16 or 17.

In one aspect, the invention provides an antibody comprising at least one, at least two or all three of the following sequences: (i) an HVR-H1 sequence comprising the sequence of SEQ ID NO: 2; (ii) comprising SEQ ID NO: The HVR-H2 sequence of the sequence of 4; (iii) the HVR-H3 sequence comprising the sequence of SEQ ID NO: 7.

In one aspect, the invention provides an antibody comprising at least one, at least two or all three of the following sequences: (i) an HVR-L1 sequence comprising the sequence of SEQ ID NO: 9; (ii) comprising SEQ ID NO: The HVR-L2 sequence of the sequence of 11; (iii) the HVR-L3 sequence comprising the sequence of SEQ ID NO: 15.

The amino acid sequences of SEQ ID NOS: 1-17 are numbered with respect to individual HVRs (i.e., H1, H2 or H3) as indicated in Figure 1, which is consistent with the Kabat numbering system as described below.

In one aspect, the invention provides an antibody comprising a heavy chain HVR sequence as described in Figure 1.

In one aspect, the invention provides an antibody comprising a light chain HVR sequence as described in Figure 1.

Some examples of antibodies of the invention comprise a humanized 4D5 antibody (huMAb4D5-8) as described in SEQ ID NO: 18 below (HERCEPTIN , Genentech, Inc., South San Francisco, CA, USA) (also referred to in US Patent No. 6,407,213 and Lee et al., J. Mol. Biol. (2004), 340(5): 1073-93) Light chain variable domain.

In one embodiment, the huMAb4D5-8 light chain variable domain sequence is at one or more positions at positions 30, 66, and 91 (Asn, Arg, and His, respectively, as indicated by the bold/italic, above) Modification. In one embodiment, the modified huMAb4D5-8 sequence comprises Ser at position 30, Gly at position 66 and/or Ser at position 91. Thus, in one embodiment, an antibody of the invention comprises a light chain variable domain comprising the sequence set forth in SEQ ID NO: 52:

The substituted residues for huMAb4D5-8 are indicated by the above bold/italic.

An antibody of the invention may comprise any suitable framework variable domain sequence, with the proviso that it substantially retains binding activity to EphB4. For example, in some embodiments, an antibody of the invention comprises a human subgroup III heavy chain framework consensus sequence. In one embodiment of such antibodies, the framework consensus sequence comprises substitutions at positions 71, 73 and/or 78. In some embodiments of such antibodies, position 71 is A, 73 is T and/or 78 is A. In one embodiment, the antibodies comprise huMAb4D5-8 (HERCEPTIN) , Genentech, Inc., South San Francisco, CA, USA) (also in U.S. Patent Nos. 6,407,213 and 5,821,337 and Lee et al, J. Mol. Biol. (2004), 340(5):1073-93 Reference to the heavy chain variable domain framework sequence. In one embodiment, the antibodies additionally comprise a human I light chain framework consistent sequence. In one embodiment, such antibodies comprise a light chain HVR sequence of huMAb4D5-8 as described in U.S. Patent Nos. 6,407,213 and 5,821,337. In one embodiment, the antibodies comprise huMAb4D5-8 (HERCEPTIN) , Genentech, Inc., South San Francisco, CA, USA) (also in U.S. Patent Nos. 6,407,213 and 5,821,337 and Lee et al, J. Mol. Biol. (2004), 340(5):1073-93 Reference is made to the light chain variable domain sequence.

In one embodiment, an antibody of the invention comprises a heavy chain variable domain, wherein the framework sequence comprises SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 The sequences of 32, 33, 34, 35, 36 and/or 37, and the HVR H1, H2 and H3 sequences are SEQ ID NOs: 9, 11 and/or 15, respectively. In one embodiment, an antibody of the invention comprises a light chain variable domain, wherein the framework sequence comprises the sequence of SEQ ID NO: 38, 39, 40 and/or 41, and the HVR L1, L2 and L3 sequences are respectively SEQ ID NO : 2, 4 and / or 7.

In one embodiment, the antibody of the invention comprises a heavy chain variable domain, wherein the framework sequence comprises the sequence of SEQ ID NO: 46, 47, 48 and/or 49, and the HVR H1, H2 and H3 sequences are respectively SEQ ID NO : 2, 4 and / or 7. In one embodiment, the antibody of the invention comprises a light chain variable domain, wherein the framework sequence comprises the sequence of SEQ ID NO: 42, 43, 44 and/or 45, and the HVR L1, L2 and L3 sequences are respectively SEQ ID NO : 9, 11 and / or 15.

In one embodiment, the antibody of the invention comprises a heavy chain variable domain, wherein the framework sequence comprises the sequences of SEQ ID NOs: 46, 47, 51 and 49, and the HVR H1, H2 and H3 sequences are SEQ ID NO: 2, respectively. , 4 and / or 7. In one embodiment, an antibody of the invention comprises a light chain variable domain, wherein the framework sequence comprises the sequences of SEQ ID NOs: 42, 43, 50, and 45, and the HVR L1, L2, and L3 sequences are SEQ ID NO: 9, respectively. , 11 and / or 15.

In one embodiment, the antibodies of the invention are affinity matured to achieve the desired targeted binding affinity. In one example, the affinity matured antibodies of the invention are contained in amino acid positions H28, H31, H34, H52a, H54, H55, H57, H58, H95, L29, L30, L31, L32, L33, L53, L55, L90 Substitution at one or more positions of L91, L92, L93, L94, L56 or L97. In one embodiment, the affinity matured antibody of the invention comprises one or more of the following substitutions: (a) in the heavy chain, T28S, G31N, I34L, P52aL, S54G, G55S, T57S or R, D58E or G, G95S; (b) In the light chain, V29S, S30E, T31I, A32F, V33L, F53N, Y55E, Q90E or K, S91T, Y92N TE or A, T93V or I, T94V or I, P96L or E or T97S.

In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:53. In one embodiment, an antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO:54. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising a sequence of SEQ ID NO: 53 and a light chain variable domain comprising SEQ ID NO: 54 sequence.

In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:55. In one embodiment, an antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO:56. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising a sequence of SEQ ID NO: 55 and a light chain variable domain comprising SEQ ID NO: 56 sequence.

In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:57. In one embodiment, an antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO:58. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising a sequence of SEQ ID NO: 57 and a light chain variable domain comprising SEQ ID NO: 58 sequence.

In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:59. In one embodiment, an antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO:60. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising a sequence of SEQ ID NO: 59 and a light chain variable domain comprising SEQ ID NO: 60 sequence.

In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:61. In one embodiment, an antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO:62. In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising a sequence of SEQ ID NO: 61 and a light chain variable domain comprising SEQ ID NO: 62 sequence.

In one aspect, the invention provides an antibody that competes with any of the above antibodies for binding to EphB4. In one aspect, the invention provides an antibody that binds to the same epitope on EphB4 as any of the above antibodies.

As is known in the art and as described in more detail below, the amino acid position/boundary depicting the hypervariable region of the antibody can vary depending on the context and various definitions known in the art (as described below). Some locations within the variable domain can be considered as heterozygous hypervariable locations because they can be considered to be in the hypervariable region under one set of criteria and outside the hypervariable region under another set of criteria. One or more of these locations may also be found in an extended hypervariable region (as further defined below).

In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the anti-system is selected from the group consisting of chimeric antibodies, affinity matured antibodies, humanized antibodies, and human antibodies. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody is Fab, Fab', Fab'-SH, F(ab') ₂ or scFv.

In one embodiment, the antibody is a chimeric antibody, eg, an antibody comprising an antigen-binding sequence from a non-human donor grafted to a heterologous non-human, human or humanized sequence (eg, a framework and/or a constant domain sequence). In one embodiment, the non-human donor is a mouse. In one embodiment, the antigen binding sequence is synthetic, such as by mutation induction (eg, phage display screening, etc.). In one embodiment, a 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 human Light chain. In one embodiment, the murine heavy chain V region is fused to the human IgGl C region.

Humanized antibodies of the invention include those having an amino acid substitution in the FR and affinity matured variants having altered in the grafted CDRs. Substituted amino acids in the CDR or FR are not limited to the amino acids present in the donor or acceptor antibody. In other embodiments, the antibodies of the invention additionally comprise alterations in amino acid residues in the Fc region that result in improved effector functions, including enhanced CDC and/or ADCC function and B-cell killing. Other antibodies of the invention include such antibodies having specific changes that improve stability. In other embodiments, an antibody of the invention comprises an alteration of an amino acid residue in the Fc region that results in reduced effector functions, such as decreased CDC and/or ADCC function and/or reduced B-cell killing. In some embodiments, an antibody of the invention is characterized by reduced binding (eg, deletion binding) to human complement factor C1q and/or human Fc receptors on natural killer (NK) cells. In some embodiments, an antibody of the invention is characterized by reduced binding (eg, deletion binding) to human FcγRI, FcγRIIA, and/or FcγRIIIA. In some embodiments, an antibody of the invention is an IgG class (eg, IgG1 or IgG4) and is in E233, L234, G236, D265, D270, N297, E318, K320, K322, A327, A330, P331, and/or P329 (according to EU The index number) contains at least one mutation. In some embodiments, the antibody comprises the mutation L234A/L235A or D265A/N297A.

In one aspect, the invention provides an anti-EphB4 polypeptide comprising any of the antigen binding sequences provided herein, wherein the anti-EphB4 polypeptide specifically binds to EphB4.

The antibodies of the invention bind (e.g., specifically bind) to EphB4, and in some embodiments, can modulate one or more aspects of EphB4-related effects, including but not limited to, EphB4 activation, EphB4 downstream molecular signaling, EphB4 coordination Activation, downstream molecular signaling of EphB4 ligands, ligands (eg, ephrin-B1, ephrin-B2, and/or ephrin-B3) bind to disruption of EphB4, EphB4 phosphorylation, and/or EphB4 multimerization and/or EphB4 ligand phosphorylation and/or disruption of any biologically relevant EphB4 and/or EphB4 ligand biological pathway, inhibition of angiogenesis and/or treatment and/or prevention of tumors, cell proliferative disorders or cancer; / or treatment or prevention of a condition associated with EphB4 performance and/or activity (eg, increased EphB4 performance and/or activity). In some embodiments, an antibody of the invention specifically binds to EphB4. In some embodiments, the antibody specifically binds to the EphB4 extracellular domain (ECD). In some embodiments, the antibody specifically binds to a polypeptide consisting of or consisting essentially of an EphB4 extracellular domain. In some embodiments, the antibody specifically binds to EphB4, wherein KD is about 50 pM or greater. In some embodiments, an antibody of the invention reduces, inhibits, and/or blocks EphB4 activity in vivo and/or in vitro. In some embodiments, the antibody reduces, inhibits, and/or blocks EphB4 autophosphorylation. In some embodiments, the antibody competes for binding to an EphB4 ligand (reducing and/or blocking EphB4 ligand binding to EphB4).

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

In one aspect, the invention provides the use of an antibody of the invention for the preparation of a medicament for inhibiting angiogenesis.

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 a nucleic acid encoding an anti-EphB4 antibody 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 carrier. 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, for example, a recombinant vector such as an expression vector. Any of a variety of host cells can 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, such as a mammalian cell (eg, a Chinese hamster ovary (CHO) cell).

In one aspect, the invention provides a method of making an antibody of the invention. For example, the invention provides a method of making an anti-EphB4 antibody (as defined herein, including full length and fragments thereof) or an immunoconjugate comprising expressing the antibody (or a fragment thereof) in a suitable host cell The recombinant vector is invented, and the antibody is recovered.

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-EphB4 antibodies of the invention. In one embodiment, the composition comprises a nucleic acid of the invention. In one embodiment, the composition comprising the antibody additionally comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment, the article of manufacture of the present invention additionally comprises instructions for administering the composition (eg, an antibody) to a patient (eg, instructions for any of the above methods).

In one aspect, the invention provides a kit comprising a first container and a second container; the first container comprising a composition comprising one or more anti-EphB4 antibodies of the invention; the second container comprising Buffer. In one embodiment, the buffer is pharmaceutically acceptable. In one embodiment, the composition comprising the antibody additionally comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment, the kit further includes instructions for administering the composition (eg, the antibody) to a patient.

In one aspect, the invention provides the use of an anti-EphB4 antibody of the invention for the preparation of a medicament for the therapeutic and/or prophylactic treatment of a condition such as a cancer, a tumor and/or a cell proliferative disorder. In some embodiments, the condition is a neuropathy or a neurodegenerative disease. In some embodiments, the condition is a pathological condition associated with angiogenesis.

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

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

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

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

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

The present invention provides methods and compositions useful for modulating disease states associated with EphB4 expression and/or activity (e.g., increased or decreased performance and/or activity or poor performance 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 performance and/or activity of EphB4, the methods comprising: administering to a patient in need of such treatment An effective amount of an anti-EphB4 antibody.

In one aspect, the invention provides methods for killing cells, such as cancer or tumor cells, comprising administering an effective amount of an anti-EphB4 antibody to a patient in need of such treatment.

In one aspect, the invention provides methods for reducing, inhibiting, blocking or preventing tumor or cancer growth, the methods comprising administering to a patient in need of such treatment an effective amount of an anti-EphB4 antibody.

In one aspect, the invention provides methods for treating or preventing a neuropathic or neurodegenerative disease or repairing damaged nerve cells, the methods comprising administering to a patient in need of such treatment an effective amount of an anti-EphB4 antibody.

In one aspect, the invention provides methods for promoting neuronal development, proliferation, maintenance, or regeneration, the methods comprising administering to a patient in need of such treatment an effective amount of an anti-EphB4 antibody.

In one aspect, the invention provides a method for inhibiting angiogenesis comprising administering to a patient in need of such treatment an effective amount of an anti-EphB4 antibody.

In one aspect, the invention provides a method for treating a pathological condition associated with angiogenesis comprising administering to a patient in need of such treatment an effective amount of an anti-EphB4 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 disorder.

The methods of the invention can be used to affect any suitable pathological condition. Exemplary disorders are described herein and include cancers selected from the group consisting of small cell lung cancer, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma.

In one embodiment, the cells targeted in the methods of the invention are cancer cells. For example, the cancer cells may be cancer cells selected from the group consisting of breast cancer cells, colorectal cancer cells, lung cancer cells, papillary cancer cells, colon cancer cells, pancreatic cancer cells, ovarian cancer cells, and cervical cancer. Cancer cells, central nervous system cancer cells, osteosarcoma cells, renal cancer cells, hepatocellular carcinoma cells, bladder cancer cells, gastric cancer cells, head and neck squamous cancer cells, melanoma cells, leukemia cells, and colon adenoma cells. In one embodiment, the cells targeted in the methods of the invention are hyperproliferative and/or proliferating cells. In one embodiment, the cells targeted in the methods of the invention are dysplastic cells. In another embodiment, the cells targeted in the methods of the invention are metastatic cells.

The methods of the invention may additionally comprise additional treatment steps. For example, in one embodiment, a method further comprises the step of exposing the targeted cells and/or tissues (eg, cancer cells) to a therapeutic or chemotherapeutic agent.

In one aspect, the invention provides a method comprising administering an effective amount of an anti-EphB4 antibody and an effective amount of another therapeutic agent (eg, an anti-angiogenic agent). For example, an anti-EphB4 antibody is used in combination with an anti-cancer agent or an anti-angiogenic agent to treat various neoplastic or non-neoplastic conditions. In one embodiment, the neoplastic or non-neoplastic condition is a pathological condition associated with angiogenesis. In some embodiments, the additional therapeutic agent is an anti-angiogenic agent, an anti-biochemical agent, and/or a chemotherapeutic agent.

The anti-EphB4 antibody can be administered in the same composition or as a separate composition, either continuously or in combination with another therapeutic agent effective to achieve the same. The anti-EphB4 antibody and another therapeutic agent (eg, an anticancer agent, an anti-angiogenic agent) can be simultaneously completed (for example) as a single composition or as two or more distinct compositions using the same or different administration routes. Dosing. Alternatively or additionally, the administration may be completed in succession in any order. Additionally or alternatively, the steps can be performed as a combination of sequential or simultaneous administration in any order. In certain embodiments, there may be a time interval ranging from a few minutes to a few days to weeks to months between administration of two or more compositions. For example, an anticancer agent can be administered first, followed by administration of an anti-EphB4 antibody. However, it is also contemplated to administer the drug simultaneously or first to the anti-EphB4 antibody. Thus, in one aspect, the invention provides a method comprising administering an anti-EphB4 antibody followed by administration of an anti-angiogenic agent (eg, an anti-VEGF antibody, such as bevacizumab). In certain embodiments, there may be a time interval ranging from a few minutes to a few days to weeks to months between administration of two or more compositions.

In certain aspects, the invention provides a method of treating a disorder (eg, a tumor, a cancer, and/or a cell proliferative disorder) by administering an effective amount of an anti-EphB4 antibody and/or an angiogenesis inhibitor and one or more chemotherapeutic agents. The method. A variety of chemotherapeutic agents can be used in the combination therapies of the invention. An illustrative and non-limiting list of chemotherapeutic agents covered is provided herein under "Definition." The administration of the anti-EphB4 antibody and the chemotherapeutic agent (for example) as a single composition or as two or more distinct compositions can be accomplished simultaneously using the same or different routes of administration. Alternatively or additionally, the administration may be completed in succession in any order. Additionally or alternatively, the steps can be performed as a combination of sequential or simultaneous administration in any order. In certain embodiments, there may be a time interval ranging from a few minutes to a few days to weeks to months between administration of two or more compositions. For example, a chemotherapeutic agent can be administered first, followed by administration of an anti-EphB4 antibody. However, it is also contemplated to administer the drug simultaneously or first to the anti-EphB4 antibody. Thus, in one aspect, the invention provides a method comprising administering an anti-EphB4 antibody followed by administration of a chemotherapeutic agent. In certain embodiments, there may be a time interval ranging from a few minutes to a few days to weeks to months between administration of two or more compositions.

In one aspect, the invention provides a method of potentiating the efficacy of an anti-angiogenic agent in a patient having a pathological condition associated with angiogenesis comprising administering to the patient an effective amount of an anti-EphB4 antibody and an anti-angiogenic agent, Thereby, the inhibitory activity of the anti-angiogenic agent is enhanced.

In another aspect, the invention provides methods for detecting EphB4, the methods comprising detecting an EphB4-anti-EphB4 antibody complex in a sample. The term "detecting" as used herein includes qualitative and/or quantitative detection (measurement content) with or without reference to a control.

In another aspect, the invention provides methods for diagnosing a disorder associated with EphB4 expression and/or activity, the methods comprising detecting EphB4- in a biological sample from a patient having or suspected of having the disorder Anti-EphB4 antibody complex. In some embodiments, EphB4 exhibits increased performance or abnormal performance. In some embodiments, the condition is a tumor, cancer, and/or a cell proliferative disorder.

In another aspect, the invention provides any of the anti-EphB4 antibodies described herein, wherein the anti-EphB4 antibody comprises a detectable label.

In another aspect, the invention provides any of the anti-EphB4 antibodies and EphB4 complexes described herein. In some embodiments, the complex is in vivo or ex vivo. In some embodiments, the complex comprises cancer cells. In some embodiments, the anti-EphB4 anti-system is detectably labeled.

In the present context, the invention provides anti-EphB4 antibodies useful for, for example, treating or preventing a disease state associated with EphB4 expression and/or activity (eg, increased performance and/or activity or poor performance and/or activity). In some embodiments, an antibody of the invention is used to treat a tumor, cancer, and/or cell proliferative disorder.

In another aspect, the anti-EphB4 antibodies of the invention are useful as agents for detecting and/or isolating EphB4, such as EphB4 in various tissues and cell types.

The invention further provides methods for making anti-EphB4 antibodies and polynucleotides encoding anti-EphB4 antibodies.

General technology

The techniques and procedures described or referenced herein are generally well known and commonly used by those skilled in the art using conventional methods, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory. Press, Cold Spring Harbor, NYCURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FMAusubel et al., (2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJMacPherson, BD Hames and Edited by GRTaylor (1995), Harlow and Lane (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

definition

An "isolated" antibody is one that has been identified and is isolated from components of the natural environment and/or recovered from components of the natural environment. The contaminating component of its natural environment is a material that can interfere with the diagnostic or therapeutic use of the antibody, and can include enzymes, hormones, and other protein or non-protein solutes. In a preferred embodiment, the antibody will be purified (1) to greater than 95% by weight of the antibody as determined by the Lowry method, and optimally greater than 99%; (2) sufficient to use a rotating cup sequencer Obtaining at least 15 residues of the N-terminal or internal amino acid sequence; or (3) by SDS- by Coomassie blue or preferably silver staining under reducing or non-reducing conditions The homogeneity shown by PAGE. An isolated antibody includes an antibody in situ within a recombinant cell, as at least one component of the antibody's natural environment will not be present. However, in general, the isolated antibody will be prepared by at least one purification step.

An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule, which is typically associated with a contaminating nucleic acid molecule in the natural source of the antibody nucleic acid. The isolated nucleic acid molecule differs from its found form or setting in nature. Thus, an isolated nucleic acid molecule is distinguished from a nucleic acid molecule present in a natural cell. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that typically expresses an antibody, wherein, for example, the nucleic acid molecule is at a chromosomal location different from the chromosomal location of the native cell.

The term "such as the variable domain residue number in Kabat" or "such as the amino acid position number in Kabat" and variations thereof refer to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National The numbering system used by the Institutes of Health, Bethesda, MD. (1991) to compile heavy chain variable domains or light chain variable domains of antibodies. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or CDR of the variable domain. For example, the heavy chain variable domain can include a single amino acid insertion after residue 52 of H2 (according to residue 52a of Kabat) and an inserted residue after heavy chain FR residue 82 (eg, according to Kabat Residues 82a, 82b, 82c, etc.). The Kabat numbering of a residue for a given antibody can be determined by aligning the homologous region of the antibody sequence to a "standard" Kabat numbering sequence.

The phrase "substantially similar" or "substantially identical" as used herein denotes a sufficiently high degree of similarity between two values (typically, one value is associated with an antibody of the invention and another value is associated with a reference/comparative antibody). Degree, so that those skilled in the art believe that the difference between the two values in the biological characteristic content measured by the equivalent value (for example, Kd value) will have little or no biology and / Or statistically significant. As a function of the reference/comparison antibody value, 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%, more Good land is less than about 10%.

"Binding affinity" generally refers to the total intensity of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). "Binding affinity" as used herein, unless otherwise indicated, refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, an antibody and an antigen). The affinity of the molecule X for its conjugate Y is usually represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies typically bind slowly to the antigen and tend to dissociate rapidly, while high affinity antibodies typically bind faster to the antigen and tend to remain bound for long term. Various methods of measuring binding affinity are known in the art, and any of these methods can be used to achieve the objectives of the present invention. Certain illustrative embodiments are described below.

In one embodiment, a "Kd" or "Kd value" according to the present invention is measured by performing a radiolabeled antigen binding assay (RIA) with a Fab version of the antibody of interest, such as the following: As determined by the assay for solution binding affinity, the assay for the affinity of the assay comprises the steps of: equilibrating the Fab with a minimal concentration of the ( ^125I ) labeled antigen in the presence of a continuous titration of the unlabeled antigen, followed by capture and antibiotic resistance. Fab antibody coated plate-bound antigen (Chen et al, (1999) J. Mol Biol 293: 865-881). To establish assay conditions, microtiter plates (Dynex) were coated overnight with 5 μg/ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then at room temperature (approximately 23 ° C) Blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours. In a non-sorbent titration plate (Nunc #269620), mix 100 pM or 26 pM [ ¹²⁵ I] antigen with serially diluted Fab of interest (eg, in accordance with the assessment of anti-VEGF antibody Fab-12, Presta et al. (1997) Cancer Res. 57: 4593-4599). Subsequently, the Fab concerned is cultivated overnight; however, the cultivation lasts for a longer period of time (eg 65 hours) to ensure a balance is achieved. Thereafter, the mixture is transferred to a capture plate at room temperature for incubation (eg, over 1 hour). Subsequently, the solution was removed and the plate was washed 8 times with 0.1% Tween-20 in PBS. When the plates were dry, 150 μl of scintillant (MicroScint-20; Packard) was added to each well, and the plates were counted for 10 minutes on a Topcount gamma counter (Packard). Each Fab providing a concentration less than or equal to 20% of the maximum binding is selected for competitive binding assays. According to another embodiment, by using the BIAcore ^TM -2000 or a ^{BIAcore TM -3000 (BIAcore, Inc.,} Piscataway, NJ) at 25 deg.] C and using a surface plasmon resonance assay (surface plasmon resonance assay) Reaction with about 10 The fixed antigen CM5 wafer of the unit (RU) measures the Kd or Kd value. Briefly, according to the supplier's instructions, N -ethyl- N' -(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N -hydroxysuccinimide ( NHS) Activated carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc.). The antigen was diluted to 5 μg/ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8), followed by injection at a flow rate of 5 μl/min to achieve about 10 reaction units (RU) of coupled protein. After the antigen was injected, 1 M ethanolamine was injected to block the unreacted groups. For kinetic measurements, Fab (0.78 nM to 500 nM) was serially diluted twice in PBS (PBST) with 0.05% Tween 20 at a flow rate of about 25 μl/min at 25 °C. The association rate ( _kon ) and dissociation rate ( _koff ) were calculated by simultaneously fitting the association and dissociation induction spectra using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software, version 3.2). The equilibrium dissociation constant (Kd) was calculated as k _off / k _on the ratio. See, for example, Chen, Y. et al., (1999) J. Mol Biol 293:865-881. The association rate obtained by the above surface plasma resonance test is greater than 10 ⁶ M ^-1 . S ^-1 can be measured in a concentration such as a spectrometer (such as a spectrophotometer equipped with an intermittent fluid (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred colorimetric tube) In the presence of an antigen, the fluorescence quenching technique (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) is measured at 25 ° C by using an increase or decrease in fluorescence intensity. Association rate of 20 nM anti-antigen antibody (Fab format) (pH 7.2) in PBS.

Or may also be used BIAcore ^TM -2000 ^{BIAcore TM -3000 (BIAcore, Inc.,} Piscataway, NJ) at 25 deg.] C similar to the above-described surface plasmon resonance assay with immobilized antigen CM5 wafer 10 about the reaction unit (RU) of technology " The association rate "or" k _on ". Briefly, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxy amber imine (IIC) were used according to the supplier's instructions. NHS) Activated carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc.). The antigen was diluted to 5 μg/ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8), followed by injection at a flow rate of 5 μl/min to achieve about 10 reaction units (RU) of coupled protein. After the antigen was injected, 1 M ethanolamine was injected to block the unreacted groups. For kinetic measurements, Fab (0.78 nM to 500 nM) was serially diluted twice in PBS (PBST) with 0.05% Tween 20 at a flow rate of about 25 μl/min at 25 °C. The association rate ( _kon ) and dissociation rate ( _koff ) were calculated by simultaneously fitting the association and dissociation induction spectra using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) was calculated as k _off / k _on the ratio. See, for example, Chen, Y. et al., (1999) J. Mol Biol 293:865-881. However, if the association rate obtained by the above surface plasma resonance test is greater than 10 ⁶ M ^-1 . S ^-1 can be measured in a concentration such as a spectrometer (such as a spectrophotometer equipped with an intermittent fluid (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred colorimetric tube) Determination of 20 nM in PBS at 25 ° C by fluorescence quenching technique (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) using an increase or decrease in fluorescence intensity Association rate of anti-antigen antibody (Fab form) (pH 7.2).

The term "vector," as used herein, is intended to mean a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. One type of vector is a "plastid", 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 in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in host cells into which such vectors are introduced (e.g., bacterial vectors having bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell after introduction into the host cell, and thereby replicate along with the host genome. In addition, certain vectors are capable of directing the performance of genes for their operability. Such vectors are referred to herein as "recombinant expression vectors" (or in short, "recombinant vectors"). In general, expression vectors for use in recombinant DNA techniques are typically in the form of plastids. In the present specification, "plastid" and "carrier" are used interchangeably because the plastid is the carrier of the most common form.

"Polynucleotide" or "nucleic acid" as used interchangeably herein, refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or analogs thereof, or may be incorporated by polymerization using DNA or RNA polymerase or by synthesis reaction. Any matrix in the material. Polynucleotides can comprise modified nucleotides, such as methylated nucleotides and analogs thereof. If modifications to the nucleotide structure are present, modifications can be made before or after assembly of the polymer. The nucleotide sequence can be interrupted by a non-nucleotide component. The polynucleotide may be further modified after synthesis, such as by binding to a label. Other types of modifications include, for example, "hats"; one or more naturally occurring nucleotides are substituted with analogs; internucleotide modifications, such as those with uncharged bonds (eg, methyl phosphonate, phosphoric acid) Triesters, phospholysines, urethanes, etc.) and those with a charge (such as phosphorothioates, phosphorodithioates, etc.), those with a subsidiary (such as proteins (eg, nucleases, Toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalating agents (eg acridine, psoralen, etc.), those containing chelating agents (eg metals, radioactive metals, boron, oxidizing metals) And the like, those containing an alkylating agent, those having a modified bond (for example, α-rotating isomeric nucleic acid, etc.); and unmodified forms of the polynucleotide. In addition, any of the hydroxyl groups normally present in the sugar may be replaced by, for example, a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages to additional nucleotides, or may be combined with a solid. Or semi-solid support bonding. The 5' and 3' terminal OH groups may be phosphorylated or partially substituted with an amine or an organic capping group having from 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized into standard protecting groups. Polynucleotides may also contain ribose or deoxyribose sugars of a similar form generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro- Or 2'-azido-ribose, carbocyclic sugar analogs, alpha-or isomers, epimers (such as arabinose, xylose or lyxose), piperanose, furan Sugars, heptasaccharides, acyclic analogs, and basic nucleoside analogs (such as methyl ribosides). One or more phosphodiester bonds can be replaced by an alternative linker. Such alternative linkers include, but are not limited to, wherein the phosphate ester is via P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR ₂ ("醯amine compounds "), P (O) R , P (O) oR ', CO or CH ₂ (" Swap embodiment methylal "), wherein R or R' are each independently contain an ether is H or as the case ( -O-) A substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. All of the bonds in the polynucleotide need not be the same. The above description applies to all polynucleotides mentioned herein, including RNA and DNA.

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

The "amino acid sequence identity percent (%)" for a peptide or polypeptide sequence is defined as the alignment of a specific peptide or polypeptide sequence and the introduction interval to achieve a maximum sequence identity percentage, then after these steps The percentage of amino acid residues present in the candidate sequences of the same amino acid residues in the sequences, and no conservative substitutions should be considered as part of sequence identity. Alignment can be achieved in a variety of ways within this technical capability to determine percent amino acid sequence identity, such as publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequence being compared. However, for the purposes of this paper, the amino acid sequence identity % value was generated using the sequence comparison computer program ALIGN-2, where the full source code for the ALIGN-2 program is provided in Table A below. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code shown in Table A below has been filed with user files in U.S. Copyright Office, Washington D.C., 20559, which is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available via Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use with the UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

In the case where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence sequence A of a given amino acid sequence A is given a certain amino acid sequence identity (which may be additionally referred to as a given amino acid). Sequence A has or contains % amino acid sequence identity with a given amino acid sequence B) calculated as follows: Fraction X/Y x 100 where X is in alignment with A and B, aligned by sequence The program ALIGN-2 scores the number of amino acid residues of the same pair, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the % identity of the amino acid sequence of A and B will not be equal to the % identity of the amino acid sequence of B and A.

Unless otherwise indicated, all amino acid sequence identity % values used herein were obtained using the ALIGN-2 computer program as described in the previous paragraph.

The term "EphB4" (alternatively referred to as "EphB4R") as used herein, unless otherwise specified or indicated in the context, refers to any natural or variant (natural or synthetic) EphB4 polypeptide. The term "native sequence" specifically encompasses naturally occurring truncated or secreted forms (eg, extracellular domain sequences), naturally occurring variant forms (eg, alternatively spliced forms), and naturally occurring allelic variants. The term "wild-type EphB4" generally refers to a polypeptide comprising an amino acid sequence of a naturally occurring EphB4 protein. The term "wild-type EphB4 sequence" generally refers to the amino acid sequence found in naturally occurring EphB4.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (eg, full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent, multivalent antibodies, multispecific An antibody (eg, a bispecific antibody, as long as it exhibits the desired biological activity), and may also include certain antibody fragments (as described in more detail herein). The antibody can be a human antibody, a humanized antibody, and/or an affinity matured antibody.

The term "variable" means that the sequences of certain portions of the variable domains between antibodies are generally different and that such portions 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 domain of the antibody. It is concentrated in three segments of the light chain and heavy chain variable domains called complementarity-determining regions (CDRs) or hypervariable regions. The higher degree of conservation in the variable domain is called the framework (FR). The variable domains of the native heavy and light chains each comprise four FR regions joined by three CDRs that are largely configured in a beta sheet, which form a loop junction and, in some cases, form a partial beta sheet structure. . The CDRs in each chain are held together in close proximity by the FR regions, and CDRs from other chains contribute to the formation of antigen binding sites for antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition). , National Institute of Health, Bethesda, MD (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as antibody involvement in antibody-dependent cellular toxicity.

Digestion of antibodies by papain produces two identical antigen-binding fragments each having a single antigen-binding site, termed a "Fab"fragment; and a residual "Fc" fragment whose name reflects its ability to crystallize readily. Pepsin treatment produces a F(ab') ₂ fragment that has two antigen combining sites and is still capable of cross-linking antigen.

"Fv" is the smallest antibody fragment containing the entire antigen recognition site and antigen binding site. In a two-chain Fv species, this region consists of a dimer of one of the heavy chain variable domains and one of the light chain variable domains that are tightly, non-covalently associated. In the single-chain Fv species, a heavy chain variable domain and a light chain variable domain can be covalently linked by an elastin linker such that the light and heavy chains can be similar to the structure of the two-chain Fv species. Poly" structural association. The three CDRs of each variable domain interact in this configuration to define the antigen binding site on the surface of the VH-VL dimer. In summary, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind antigen, although its binding affinity to the antigen is lower than the affinity of the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues at the carboxy terminus comprising the heavy chain CH1 domain from one or more of the cysteine regions of the antibody. Fab'-SH is a Fab' designation herein in which the constant domain cysteine residue contains a free thiol group. The F(ab') ₂ antibody fragment was originally produced as a Fab' fragment pair with hinged cysteine in between. Other chemical couplings of antibody fragments are also known.

The "light chain" of antibodies (immunoglobulins) from any vertebrate species can be assigned to two completely distinct types based on the amino acid sequence of the constant domain (referred to as And one of λ).

The immunoglobulins can be assigned to different species depending on the amino acid sequence of the heavy chain constant domain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of different types of immunoglobulins are well known.

An "antibody fragment" comprises only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all of the functions normally associated with the portion when present in the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments; bifunctional antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment comprises the antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment (eg, 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, eg, FcRn binding, antibody half-life regulation, ADCC function, and Complement binding. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to the intact antibody. For example, such antibody fragments can comprise an antigen binding arm linked to an Fc sequence that confers in vivo stability to the fragment.

The term "hypervariable region", "HVR" or "HV" as used herein, refers to a region of an antibody variable domain that is hypervariable in sequence and/or forms a structurally defined loop. Typically, the antibody comprises six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Many hypervariable region depictions are used and covered herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest , Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia pointed out the location of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used in Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable region is based on an analysis of available composite crystalline structures. The residues from each of the hypervariable regions are as follows.

The hypervariable region may comprise the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL; 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. Variable domain residues are numbered according to Kabat et al. above for each of these definitions.

"Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

A "humanized" form of a non-human (eg, murine) antibody is a chimeric antibody containing minimal sequence derived from a non-human immunoglobulin. Humanized antibodies are typically human immunoglobulins (receptor antibodies) in which residues from the hypervariable region of the receptor are subjected to residues from the hypervariable region of a non-human species (donor antibody) having the desired specificity, affinity and ability. Replacement, such non-human species such as mice, rats, rabbits or non-human primates. In some cases, the framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will comprise substantially all sequences of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to their sequences of non-human immunoglobulins, and all or substantially All FRs are their sequences of human immunoglobulin sequences. The humanized antibody will also, depending on the situation, comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For more details, see Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr.Op.Struct.Biol. 2:593-596 (1992). See also the following review articles and in the references cited: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol 1 : 105-115 (1998); Harris, Biochem.Soc.Transactions 23:. 1035-1038 (1995) Hurle and Gross, Curr. Op . Biotech . 5: 428-433 (1994).

A "chimeric" antibody (immunoglobulin) has a portion of a heavy chain and/or a light chain that is identical or homologous to a corresponding sequence derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the strand is derived from The corresponding sequence of another species or another antibody class or subclass and fragments of such antibodies is identical or homologous as long as it exhibits the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad). .Sci. USA 81:6851-6855 (1984)). Humanized antibodies as used herein are a subset of chimeric antibodies.

A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein such domains are present in a single polypeptide chain. In general, the scFv polypeptide additionally comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies , Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

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

The term "bifunctional antibody" refers to a small antibody fragment having two antigen binding sites comprising a heavy chain variable domain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL) (VH). By using a linker that is too short to be paired between the two domains on the same strand, the domains are forced to pair with the complementary domains of the other strand and create two antigen-binding sites. Bifunctional antibodies are described in more detail in, for example, EP 404,097, WO 93/11161 and Hollinger et al, Proc. Natl. Acad. Sci. USA , 90 :6444-6448 (1993).

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

An "affinity mature" anti-system has one or more altered antibodies in one or more HVRs that result in improved affinity of the antibody for the antigen compared to parental antibodies that do not have such changes. Preferred affinity matured antibodies should have a sensitivity of several nanomolar or even picomolar for the targeted antigen. Affinity mature anti-systems are produced by procedures known in the art. Affinity maturation caused by VH and VL domain shuffling is described by Marks et al, Bio/Technology 10:779-783 (1992). Random mutation induction of CDR and/or framework residues is described in Barbas et al, Proc Nat . Acad. Sci. USA 91: 3809-3813 (1994); Schier et al, Gene 169: 147-155 (1995) Yelton et al. , J. Immunol. 155: 1994-2004 (1995); Jackson et al. , J. Immunol. 154(7): 3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226: 889-896 (1992).

Antibody "effector function" refers to the biological activity attributable to the Fc region of an antibody (the native sequence Fc region or the amino acid sequence variant Fc region) and which varies with antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg, B cell receptors); And B cell activation.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted Ig binds to certain cytotoxic cells (eg, natural killer (NK) cells, neutrophils, and giants). The Fc receptor (FcR) on the cytopathic cells allows these cytotoxic effector cells to specifically bind to the targeted cells carrying the antigen and subsequently kill the targeted cells with cytotoxin. Antibodies "arm" cytotoxic cells, and antibodies are absolutely necessary for such killing effects. Primary cells (NK cells) that mediate ADCC exhibit only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu . Rev. Immunol 9:457-92 (1991). For the analysis of the ADCC activity of the molecule of interest, an in vitro ADCC assay such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, or the disclosure of U.S. Patent No. 6,737,056. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Additionally or alternatively, the ADCC activity of the molecule of interest can be analyzed in vivo in an animal model such as that disclosed in Clynes et al, PNAS (USA) 95:652-656 (1998).

A "human effector cell" is a white blood cell that exhibits one or more FcRs and performs effector functions. Preferably, the cells exhibit at least FcyRIII and perform an 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; PBMCs and NK cells are preferred. Effector cells can be isolated from natural sources (eg, blood).

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferably, the FcR is a native sequence human FcR. Furthermore, preferred FcRs are FcRs that bind to IgG antibodies (gamma receptors) and include receptors for the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of such receptors. Fc[gamma]RII receptors include FcyRIIA ("Activated Receptor") and FcyRIIB ("Inhibition Receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domain. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor-based tyrosine-based inhibitory element (ITIM) in its cytoplasmic domain. (See M.Da Ron , Annu. Rev. Immunol. 15: 203-234 (1997).) FcR is reviewed in Ravetch and Kinet, Annu . Rev. Immunol 9:457-92 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. : 330-41 (1995). The term "FcR" herein encompasses other FcRs, including those FcRs that will be recognized in the future. The term also encompasses 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 regulate the homeostasis of immunoglobulins. WO 00/42072 (Presta) describes antibody variants with improved or reduced binding to FcR. The content of the patent publication is specifically 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 (for example, see Ghetie 1997, Hinton 2004). For example, in vivo binding to human FcRn and high affinity binding of human FcRn can be assayed in transgenic mouse or transfected human cell lines expressing human FcRn or in primates administered with Fc variant polypeptides. The serum half-life of the polypeptide.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of a targeted cell in the presence of complement. Activation of the classical complement pathway is initiated by binding the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To analyze complement activation, a CDC assay can be performed (e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996)).

Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1 and WO 99/51642. The contents of their patent publications are specifically incorporated herein by reference. See also Idusogie et al, J. Immunol. 164: 4178-4184 (2000).

The term "polypeptide comprising an Fc region" refers to a polypeptide comprising an Fc region, such as an antibody or immunoadhesin (see definition below). For example, the C-terminal amide acid of the Fc region (residue 447 according to the EU numbering system) can be removed during purification of the polypeptide or by recombinant engineering of the nucleic acid encoding the polypeptide. Thus, a composition of the invention comprising a polypeptide having an Fc region may comprise a polypeptide having K447, a polypeptide in which K447 has been removed, or a mixture having and without K447 residues.

A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferably, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen.

An "agonist antibody" as used herein is an antibody that mimics at least one functional activity of a polypeptide of interest.

The "receptor human framework" for the purposes herein is a framework comprising amino acid sequences derived from the human immunoglobulin framework or from the VL or VH framework of the human consensus framework. The acceptor human framework "derived from" the human immunoglobulin framework or the human consensus framework may comprise the same amino acid sequence, or may contain an amino acid sequence change already present. If there is an alteration of the existing amino acid, there is preferably no more than 5 and preferably 4 or less or 3 or less of the existing amino acid changes. If an existing amino acid change is present in the VH, preferably the changes are only at three, two or one of positions 71H, 73H and 78H; for example, amino acid residues at their positions It can be 71A, 73T and/or 78A. In one embodiment, the sequence of the VL receptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

"Human consensus framework" refers to the framework of the most frequently occurring amino acid residues in the selected human immunoglobulin VL or VH framework sequences. In general, the human immunoglobulin VL or VH sequence is selected from a subpopulation of variable domain sequences. In general, a subgroup of sequences is a subgroup as described by Kabat et al. In one embodiment, for VL, the subgroup is a subgroup as described by Kabat et al. I. In one embodiment, for VH, the subpopulation is subgroup III as described by Kabat et al.

The "VH subgroup III consensus framework" comprises a consensus sequence obtained from the amino acid sequence of the 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 or all of each 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).

"VL Subgroup I Consistent Framework" contains variable light chains from Kabat et al. A consensus sequence obtained from the amino acid sequence in subgroup I. In one embodiment, the VL subgroup I consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 46)-L1-WYQQKPGKAPKLLIY (SEQ ID NO: 47)- L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 48)-L3-FGQGTKVEIK (SEQ ID NO: 49).

"Disease" or "disease" is any condition that would benefit from treatment with a substance/molecule or method of the invention. The term includes chronic and acute conditions or diseases, including those pathological conditions that tend to cause the mammal to have the condition in question. Non-limiting examples of conditions to be treated herein include malignant and benign tumors, carcinoma, blastoma, and sarcoma.

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

As used herein, "tumor" refers to all malignant or benign neoplastic cell growth and proliferation and all precancerous and cancerous cells and tissues. The terms "cancer", "cancer", "cell proliferative disorder", "proliferative disorder" and "tumor" are not excluded from each other when referred to herein.

The terms "cancer" and "cancerous" refer to or describe a mammalian physiological condition 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 carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma , cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, genital cancer, thyroid cancer, liver cancer , stomach cancer, melanoma and various types of head and neck cancer. Deregulation of angiogenesis can result in a variety of conditions that can be treated by the compositions and methods of the invention. These conditions include non-neoplastic and neoplastic conditions. The neoplastic conditions include, but are not limited to, the conditions described above. Non-neoplastic conditions include, but are not limited to, poor or abnormal hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriasis plaques, sarcoma-like sarcoma, atherosclerosis, atherosclerotic plaque, Diabetic and other proliferative retinopathy (including retinopathy of prematurity, post-lens fibrous tissue hyperplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal transplantation, neovascularization, cornea Transplant rejection, retinal/choroidal neovascularization, angina neovascularization (iris redness), ocular neovascular disease), vascular restenosis, arteriovenous malformation (AVM), meningioma, hemangioma, angiofibroma Thyroid hyperplasia (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, pulmonary inflammation, acute lung injury/ARDS, sepsis, primary pulmonary hypertension, malignant lung exudation, cerebral edema (eg Associated with acute stroke/closed head injury/trauma), synovial inflammation, vasospasm in RA, ossifying myositis, hypertrophic bone formation, bone Arthritis (OA), refractory ascites, polycystic ovarian disease, endometriosis, third interval of body fluid disease (pancreatitis, compartment syndrome, burning, bowel disease), uterine fibroids, premature delivery, such as Chronic inflammation of IBD (Crohn's disease and ulcerative colitis), renal allograft rejection, inflammatory bowel disease, renal disease, abnormal or abnormal tissue growth (non-cancer), bloodthirsty joints Hypertrophic scar, hair growth inhibition, Osler-Weber syndrome, purulent granuloma, posterior fibrous tissue hyperplasia, scleroderma, trachoma, vascular adhesion, synovitis, dermatitis, preeclampsia, ascites, pericardium Exudation (eg, exudation associated with pericarditis) and pleural effusion.

"Treatment," as used herein, refers to a clinical intervention that attempts to alter the natural course of the individual or cell being treated, and can be performed during prophylaxis or clinical pathology. The desired therapeutic effect includes preventing the occurrence or recurrence of the disease, alleviating the symptoms, reducing any direct or indirect pathological findings of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis. In some embodiments, an antibody of the invention is used to delay the progression of a disease or condition.

The terms "neurodegenerative disease" and "neurodegenerative disorder" are used in the broadest sense to include all pathological conditions involving neuronal degeneration and/or dysfunction, including but not limited to peripheral neuropathy; motor neuron disorders, For example, amyloidosis lateral sclerosis (ALS, Lou Gehrig's disease), Bell's palsy, and various conditions involving spinal muscular atrophy or paralysis; and other human neurodegenerative diseases, For example, Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's chorea, Down's Syndrome, neurological deafness, and Meniere's disease.

"Peripheral neuropathy" is a neurodegenerative disorder that affects peripheral nerves, most often manifested as one or a combination of motor, sensory, sensorimotor, or autonomic dysfunction. Peripheral neuropathy can, for example, be a genetically acquired disease that can result from a systemic disease or can be induced by a toxic agent such as a neurotoxic agent (eg, an anti-biochemical or industrial or environmental contaminant). "Surnal sensory neuropathy" is characterized by degeneration of peripheral sensory neurons, which may be idiopathic, due to, for example, diabetes (diabetic neuropathy), cytostatic drug therapy in cancer (eg, in, for example, Changchun New Base, cisplatin, methotrexate, 3'-azido-3'-deoxythymidine or taxane (eg paclitaxel [TAXOL] , Bristol-Myers Squibb Oncology, Princeton, NJ] and doxetaxel [TAXOTERE] , Rh Ne-Poulenc Rorer, Antony, France]) chemotherapeutic treatment), alcoholism, acquired immunodeficiency syndrome (AIDS) or genetic predisposition. Genetically acquired peripheral neuropathy includes, for example, Refsum's disease, Krabbe's disease, metachromatic white matter disorder, Fabry's disease, and Dynaecone Dejerine-Sottas syndrome, Abetalipoproteinemia, and Charcot-Marie-Tooth Disease (also known as diaphragmatic atrophy or hereditary motor sensory neuropathy) (HMSN)). Most types of peripheral neuropathy develop slowly over months or years. In clinical practice, these neuropathies are called chronic. Sometimes, peripheral neuropathy develops rapidly over a period of several days and is called acute. Peripheral neuropathy usually affects sensory and motor nerves together to cause mixed sensory and motor neuropathy, but pure sensory and pure motor neuropathy are also known.

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

For the purposes of treatment, "mammal" means any animal classified as a mammal, including humans, farm animals and zoo animals, competitive animals or pets such as dogs, horses, cats, cows, etc. . Preferably, the mammal is a human.

"Effective amount" means the amount of time and time necessary to effectively achieve the desired therapeutic or prophylactic result.

A "therapeutically effective amount" of a substance/molecule, agonist or antagonist of the invention may be capable of eliciting a desired response in an individual, such as the individual's disease state, age, sex and weight, and the substance/molecule, agonist or antagonist. And other factors change. A therapeutically effective amount is also one in which the beneficial effects of the substance/molecule, agonist or antagonist are greater than any toxic or detrimental effects. "Prophylactically effective amount" means the amount of time and time necessary to effectively achieve the desired result of prevention. Since the prophylactic dose is administered before or at the onset of the disease, the prophylactically effective amount is usually (but not necessarily) less than the therapeutically effective amount.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cell destruction. The term is intended to include radioisotopes (eg, radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ^{32 ,} and Lu), chemotherapeutic agents (eg, methotrexate) , adriamicin, vinca alkaloid (vincristine, vinblastine, etoposide), 14-hydroxy daunorubicin (doxorubicin), melphalan, mites Minomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof (eg nuclear decomposing enzymes), antibiotics and toxins (such as bacteria, fungi, plants) Or animal-derived small molecule toxins or enzymatically active toxins, including fragments and/or variants thereof, and various anti-tumor or anti-cancer agents disclosed below. Other cytotoxic agents are described below. Tumor-killing agents cause tumor cell destruction .

A "chemotherapeutic agent" is a chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN Cyclophosphamide; alkyl sulfonate such as busulfan sulfate, improsulfan and piposulfan; aziridine such as benzodopa, kappa Carboquone, meturedopa and uredopa; methyl iminoamine and methylamelamine, including altretamine, tri-ethyl melamine, Tri-ethyl phosphamide, tri-ethyl thiophosphonamide and trimethylolomelamine; acetogenin (especially bullatacin and bullatacinone) ));δ-9-tetrahydrocannabinol (dronabinol, MARINOl) Beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogue topotecan) Analogue topotecan)(HYCAMTIN ), CPT-11 (irinotecan, CAMPTOSAR) ), acetyl camptothecin, scopolectin and 9-aminocamptothecin; bryostatin; clallystatin; CC-1065 (including its adoline ( Adozelesin), carzelesin and bizelesin synthetic analogue; podophyllotoxin; podophyllinic acid; teniposide; nymphal Cyclopeptide (cryptophycin) (specifically, Candida cyclic peptide 1 and Nostoccal cyclic peptide 8); dolastatin; doocarmycin (including synthetic analogues, KW-2189 and CB1-TM1) ); eleutherobin; pancastatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil, naphthyl mustard Chlornaphazine), cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, Novembichin, phenesterine, Prednimustine, trofosfamide, uracil mustard; nitrosurea, such as carmustine, chlorozotocin, florol Fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (eg, calicheamicin) ), especially calicheamicin γ1l and calicheamicin ΩI1) (for example, see Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including Dini Dynemicin A; esperamicin; and neocarzinostatin chromophore and related chromophore bismuth chromophores, alkenomycin (aclacinomysin), actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin (carabicin) ), carminomycin, carzinophilin, chromomycinis, actinomycetes Dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-glycinic acid, doxorubicin 14-hydroxydaunorubicin [ADRIAMYCIN (doxorubicin)] (including morpholinyl-14-hydroxydaunorubicin, cyanomorpholinyl-14-hydroxydaunorubicin, 2-pyrrolineyl-14-hydroxydaunorubicin and 14-hydroxydeoxygenation Deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (mitomycin) Such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, sputum Puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex ), ketostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denoptinin, Methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-巯嘌呤Thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-thiazolidine, carmofur, arsenic Cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calulsterone, propionic acid Dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenalin, such as aminoglutethimide, mitotane, Trilostane; folic acid replenisher, such as (frolinic acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid Acid); 5-ilynuracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; Damine (demecolcine); mantle (diaziqu One); elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; Lentinan); lonidainine; maytansinoid, such as maytansine and ansamitocin; mitoguazone; mitoxantrone Mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; -ethyl hydrazine; procarbazine; PSK Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid Triaziquone; 2,2',2"-trichlorotriethylamine; trichothecene (especially T-2 toxin, (verracurin A), laccase A) Roryin A) and (anguidine); urethan; vindesine (ELDISINE) FILDESIN ); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; guacittosine ; arabinoside ("Ara-C");thiotepa; taxoids, such as TAXOL Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANETM Cremophor-free, paclitaxel albumin engineered nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Illinois) and TAXOTERE Doxetaxel (Rh ne-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR) 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinorelbine (NAVELBINE) ); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN) ); oxaliplatin; leucovovin; vinorelbine (NAVELBINE) ;;novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA) a pharmaceutically acceptable salt, acid or derivative of any of the above; and a combination of two or more of the foregoing, such as CHOP (i.e., cyclophosphamide, 14-hydroxy daunorubicin (doxorubicin) ), abbreviations for the combination therapy of vincristine and prednisolone, and FOLFOX (abbreviated as a treatment with a new combination of ELOXATINTM and 5-FU and Rockow).

This definition also includes anti-hormonal agents for modulating, reducing, blocking or inhibiting the action of hormones that promote cancer growth and which are typically in the form of systemic or whole body treatments. It can be the hormone itself. Examples include anti-estrogen and selective estrogen receptor modulators (SERMs) including, for example, tamoxifen (including NOLVADEX) Tamoxifen), EVISTA Raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON Toremifene; antiprogestin; estrogen receptor downregulator (ERD); an agent for inhibiting or preventing ovaries, for example, a luteinizing hormone releasing hormone (LHRH) agonist, such as LUPRON And ELIGARD Leuprolide acetate, goserelin acetate, buserelin acetate, and tripterelin; other antiandrogens, such as flutamide, Nilutamide and bicalutamide; and aromatase inhibitors that inhibit aromatase production of estrogen in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, MEGASE Megestrol acetate, AROMASIN Exemestane, formestanie, fadrozole, RIVISOR Vorozole, FEMARA Letrozole and ARIMIDEX Anmeta ingot (anastrozole). In addition, this chemotherapeutic definition includes bisphosphonates such as clodronate (eg, BONEFOS) Or OSTAC ), DIDROCAL Etidronate, NE-58095, ZOMETA Zoledronic acid/zoledronate, FOSAMAX Alendronate, AREDIA Pamidronate, SKELID Tiluronate or ACTONEL Risedronate; and troxacitabine (1,3-dioxolan cytidine analog); antisense oligonucleotides, particularly signals that are associated with abnormal cell proliferation Gene expression in the transmission pathway, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE Vaccine and gene therapy vaccines (eg ALLOVECTIN) Vaccine, LEUVECTIN Vaccine and VAXID Vaccine; LURTOTECAN Topoisomerase 1 inhibitor; ABARELIX rmRH; lapatinib ditosylate (ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor, also known as GW572016); and any of the above substances may be pharmaceutically Accepted salts, acids or derivatives.

"Growth inhibitory" as used herein refers to a compound or composition that inhibits the growth of cells (eg, cells expressing EphB4) in vitro or in vivo. Thus, growth inhibitors can be inhibitors that significantly reduce the percentage of cells in the S phase, such as cells expressing EphB4. Examples of growth inhibitors include agents that block cell cycle progression (at the non-S phase), such as agents that induce G1 arrest and M arrest. Traditional M-stage blockers include vencar (vincas, vincristine and vinblastine), taxanes and such as 14-hydroxydaunorubicin, epirubicin, daunorubicin, etoposide and Bole Topoisomerase II inhibitor ofmycin. The agents that arrest G1 also overflow into the S phase arrest, for example, such as tamoxifen, prednisone, dacarbazine, dichloroethyl methylamine, cisplatin, methotrexate, 5-fluorouracil and ara-C DNA alkylating agent. Further information can be found in Murakami et al., The Molecular Basis of Cancer, Mendelsohn and Israel, ed., titled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (WB Saunders: Philadelphia, 1995), especially page 13. Taxanes (pacific paclitaxel and docetaxel) are anticancer drugs derived from yew trees. Docetaxel (TAXOTERE) , Rhone-Poulenc Rorer) from European yew (European yew), Pacific Paclitaxel (TAXOL) , semi-synthetic analog of Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in inhibition of mitosis in cells.

"14-hydroxydaunorubicin" is an anthracycline antibiotic. The complete chemical name of 14-hydroxydaunorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lais-hexapipetose) Oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyethenyl)-1-methoxy-5,12-nonanedione.

The term "anti-neoplastic composition" refers to a composition useful for treating cancer comprising at least one active therapeutic agent, such as an "anti-cancer agent." Examples of therapeutic agents (anticancer agents, also referred to herein as "anti-stimulants") include, but are not limited to, for example, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiation therapy, anti-drugs Angiogenesis agents, apoptotic agents, anti-tubulin agents, toxins, and other agents for treating cancer, such as anti-VEGF neutralizing antibodies, VEGF antagonists, anti-HER-2, anti-CD20, epidermal growth factor receptor (EGFR) Antagonists (eg, tyrosine kinase inhibitors), HER1/EGFR inhibitors, erlotinib, COX-2 inhibitors (eg, celecoxib), interferons, cytokines, binding to Antagonists of one or more of ErbB2, ErbB3, ErbB4 or VEGF receptors (eg neutralizing antibodies), inhibitors of platelet-derived growth factor (PDGF) receptor tyrosine kinase and/or stem cell factor (SCF) (eg imatinib mesylate (Gleevec) Novartis)), TRAIL/Apo2 and other biologically active and organic chemicals.

The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance which is less cytotoxic to tumor cells than the parent drug and which can be activated or converted by the enzyme to be more Active parent drug form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986); and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al. (eds.), pp. 247-267, Humana Press (1985). Prodrugs of the invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, and glycosylation Medicine, prodrug containing β-indoleamine, prodrug containing phenoxyacetamide as appropriate or substituted with phenylacetamide as required, 5-fluorocytosine and other 5-fluoro A uridine prodrug that can be converted to a more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into prodrug forms for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.

"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 directly or indirectly inhibits angiogenesis , angiogenesis or poor vascular permeability. For example, an anti-angiogenic agent is an antibody or other antagonist of an angiogenic agent as defined above, such as an anti-VEGF antibody, an anti-VEGF receptor antibody, a small molecule that blocks VEGF receptor signaling (eg, PTK787/ ZK2284, SU6668, SUTENT/SU11248 (sunitinib malate), AMG706). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin, endostatin, and the like. See, for example, Klagsbrun and D'Amore, Annu. Rev. Physiol., 53: 217-39 (1991); Streit and Detmar, Oncogene, 22: 3172-3179 (2003) (eg Table 3, listing malignancy) Anti-angiogenic therapy in melanoma); Ferrara and Alitalo, Nature Medicine 5(12): 1359-1364 (1999); Tonini et al, Oncogene, 22: 6549-6556 (2003) (eg Table 2, column Anti-angiogenic factors; and Sato Int. J. Clin. Oncol., 8:200-206 (2003) (for example, Table 1, which lists anti-angiogenic agents used in clinical trials).

Composition of the present invention and method of producing the same

The invention encompasses compositions comprising a pharmaceutical composition comprising an anti-EphB4 antibody and a polynucleotide comprising a sequence encoding an anti-EphB4 antibody. As used herein, a composition comprises one or more antibodies that bind to EphB4 and/or one or more polynucleotides comprising a sequence encoding one or more antibodies that bind to EphB4. Such compositions may additionally comprise a suitable carrier, such as a pharmaceutically acceptable excipient including a buffer, which is well known in the art.

Examples of isolated antibodies and polynucleotides are also contemplated by the invention. The invention also encompasses substantially pure antibody and polynucleotide embodiments.

The anti-EphB4 antibody of the present invention is preferably a monoclonal antibody. Fab, Fab', Fab'-SH and F(ab') ₂ fragments of the anti-EphB4 antibodies provided herein are also encompassed within the scope of the invention. Such antibody fragments can be produced by conventional means such as enzymatic digestion, or can be produced by recombinant techniques. Such antibody fragments can be chimeric or humanized. These fragments can be used for the diagnostic and therapeutic purposes set forth below.

The monoclonal antibodies are obtained from a population of substantially homologous 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 "single plant" indicates that the antibody is characterized in that it is not a mixture of discrete antibodies.

The anti-EphB4 monoclonal antibody of the present invention can be produced by the fusion tumor method first described by Kohler et al., Nature, 256 :495 (1975), or can be obtained by recombinant DNA method (U.S. Patent No. 4,816,567).

In the fusion tumor method, a mouse or other appropriate host animal such as a hamster is immunized to cause lymphocytes that produce or produce an antibody that specifically binds to a protein for immunization. Antibodies to EphB4 are typically produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of EphB4 and adjuvants. EphB4 can be prepared using methods well known in the art, some of which are additionally described herein. For example, the recombination of EphB4 is described below. In one embodiment, the animal is immunized with an EphB4 derivative comprising an EphB4 extracellular domain (ECD) fused to the Fc portion of the immunoglobulin heavy chain. In a preferred embodiment, the animal is immunized with an EphB4-IgG1 fusion protein. Typically an immunogenic conjugate of EphB4 with monophosphoryl lipid A (MPL) / diculose collicum (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT) or The derivative immunizes the animal and is injected intradermally at multiple sites. Two weeks later, the animals were bolused. After 7 to 14 days, the animals were bled and analyzed for serum anti-EphB4 titers. The animal is bounced until the titer reaches a steady state.

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

The fusion tumor cells thus prepared are inoculated and grown in a suitable medium which preferably contains one or more substances which inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the hypoxanthohumolin porphyrin phosphoribosyltransferase (HGPRT or HPRT), the medium used for the fusion tumor should generally include hypoxanthine, aminopterin and thymidine (HAT medium). ), these substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those which are efficiently fused, stably produced to a high degree by the selected antibody-producing cell-supporting antibody, and which are sensitive to a medium such as HAT medium. Among these cells, preferred myeloma cell lines are murine myeloma cell lines, such as those from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California USA). A murine myeloma cell line and SP-2 or X63-Ag8-653 cells (available from American Type Culture Collection, Rockville, Maryland USA). Human myeloma and mouse-human hybrid myeloma 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 , Pages 51-63 (Marcel Dekker, Inc., New York, 1987)).

The medium in which the fusion tumor cells were grown was assayed for the production of monoclonal antibodies against EphB4. The binding specificity of the monoclonal antibodies produced by the fusion tumor cells should preferably be determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al, Anal. Biochem. , 107 : 220 (1980).

After identifying the fusion tumor cells that produce the desired specificity, affinity and/or activity of the antibody, the pure lines can be sub-selected by limiting the dilution procedure and allowed to grow by standard methods ( Going, Monoclonal Antibodies: Principles and Practice) , pp. 59-103 (Academic Press, 1986)). Suitable media for achieving this include, for example, D-MEM or RPMI-1640 medium. In addition, the fusion tumor cells can be grown in vivo as ascites tumors in animals.

According to conventional immunoglobulin purification procedures such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, appropriate separation from culture medium, ascites fluid or serum A monoclonal antibody secreted by a sub-pure.

The anti-EphB4 antibodies of the invention can be prepared by screening combinatorial libraries for the pure lines of synthetic antibodies having the desired activity. In principle, a synthetic antibody-derived line is selected by screening a phage-containing phage library that exhibits various fragments (Fv) of the antibody variable region fused into the phage sheath protein. These phage libraries are screened by affinity chromatography of the desired antigen. A pure lineage of an Fv fragment that is capable of binding to the desired antigen is adsorbed by the antigen and is therefore isolated from the non-binding pure line in the library. Subsequently, the pure line is bound by antigen solubilization, and the bound pure lines can be further enriched by an additional antigen adsorption/dissolution cycle. An appropriate antigen screening procedure is designed to select the phage-pure line of interest, followed by the Fv sequence from the pure line of phage of interest and Kabat et al., Sequences of Proteins of Immunological Interest , 5th Edition, NIH Publication 91-3242, Bethesda MD ( 1991, a suitable constant region (Fc) sequence as described in Volumes 1-3 to construct a full length anti-EphB4 antibody, whereby any of the anti-EphB4 antibodies of the invention can be obtained.

The antigen binding domain of the antibody is formed by two variable (V) regions of about 110 amino acids, each from the light chain (VL) and the heavy chain (VH), both presenting three Hypervariable loop or complementarity determining region (CDR). The variable domain can be functionally presented on the phage as a single-chain Fv (ScFv) fragment in which VH and VL are covalently linked via a short, curved peptide, or as a Fab fragment each fused to a constant domain and non-covalently interacting As described in Winter et al ., Ann. Rev. Immunol . , 12: 433-455 (1994). As used herein, a bacteriophage-derived ScFv and a phage-derived Fab are collectively referred to as "Fv phage pure line" or "Fv pure line".

The VH and VL gene lineages can be individually selected by polymerase chain reaction (PCR) and randomly recombined into a phage library, followed by Winter et al., Ann. Rev. Immunol . , 12: 433-455 (1994) The antigen binding pure lines of these phage libraries are sought as described in . The library from the immunogenic source provides antibodies with high affinity to the immunogen without constructing the fusion tumor. Alternatively, the natural lineage can be selected to provide a single source of human antibodies against various non-autoantigen and autoantigens without any immunization, as in Griffiths et al , EMBO J, 12 : 725-734 (1993). Said in the middle. Finally, a natural library, such as Hoogenboom, can also be synthesized synthetically by autologously selecting a non-rearranged V gene fragment from a stem cell and using a PCR primer containing a random sequence to encode a highly variable CDR3 region and achieving in vitro rearrangement. Winter, J. Mol. Biol. , 227: 381-388 (1992).

Filamentous phage were used to present antibody fragments by fusion to the microsphingin pIII. Antibody fragments can be presented as single-chain Fv fragments in which the VH and VL domains are linked to the same polypeptide chain by a curved polypeptide spacer, for example, as in Marks et al , J. Mol. Biol., 222 : 581-597 (1991). Said; or presented as a Fab fragment in which one strand is fused to pIII and the other strand is secreted in the interstitium of the bacterial host cell, assembled in the intercellular substance by displacement of certain wild-type sheath proteins The Fab sheath protein structure on the surface of the phage is described, for example, in Hoogenboom et al., Nucl. Acids Res. , 19 : 4133-4137 (1991).

In general, nucleic acids encoding antibody gene fragments are obtained from immune cells collected from humans or animals. If it is desired to favor a library rich in anti-EphB4 pure lines, human or animal is immunized with EphB4 to generate an antibody response, and spleen cells and/or circulating B cells or other peripheral blood lymphocytes (PBL) are recovered for library construction. In a preferred embodiment, an anti-EphB4 antibody response is produced in a transgenic mouse carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system), thereby causing EphB4 immunity to cause B The cells produce human antibodies against EphB4, thereby obtaining a library of human antibody gene fragments biased to be rich in anti-EphB4 pure lines. The production of transgenic mice producing human antibodies is described below.

EphB4-specific membranes can be isolated by using appropriate screening procedures (for example, by isolating cells with EphB4 affinity chromatography, or by absorbing fluorescent dye-labeled EphB4 followed by flow-activated cell sorting (FACS)). Additional enrichment of anti-EphB4 reactive cell populations was obtained by binding to B cells of the antibody.

Alternatively, spleen cells and/or B cells or other PBLs from unimmunized donors are used to allow for better presentation of possible antibody lineages, and any animal (human or non-human) species in which EphB4 is not antigenic is also permitted. To construct an antibody library. For libraries incorporated into the construction of an in vitro antibody gene, stem cells from such animals are harvested to provide a nucleic acid encoding an unrearranged antibody gene segment. The immune cells of interest can be obtained from a variety of animal species, such as humans, mice, rats, rabbits, lupines, canines, felines, pigs, cattle, horses, and avian species.

Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. In the case of rearranged VH and VL gene libraries, genomic DNA or mRNA can be isolated from lymphocytes, followed by polymerase chaining with primers that match the 5' and 3' ends of the rearranged VH and VL genes. Reaction (PCR) (as described in Orlandi et al , Proc. Natl. Acad. Sci. (USA), 86 : 3833-3837 (1989)), thereby obtaining different V gene lineages for expression, thereby obtaining The DNA you want. The V gene can be amplified by cDNA and genomic DNA, wherein the backward primer encodes the 5' end of the mature V domain and the forward primer is in the J segment, such as Orlandi et al. (1989) and Ward et al. Human, Nature , 341 : 544-546 (1989). However, for cDNA amplification, the backward primer can also be in the exon of the leader sequence, as described in Jones et al, Biotechnol. , 9 : 88-89 (1991); and the forward primer is constant. Within the zone, as described in Sastry et al, Proc. Natl. Acad. Sci. (USA) , 86 : 5728-5732 (1989). To maximize complementarity, degeneracy can be incorporated into the primer as described by Orlandi et al. (1989) or Sastry et al. (1989). Preferably, library diversity is maximized by using PCR primers that target each V gene family such that all available VH and VL sequences present in the nucleic acid nucleic acid sample are amplified, for example, such as Marks et al. , described in the method of J. Mol. Biol. , 222 : 581-597 (1991) or as described by Orum et al ., Nucleic Acids Res. , 21 : 4491-4498 (1993). In order to select the amplified DNA into the expression vector, a rare restriction site can be introduced into the PCR primer as a label for one end (as described by Orlandi et al. (1989)), or by using a labeled primer. PCR amplification is performed and introduced as a label for one end (as described in Clackson et al, Nature, 352:624-628 (1991)).

A synthetic rearranged V gene lineage can be obtained from a V gene segment in vitro. Most human VH gene segments have been cloned and sequenced (reported in Tomlinson et al , J. Mol. Biol. , 227 :776-798 (1992)) and mapped (reported in Matsuda et al , Nature Genet). . 3 :88-94 (1993); these selected colonies (including all major configurations in the H1 and H2 loops) can be used to encode PCR primers of H3 loops of different sequences and lengths. Different VH gene lineages are generated as described in Hoogenboom and Winter, J. Mol. Biol., 227 :381-388 (1992). It is also possible to produce a VH lineage in which all sequence diversity is concentrated on a long H3 loop of a single length, as described in Barbas et al , Proc. Natl. Acad. Sci. USA , 89 : 4457-4461 (1992). Human V And the Vλ segment has been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol. , 23 : 1456-1461 (1993)) and can be used to make synthetic light chain lineages. Synthetic V gene lineages based on various VH and VL folding folds and L3 and H3 lengths will encode antibodies with considerable structural diversity. After amplification of the DNA encoding the V gene, the germline V gene segment can be rearranged in vitro according to the method of Hoogenboom and Winter, J. Mol. Biol., 227 :381-388 (1992).

The lineage of antibody fragments can be constructed by combining the VH and VL gene lineages in a variety of ways. Each lineage can be established with a different vector, and the vectors are in vitro by a combination of infections (eg, as described in Hogrefe et al , Gene , 128 : 119-126 (1993)) or in vivo (eg, Waterhouse et al. Recombination of the loxP system described in Human , Nucl. Acids Res., 21 : 2265-2266 (1993). The in vivo recombination method utilizes the two-strand nature of the Fab fragment to overcome the limitations regarding the size of the library imposed by E. coli transformation efficiency. The native VH and VL lineages are individually selected, one is selected for phagemid and the other is selected for phage vector. Subsequently, the two libraries were combined by phage infection of bacteria containing phagemids such that each cell contained a different combination and the library size was limited only by the number of cells present (about 10 ¹² pure lines). Both vectors contain an in vivo recombination signal such that the VH and VL genes are recombined on a single replicon and co-packaged in phage virions. Such huge libraries provide a large number of good affinity (K _d ^-1 of about 10 ^-8 M) of different antibodies.

Alternatively, the lineages can be sequentially cloned into the same vector, for example as described by Barbas et al , Proc. Natl. Acad. Sci. USA, 88 : 7978-7982 (1991); or assembled by PCR. Colonization is carried out together and subsequently, for example as described in Clackson et al , Nature , 352: 624-628 (1991). PCR assembly can also be used to link VH and VL DNA to DNA encoding a curved peptide spacer to form a single chain Fv (scFv) lineage. In another technique, "intracellular PCR assembly" is used to combine VH and VL genes into lymphocytes by PCR and subsequent selection of lineages of linked genes, such as Embleton et al., Nucl. Acids Res. , 20 : Said in 3831-3837 (1992).

Of an antibody (natural or synthetic) can be of the untreated libraries generated with a moderate affinity (K _d ^-1 of about ¹⁰⁶ to 10 ⁷ M ^-1), but also by constructing the library and from the secondary re-selection in vivo The external analog affinity matures as described by Winter et al. (1994). For example, by using a misproducible polymerase (reported in Leung et al ., Technique, 1: 11-15 (1989)) by Hawkins et al , J. Mol. Biol., 226 : 889-896 (1992) The method is either introduced randomly in vitro by the method of Gram et al ., Proc. Natl. Acad. Sci USA , 89 : 3576-3580 (1992). In addition, affinity maturation can also be performed, for example, by PCR using a primer carrying a random sequence spanning the CDR of interest to randomly mutate one or more CDRs in a selected individual Fv pure line and screen for a pure line with higher affinity. . WO 9607754 (published on Mar. 14, 1996) describes a method for inducing mutation induction in a complementarity determining region of an immunoglobulin light chain to establish a light chain gene library. Another effective method is to recombine the VH or VL domain selected by phage display and the naturally occurring V domain variant obtained from the unimmunized donor, and the screening is higher in several rounds of strand shuffling. Affinity, as described by Marks et al ., Biotechnol., 10:779-783 (1992). This technique allows the production of antibodies and antibody fragments with affinities in the range of ^10-9 M.

EphB4 nucleic acid and amino acid sequences are known in the art. The nucleic acid sequence encoding EphB4 can be designed using the amino acid sequence of the desired EphB4 region. Alternatively, the cDNA sequence of GenBank Accession No. NM_004444 (or a fragment thereof) or the one disclosed in U.S. Patent No. 635,177 can be used. DNA encoding EphB4 can be prepared by a variety of methods known in the art. Such methods include, but are not limited to, chemical synthesis, such as triesters, phosphites, according to any of the methods described in Engels et al, Agnew. Chem. Int. Ed. Engl. , 28 : 716-734 (1989). , Aminophosphate and H-phosphonate methods. In one embodiment, the host cell expresses a preferred codon for designing a DNA encoding EphB4. Alternatively, DNA encoding EphB4 can be isolated from a genomic or cDNA library.

After constructing a DNA molecule encoding EphB4, the DNA molecule is operably linked to a expression control sequence, such as a plastid expression vector, wherein the control sequence is recognized by a host cell transformed with the vector. In general, plastid vectors contain replication and control sequences derived from species compatible with the host cell. Vectors typically carry a replication site and a sequence encoding a protein that provides phenotypic selection in a transformed cell. Vectors suitable for expression in prokaryotic and eukaryotic host cells are known in the art and some are further described herein. Cells such as yeast eukaryotic organisms or cells derived from multicellular organisms such as mammals can be used.

Optionally, the DNA encoding EphB4 is operably linked to a secretory leader sequence, resulting in the expression product being secreted by the host cell into the culture medium. Examples of secretory leader sequences include stII, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and alpha factor. The 36 amino acid leader sequence of Protein A (Abrahmsen et al., EMBO J. , 4 :3901 (1985)) is equally applicable herein.

Transfecting a host cell with the above-described expression vector or selection vector of the present invention and preferably transforming it, and a conventional nutrient medium modified to be suitable for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence The cells are cultured in the medium.

Transfection refers to the uptake of a performance vector by a host cell, regardless of whether or not any coding sequence is actually expressed. A variety of transfection methods are known to those of ordinary skill in the art, such as CaPO ₄ precipitation and electroporation. When any indication for manipulation of this vector occurs within a host cell, it is generally recognized as successful transfection. Transfection methods are well known in the art, and some methods are further described herein.

Transformation means introducing DNA into an organism so that the DNA can be replicated as an extrachromosomal element or by a chromosomal element. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Transformation methods are well known in the art, and some methods are further described herein.

Prokaryotic host cells for the production of EphB4 can be cultured as described generally by Sambrook et al.

Mammalian host cells for the production of EphB4 can be cultured in a variety of media, which are well known in the art and some of which are described herein.

Host cells referred to in this disclosure encompass cells in an in vitro culture medium as well as cells in a host animal.

Purification of EphB4 can be achieved using methods recognized in the art, some of which are described herein.

Purified EphB4 can be attached to such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, methacrylate methacrylate gels, polyacrylic acid and polymethacrylic copolymers Suitable substrates for nylon, neutral and ionic carriers and their analogs for phage display in pure affinity chromatography. Attachment of the EphB4 protein to the substrate can be achieved by the method described in Methods in Enzymology , Vol. 44 (1976). A common technique for attaching a protein ligand to a polysaccharide matrix such as agarose, dextran or cellulose involves coupling the carrier with a cyanogen halide and subsequently coupling the primary aliphatic or aromatic amine of the peptide ligand to the activated The matrix.

Alternatively, EphB4 can be used to coat the pores of the adsorption disk, either on the host cells immobilized on the adsorption pad or for cell sorting, or in combination with biotin for streptavidin-coated beads, or It is used in any of the other methods known in the art for screening phage display libraries.

The phage library sample is contacted with immobilized EphB4 under conditions suitable for binding at least a portion of the phage particles to the adsorbent. Typically, conditions (including pH, ionic strength, temperature, and the like) are selected to mimic physiological conditions. The phage bound to the solid phase is washed and then eluted by acid (for example, as described in Barbas et al ., Proc. Natl. Acad. Sci USA , 88 : 7978-7982 (1991)), or by a base. Dissolution (for example, as described in Marks et al , J. Mol. Biol., 222 : 581-597 (1991)), or by EphB4 antigen competition (for example, with Clackson et al , Nature , 352 :624). -628 (1991) The procedure for antigen competition is similar). Enrichment of 20-1,000 fold phage in a single round of selection. In addition, the enriched phage can be grown in bacterial culture and subjected to other rounds of selection.

The efficiency of selection depends on a number of factors, including the dissociation kinetics during washing and whether multiple antibody fragments on a single phage can simultaneously bind to the antigen. Antibodies with rapid dissociation kinetics (and weak binding affinity) can be retained by using short washes, multivalent phage presentation, and high coating density of the antigen in the solid phase. The high density not only stabilizes the phage via multivalent interactions, but also facilitates recombination of the dissociated phage. It can be presented by using long-term 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 (eg, Marks et al ., Biotechnol. , 10 :779-783 (1992)) to promote the selection of antibodies with slow dissociation kinetics.

It is possible to choose between phage antibodies that have different affinities for EphB4, even with slightly different affinities. However, random mutations in selected antibodies (e.g., mutations in certain affinity maturation techniques as described above) may result in a variety of mutants, most of which bind to the antigen, and some have higher affinity. The use of restricted EphB4 competitively eliminates a few high-affinity phage. To retain all mutants with higher affinity, phage can be incubated with excess biotin-conjugated EphB4, but biotin-conjugated EphB4 has a lower concentration of constant target molar concentration with a lower molar concentration than EphB4. Subsequently, the high affinity bound phage can be captured by the streptavidin coated paramagnetic beads. This "equilibrium capture" enables the selection of antibodies with sensitivity based on the binding affinity of the antibody, which allows for the isolation of mutant lines with only twice the high affinity from a large number of excess phage with lower affinity. Conditions for washing the phage bound to the solid phase can also be manipulated to distinguish based on dissociation kinetics.

The anti-EphB4 pure line can be selected according to the activity. In one embodiment, the invention provides for blocking the binding between an EphB4 ligand (eg, ephrin-B1, ephrin-B2, and/or ephrin-B3) and EphB4 but does not block the EphB4 ligand from the second protein ( For example, an anti-EphB4 antibody that binds between EphB1, EphB3, EphB4, EphB5, and/or EphB6). The Fv pure line corresponding to these anti-EphB4 antibodies can be selected by the following steps: (1) separating the anti-EphB4 pure line from the phage library as described above, and optionally expanding into a population in a suitable bacterial host. Enhance the phage pure line population isolated; (2) select EphB4 and second protein that require blocking and non-blocking activities, respectively; (3) adsorb the anti-EphB4 phage pure line to the immobilized EphB4; (4) use excess second protein To dissociate any poorly pure lines that recognize the EphB4 binding determinant of the binding determinant of the second protein or the binding determinant of the second protein; and (5) the solvate remains after the step (4) Keep the pure line of adsorption. Optionally, the pure line with the desired blocking/non-blocking properties can be further enriched by repeating the selection procedure described herein one or more times.

The DNA or phage encoding the fusion cell-derived monoclonal antibody of the present invention exhibits an easy separation of the Fv pure line, and is easy to use conventional procedures (for example, by using a specific weight designed to be specifically amplified by a fusion tumor or phage DNA template) The oligonucleotide primers of the strand and light chain coding regions are sequenced. After isolation, the DNA can be placed in an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin (such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma). In the cell, the synthesis of the desired monoclonal antibody is obtained in a recombinant host cell. A literature review on recombinant expression in bacterial DNA encoding antibodies includes Skerra et al , Curr. Opinion in Immunol. , 5 : 256 (1993) and Pluckthun, Immunol. Revs , 130 : 151 (1992).

The DNA encoding the Fv pure line of the invention can be combined with a DNA sequence known to encode a heavy chain and/or a light chain constant region (for example, a suitable DNA sequence can be obtained as described above by Kabat et al.) to form a full length or partial length. A pure line of heavy and/or light chains. It will be appreciated that any isotype constant region can be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and such constant regions can be obtained from any human or animal species. As used herein, a "chimeric" antibody and a "hybridization" antibody include variable domain DNA derived from an animal (such as a human) species and subsequently fused to constant region DNA of another animal species to form a "hybridization" code. Fv pure line of sequence (full length heavy chain and/or light chain). In a preferred embodiment, an Fv pure line derived from human variable DNA is fused to human constant region DNA to form a full human coding sequence, i.e., a full length or partial length heavy and/or light chain.

For example, DNA encoding an anti-EphB4 antibody from a fusion tumor of the invention can also be modified by substituting a homologous murine sequence from a fusion tumor homologous line with a coding sequence of the human heavy and light chain constant domains (eg, such as Morrison) Et al., Proc. Natl. Acad. Sci. USA , 81 : 6851-6855 (1984). DNA encoding an antibody or fragment derived from a fusion tumor or Fv pure line may be further modified by covalent ligation to an immunoglobulin coding sequence, all or part of which is used to encode a non-immunoglobulin polypeptide. In this manner, a "chimeric" or "hybrid" antibody having the binding specificity of an antibody of the Fv pure line or fusion line of the invention is prepared.

Antibody fragment

The invention encompasses antibody fragments. In some cases, it may be advantageous to use antibody fragments rather than whole antibodies. Fragments of smaller size can be quickly cleared and can result in improved access to solid tumors.

Various techniques have been developed for the production of antibody fragments. Typically, such fragments are obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al, Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al, Science, 229: 81 (1985)) . However, such fragments can now be produced directly by recombinant host cells. Both Fab, Fv and ScFv antibody fragments can be expressed in and secreted by E. coli, and thus a large number of such fragments can be easily produced. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') ₂ fragments (Carter et al, Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab') ₂ fragments can be isolated directly from recombinant host cell culture. Fab and F(ab') ₂ fragments comprising a salvage receptor binding epitope residue having an increased in vivo half-life are described in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments should be apparent to those skilled in the art. In other embodiments, the antibody selected is a single chain Fv fragment (scFv). See WO 93/16185, U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and sFv are the only species that lack a constant region but still have a complete combinatorial site; therefore, they are suitable for reduced non-specific binding during in vivo use. The sFv fusion protein can be constructed to produce fusion of effector proteins at the amino or carboxy terminus of the sFv. See Antibody Engineering, edited by Borrebaeck, supra. The antibody fragment can also be a "linear antibody", for example, as described in U.S. Patent No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

Humanized antibody

The invention encompasses humanized antibodies. A variety of methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced therein from a non-human source. Such non-human amino acid residues are often referred to as "input" residues, which are typically taken from the "input" variable domain. Basically according to Winter and colleagues (Jones et al., (1986) Nature 321:522-525; Riechmann et al ., (1988) Nature 332:323-327; Verhoeyen et al ., (1988) Science 239:1534-1536 The method is humanized by substituting the corresponding sequence of the human antibody with a hypervariable region sequence. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein sequences substantially smaller than the entire human variable domain have been replaced by corresponding sequences from non-human species. In fact, humanized antibodies are typically human antibodies in which certain hypervariable region residues and possibly certain FR residues are substituted with residues from analogous sites in rodent antibodies.

The selection of human variable domains (light and heavy) for the production of humanized antibodies is extremely important to reduce antigenicity. The variable domain sequences of rodent antibodies are screened against known human variable domain sequences according to the so-called "best fit" method. Subsequently, the human sequence closest to the rodent sequence is accepted as the human framework for humanized antibodies (Sims et al., (1993) J. Immunol. 151:2296; Chothia et al., (1987) J. Mol. Biol. 196 :901). Another method uses a specific framework derived from a consensus sequence of all human antibodies with a particular light chain or heavy chain subpopulation. The same framework can be used for several different humanized antibodies (Carter et al , (1992) Proc. Natl. Acad. Sci. USA , 89: 4285; Presta et al , (1993) J. Immunol. , 151: 2623).

More importantly, antibodies that are humanized to maintain high affinity for antigens and other beneficial biological properties are often required. To achieve this goal, humanized antibodies are prepared according to one method by analyzing the parental sequences and various conceptual humanized products using a three-dimensional model of the parental sequence and the humanized sequence. Three-dimensional immunoglobulin models are commonly available and are well known to those skilled in the art. A computer program describing and presenting the approximate three-dimensional configuration of the selected candidate immunoglobulin sequences can be used. Examination of such presentations allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this manner, FR residues can be selected from the receptor and input sequences and combined to achieve desired antibody characteristics, such as increased affinity for targeted antigens. In general, the effects on antigen binding are directly and substantially entirely related to hypervariable region residues.

Human antibody

The human anti-EphB4 antibody of the present invention can be constructed by combining an Fv pure line variable domain sequence selected from a human-derived phage display library with a known human constant domain sequence as described above. Alternatively, the human monoclonal anti-EphB4 antibody of the present invention can be produced by the fusion tumor method. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies have been described by, for example, 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 Boerner et al., J.Immunol, 147: 86 (1991 )..

It is now possible to produce a transgenic animal (e.g., a mouse) that is capable of producing a whole human antibody lineage immediately after immunization without producing endogenous immunoglobulin. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into such germline mutant mice will result in the production of human antibodies following antigen challenge. See, for example, Jakobovits et al , Proc. Natl. Acad. Sci USA, 90 : 2551 (1993); Jakobovits et al , Nature, 362 : 255 (1993); Bruggermann et al , Year in Immunol., 7: 33 (1993). ).

Gene shuffling can also be used to obtain human antibodies from non-human (e.g., rodent) antibodies, wherein the human antibodies have similar affinities and specificities as the original non-human antibodies. According to this method (also referred to as "epitope imprinting"), the human V domain gene lineage is substituted for a heavy or light chain variable of a non-human antibody fragment obtained by the phage display technique as described above. Region, thereby producing a non-human chain/human chain scFv or Fab chimera population. Selection with an 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 that is damaged when the primary phage exhibits the corresponding non-human chain in the pure line, ie, the antigen is determined. Base decision (imprint) for the choice of human chain collocation. Human antibodies are obtained when the process is repeated to replace the remaining non-human chains (see PCT WO 93/06213, published Apr. 1, 1993). Unlike traditional humanized non-human antibodies by CDR grafting, this technology provides fully human antibodies that do not have FR or CDR residues of non-human origin.

Bispecific antibody

A bispecific antibody is a monoclonal antibody having binding specificity for at least two different antigens, preferably a human or humanized antibody. In the context of the present invention, one of the binding specificities is the binding specificity for EphB4 and the other is the binding specificity for any other antigen. An exemplary bispecific antibody can bind to two different epitopes of the EphB4 protein. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express EphB4. These antibodies have an EphB4 binding arm and a binding cytotoxic agent (eg, saporin, anti-interferon-alpha, vinblastine, ricin A chain, methotrexate or radioisotope hapten) ) The arm. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F(ab') ₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific anti-systems has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature , 305 :537 (1983)). . Due to the random grouping of immunoglobulin heavy and light chains, these fusion tumors (hybridomas) produce a potential mixture of 10 different antibody molecules, of which only one has the appropriate bispecific structure. Purification of the appropriate molecule, usually by affinity chromatography steps, is quite cumbersome and the product yield is low. A similar procedure is disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al, EMBO J., 10: 3655 (1991).

The antibody variable domain having the desired binding specificity (antibody antigen binding site) is fused to an immunoglobulin constant domain sequence according to a different and better method. Preferably, the fusion is carried out with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, the CH2 and CH3 regions. Preferably, the first heavy chain constant region (CH1) comprising a site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. In the examples where three polypeptide chains of unequal ratios are used in construction to provide optimal yield, this provides great flexibility in the adjustment of the mutual ratio of the three polypeptide fragments. However, when at least two polypeptide chains are expressed in equal ratios to produce high yields or when such ratios are not of particular importance, it is possible to insert the coding sequences of two or all three polypeptide chains into one expression vector.

In a preferred embodiment of the method, the bispecific anti-system consists of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm ( A second binding specificity is provided to constitute. It has been discovered that since the immunoglobulin light chain is only present in half of the bispecific molecule to provide a convenient means of isolation, the asymmetric structure promotes the separation of the desired bispecific compound from the undesirable immunoglobulin combination. This method is disclosed in WO 94/04690. For additional details regarding the production of bispecific antibodies, see, for example, Suresh et al, Methods in Enzymology , 121:210 (1986).

According to another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from the recombinant cell culture. The preferred interface comprises at least a portion of the _CH3 domain of the antibody constant domain. In this way, one or more of the small amino acid side chains from the first antibody molecule interface are replaced with a larger side chain (eg, tyrosine or tryptophan). Compensating for the same or similar size to the large side chain at the interface of the second antibody molecule by replacing the large amino acid side chain with a small amino acid side chain (eg, alanine or threonine) Hole". This provides a mechanism to increase the yield of heterodimers over other undesirable end products, such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the heteroconjugates can be conjugated to an antibiotic protein and the other antibody to biotin. For example, such antibodies have been suggested to target immune system cells against undesirable cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/00373 and EP 03089). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 and the numerous cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, a bispecific antibody can be prepared using a chemical bond. The procedure for proteolytic cleavage of intact antibodies to produce F(ab') ₂ fragments is described in Brennan et al, Science , 229 :81 (1985). The fragments are reduced in the presence of the dithiol conjugate sodium arsenite to stabilize the adjacent dithiol and prevent intermolecular disulfide formation. Subsequently, the resulting Fab' fragment is converted to a thionitrobenzoate (TNB) derivative. Subsequently, a Fab'-TNB derivative is reconverted to a Fab'-thiol by reduction with mercaptoethylamine and mixed with another Fab'-TNB derivative of the same molar amount to form a bispecific antibody. . The bispecific antibody produced can be used as an agent for selectively immobilizing an enzyme.

Recent developments 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 the production of a fully humanized bispecific antibody F(ab') ₂ molecule. Each Fab' fragment was secreted separately by E. coli and subjected to in vitro directed chemical coupling to form a bispecific antibody. The bispecific antibody thus formed is capable of binding to cells overexpressing the HER2 receptor and normal human T cells, and also triggers the lytic activity of human cytotoxic lymphocytes on human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al., J. Immunol. , 148(5): 1547-1553 (1992). The leucine zipper peptide from the Fos and Jun proteins was ligated to the Fab' portion of two different antibodies by gene fusion. The antibody that restores the hinge region is homodimerized to form a monomer, and then reoxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. The "bifunctional antibody" technique described by Hollinger et al, Proc. Natl. Acad. Sci. USA , 90 :6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) joined to the light chain variable domain (VL) by a linker which is too short to allow pairing of the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. , 152: 5368 (1994).

Covers antibodies greater than two valences. For example, a trispecific antibody can be prepared. Tutt et al. , J. Immunol. 147 : 60 (1991).

Multivalent antibody

The multivalent antibody can be internalized (and/or catabolized) by the cells expressing the antigen bound to the antibody faster than the bivalent antibody. The antibody of the present invention may be a multivalent antibody having three or more antigen binding sites (which is different from an antibody of the IgM species) (for example, a tetravalent antibody), which can easily encode an antibody polypeptide by recombinant expression. The nucleic acid of the chain is manufactured. A multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of), for example, an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites at the amino terminus of the Fe region. Preferred multivalent antibodies herein comprise, for example, from three to about eight but preferably four antigen binding sites (or consist of). A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the polypeptide chain can comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is the first variable domain, VD2 is the second variable domain, and Fc is a polypeptide chain of the Fc region, X1 and X2 represent an amino acid or a polypeptide, and n is 0 or 1. For example, the polypeptide chain can comprise: a VH-CH1-bend linker-VH-CH1-Fc region chain or a VH-CH1-VH-CH1-Fc region chain. Herein, the multivalent antibody may additionally comprise at least two (and preferably four) light chain variable domain polypeptides. Herein, a multivalent antibody can, for example, comprise from about two to about eight light chain variable domain polypeptides. A light chain variable domain polypeptide encompassed herein comprises a light chain variable domain and, as the case may be, additionally comprises a CL domain.

Antibody variant

In certain 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 the antibody. Amino acid sequence variants of antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, with the proviso that the final construct has the desired characteristics. Amino acid changes can be introduced into the target antibody amino acid sequence at the time of manufacture of the sequence.

A method suitable for recognizing certain residues or regions of an antibody, which is a preferred mutation-inducing position, is called "alanine scanning mutagenesis", such as Cunningham and Wells (1989) Science , 244:1081- Said 1085. Thus, residues or groups of the targeting residues are recognized (eg, charged residues such as arg, his, lys, and glu) and are neutralized with a neutral or negatively charged amino acid (most preferably alanine or Polyalanine) substitutions affect the interaction of the amino acid with the antigen. Subsequently, the position of the amino acid which is confirmed to be functionally sensitive to the substitution is improved by introducing additional or other variants at the substitution site or introducing additional or other variants as substitution sites. Therefore, although the site of the change in the amino acid sequence has been scheduled to be introduced, the nature of the mutation itself does not need to be predetermined. For example, to analyze the potency of a mutation at a given site, an ala scan or random mutation induction is performed at the targeting codon or region, and the immunoglobulin exhibited by the desired activity is screened.

Amino acid sequence insertions include amino terminus and/or carboxy terminus fusions ranging from one residue to a polypeptide containing one hundred or more residues, and sequences within single or multiple amino acid residues insert. Examples of terminal insertions include antibodies having N-terminal methionine residues or antibodies fused to cytotoxin polypeptides. Other insertion variants of the antibody molecule include fusions of the N-terminus or C-terminus of the antibody with an enzyme (eg, for ADEPT), or a polypeptide that increases the serum half-life of the antibody.

Polypeptide glycosylation is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an aspartic acid residue. Tripeptide sequence aspartic acid-X-serine and aspartic acid-X-threonine (where X is any amino acid other than proline) is enzymatically attached to the carbohydrate part The recognition sequence of the protamine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide results in a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetyl galactosamine, galactose or xylose to a hydroxyl amino acid, which is most often seric acid or sulphite However, 5-hydroxyproline or 5-hydroxy lysine may also be used.

The amino acid sequence is altered to contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites), thereby facilitating the addition of a glycosylation site to the antibody. Alterations can also be made by adding one or more serine or threonine residues to the original antibody sequence or substituting the residues (for O-linked glycosylation sites).

If the antibody comprises an Fc region, the carbohydrate attached thereto can also be modified. For example, an antibody having a mature carbohydrate structure lacking trehalose attached to the Fc region of an antibody is described in US Patent Application No. 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). </ RTI></RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt; An antibody having at least one galactose residue in an oligosaccharide attached to the Fc region of an antibody is reported in WO 1997/30087 to Patel et al. See also WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.), which focus on antibodies having altered carbohydrates attached to the Fc region of an antibody. See also US 2005/0123546 (Umana et al. ) for antigen binding molecules having modified glycosylation.

Preferred glycosylation variants herein comprise an Fc region in which the carbohydrate structure attached to the Fc region lacks trehalose. These variants have improved ADCC function. Optionally, the Fc region additionally comprises one or more amino acid substitutions that further modify ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (Eu numbering of residues). Examples of publications relating to "de-alcoholylation" or "trehalose-deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/ 0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al, Biotech. Bioeng. 87:614 (2004). Examples of cell lines that produce de-fucosylated antibodies include Lec13 CHO cells lacking in protein trehalylation (Ripka et al, Arch. Biochem. Biophys. 249: 533-545 (1986); US Patent Application No. US 2003 /0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al, especially Example 11) and gene knockout cell lines, such as α-1,6- trehalyltransferase gene, FUT8 , gene knockout CHO cell (. Yamane-Ohnuki et al., Biotech.Bioeng 87: 614 (2004) ).

Another type of variant is an amino acid substitution variant. In these variants, at least one amino acid residue in the antibody molecule is replaced by a different residue. The most interesting sites for substitution mutation induction include hypervariable regions, but also cover FR changes. The conservative substitutions are shown in Table 1 under the heading "Better Replacement". If such substitutions result in a change in biological activity, a more substantial change referred to in Table 1 as "exemplary substitution" or as further described below with reference to the amino acid species can be introduced and the product screened.

Substantial modifications to the biological properties of an antibody are achieved by selective substitutions that differ significantly in their effect on the maintenance of: (a) the structure of the polypeptide backbone in the substitution region, for example, in the form of a sheet or a helix Configuration; (b) charge or hydrophobicity at the molecular targeting site; or (c) overall side chain. Naturally occurring residues can be grouped based on overall side chain properties: (1) hydrophobic residues: n-leucine, met, ala, val, leu, ile; (2) neutral hydrophilic residues: Cys, Ser, Thr, Asn, Gln; (3) acidic residues: asp, glu; (4) basic residues: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) ) aromatic residues: trp, tyr, phe.

Non-conservative substitutions will require members of one of these categories to be exchanged with another species.

One type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (eg, a humanized antibody or a human antibody). In general, the resulting variants selected for further development should have improved biological properties relative to the parent antibody from which they are produced. A convenient way to generate such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each point. When fused to the M13 gene III product packaged in each filamentous phage particle, the antibodies thus produced by the particles are presented. Subsequently, the phage display variants with respect to the biological activity (e.g., binding affinity) of the antibodies as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutation induction can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Additionally or alternatively, it may be useful to analyze the crystal structure of the antigen-antibody complex to recognize the point of contact between the antibody and the antigen. These contact residues and adjacent residues are candidates that are substituted according to the techniques described in detail herein. Following generation of such variants, the population of variants is subjected to screening as described herein, and antibodies with superior properties can be selected for further development in one or more relevant assays.

Nucleic acid molecules encoding antibody amino acid sequence variants are prepared by a variety of methods known in the art. Such methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or mediated by previously prepared antibody variants or non-variant-type oligonucleotides (or Site-directed mutagenesis, PCR mutation induction, and cassette mutagenesis preparation.

It may be desirable to introduce one or more amino acid modifications in the Fc region of an immunoglobulin polypeptide of the invention, thereby producing an Fc region variant. An Fc region variant may comprise a human Fc region sequence comprising an amino acid modification (eg, a substitution) at one or more amino acid positions (including the position of hinge cysteine) (eg, human IgGl, IgG2, IgG3) Or IgG4 Fc region).

Based on this description and the teachings of the art, it is contemplated that in certain embodiments, an antibody for use in a method of the invention may comprise one or more alterations, such as in the Fc region, as compared to a wild-type counterpart antibody. However, such antibodies will retain the characteristics required for substantially the same therapeutic utility as their wild-type counterparts. For example, it is believed that certain changes that would result in C1q binding and/or complement dependent cytotoxicity (CDC) alteration (ie, improvement or reduction) can be made in the Fc region, eg, as described in WO 99/51642 Said. See also the following documents relating to other examples of Fc region variants: Duncan & Winter Nature 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351. WO 00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with improved or reduced binding to FcR. The contents of these patent publications are specifically incorporated herein by reference. See also Shields et al, J. Biol. Chem. 9(2): 6591-6604 (2001). Has an increased half-life and improved binding to the neonatal Fc receptor (FcRn) (which causes maternal IgG transfer to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al), J The antibody of Immunol. 24: 249 (1994) is described in US 2005/0014934 A1 (Hinton et al.). Such antibodies comprise an Fc region in which one or more substitutions modify the binding of the Fc region to FcRn. Polypeptide variants having altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1, WO 99/51642. The contents of their patent publications are specifically incorporated herein by reference. See also Idusogie et al, J. Immunol. 164: 4178-4184 (2000).

Antibody derivative

The antibodies of the invention may be further modified to contain additional non-protein portions known in the art and readily available. Preferably, the moiety suitable for derivatizing 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone , poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and Glycans or poly(n-vinylpyrrolidone) polyethylene glycols, propylene glycol homopolymers, polyoxypropylene/ethylene oxide copolymers, polyethoxylated polyols (such as glycerin), polyvinyl alcohols, and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, the polymers can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, specific characteristics or functions of the antibody to be modified, whether the antibody derivative will be used in a given condition, and the like. .

Screen for antibodies with the desired properties

The physical/chemical properties and biological functions of the antibodies of the invention can be characterized by various assays known in the art. In some embodiments, reducing or blocking EphB4 activation, reducing or blocking EphB4 downstream molecular signaling, reducing or blocking EphB4 ligand activation, reducing or blocking EphB4 ligand downstream molecular signaling, disruption or resistance A mismatched group (eg, ephrin-B1, ephrin-B2, and/or ephrin-B3) binds to EphB4, EphB4 phosphorylation and/or EphB4 multimerization and/or EphB4 ligand phosphorylation and/or treatment and/or The prevention of a tumor, a cell proliferative disorder or cancer and/or treatment or prevention of any one or more of the conditions associated with EphB4 expression and/or activity (eg, increased EphB4 expression and/or activity) to characterize the antibody.

Purified antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, invariant size exclusion, high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and Papaya enzyme digestion.

In certain embodiments of the invention, the biological activities of the antibodies produced herein are analyzed. In some embodiments, the antigen binding activity of an antibody of the invention is tested. Antigen binding assays known in the art and useful herein include, but are not limited to, the use of, for example, western dot methods, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays , direct or competitive binding assays for fluorescein immunoassays and protein A immunoassays. An illustrative antigen binding assay is provided below in the Examples section.

In another embodiment, the invention provides an anti-EphB4 monoclonal antibody that competes for binding to EphB4 with a 30.35, 30.35.1D2 and/or 30.35.2D8 antibody. Such competing antibodies include antibodies that recognize the EphB4 epitope that is identical or overlapping with the EphB4 epitope recognized by antibodies 30.35, 30.35.1D2 and/or 30.35.2D8. The competing antibodies can be obtained by screening anti-EphB4 fusion tumor supernatants to compete for binding to the immobilized EphB4 with the labeled 30.35, 30.35.1D2 and/or 30.35.2D8 antibodies. The fusion antibody supernatant containing the competing antibody will reduce the binding detected in the competitive binding mixture of the present invention compared to the amount of bound, labeled antibody detected in the control binding mixture containing the unrelated (or no) antibody. The amount of labeled antibody. Any of the competitive binding assays described herein are applicable to the foregoing procedures.

In another aspect, the invention provides an anti-EphB4 monoclonal antibody comprising one or more (eg, 2, 3, 4, 5, and/or 6) HVRs of 30.35, 30.35.1D2, or 30.35.2D8 antibodies. Porting one or more HVRs of 30.35, 30.35.1D2 or 30.35.2D8 to a template antibody sequence (eg, a human antibody sequence that corresponds to the corresponding murine sequence of the parent antibody, or a specific subpopulation of the parent antibody light or heavy chain) And the resulting chimeric light chain and/or heavy chain variable region sequences in the recombinant host cell, with or without accompanying constant region sequences, as described herein, thereby constructing An anti-EphB4 monoclonal antibody comprising one or more HVRs of 30.35, 30.35.1D2 or 30.35.2D8.

An anti-EphB4 antibody of the invention having the unique properties described herein can be obtained by screening a pure line of an anti-EphB4 fusion tumor having the desired properties according to any convenient method. For example, if it is desired to block or not block the EphB4 ligand binding to EphB4 against an EphB4 monoclonal antibody, the candidate antibody can be tested in a binding competition assay such as a competitive binding ELISA in which the plate is coated with EphB4 a well, and stratifying the antibody solution in excess relative to the Eph ligand of interest on the coated culture plate, and enzymatically detecting the bound antibody, eg, the bound antibody and the HRP-binding anti-Ig antibody or organism The anti-Ig antibody contacts and develops HRP color response, for example, by developing the culture disk with streptavidin-HRP and/or hydrogen peroxide and by spectrophotometer using an ELISA disk reader This was achieved by detecting the HRP color response at 490 nm.

If an anti-EphB4 antibody that inhibits or activates EphB4 activation is desired, the candidate antibody can be tested for EphB4 phosphorylation assay. Such assays are known in the art and one such assay is described in the Examples section.

If an anti-EphB4 antibody that inhibits cell growth is desired, the candidate antibody can be tested in an in vivo and/or in vitro assay for measuring inhibition of cell growth. Such assays are known in the art and are further described and exemplified herein.

In one embodiment, the invention encompasses an altered antibody that has some, but not all, of the effector functions, which makes it possible that the half-life of the antibody in vivo is extremely important while certain effector functions (such as complement and ADCC) are Candidate antibodies that are not desirable in a variety of applications that are not necessary or harmful. In certain embodiments, the Fc activity of the produced immunoglobulin is measured to ensure that only the desired properties are maintained. In vitro and/or in vivo cytotoxicity assays can be performed to determine the reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcyR binding (and thus may lack ADCC activity) but still retains FcRn binding ability. The primary cells (NK cells) that mediate ADCC exhibit only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu . Rev. Immunol 9:457-92 (1991). An example of an in vitro assay to analyze the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Additionally or alternatively, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model (such as those disclosed in Clynes et al, PNAS (USA) 95:652-656 (1998)). A C1q binding assay can also be performed to determine that the antibody is unable to bind to C1q and thus lacks CDC activity. To analyze complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al ., J. Immunol. Methods 202: 163 (1996). FcRn binding and in vivo clearance/half life assays can also be performed using methods known in the art.

Vector, host cell and recombinant method

For recombinant production of an antibody of the invention, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further selection (amplification of DNA) or expression. The DNA encoding the antibody is readily separable and can be readily sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). A variety of carriers are available. The choice of vector will depend, in part, on the host cell to be used. Preferred host cells are typically cells of prokaryotic or eukaryotic (usually mammalian) origin. It will be appreciated that any isotype constant region can be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and such constant regions can be obtained from any human or animal species.

a. Production of antibodies using prokaryotic host cells: i. Vector construction A polynucleotide sequence encoding a polypeptide component of an antibody of the invention can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated from the antibody-producing cells (such as fusion tumor cells) and sequenced. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR technique. After obtaining the sequence encoding the polypeptide, it is inserted into a recombinant vector capable of replicating in a prokaryotic host and expressing the heterologous polynucleotide. The objects of the invention can be achieved using a variety of carriers that are available and known in the art. The choice of appropriate vector will depend primarily on the size of the nucleic acid inserted into the vector and the particular host cell into which the vector is transformed. Each vector contains multiple components depending on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the particular host cell with which it is retained. Vector components typically 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, plastid vectors containing replicons and control sequences derived from species compatible with the host cell are used in connection with such hosts. Vectors generally have a replication site and a marker sequence capable of providing phenotypic selection in transformed cells. For example, pBR322, a plastid derived from E. coli species, is typically used to transform E. coli. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides a convenient means of identifying transformed cells. pBR322, its derivatives or other microbial plastids or phages may also contain or be modified to contain a promoter which can be used by a microbial organism to express an endogenous protein. An example of a pBR 322 derivative for the expression of a specific antibody is described in detail in U.S. Patent No. 5,648,237 to Carter et al.

In addition, phage vectors containing replicons and control sequences compatible with the host microorganisms associated with such hosts can also be used as transformation vectors. For example, phage such as λGEM.TM.-11 can be used to make recombinant vectors useful for transformation of susceptible host cells, such as E. coli LE392.

The expression vector of the present invention may comprise two or more promoter-cistronic pairs encoding each polypeptide component. A promoter is an untranslated regulatory sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are generally divided into two categories, namely, inducible and constitutive. Inducible promoters are promoters that respond to changes in culture conditions (eg, the presence or absence of nutrients or changes in temperature) and, under their control, initiate cistron transcription at increased levels.

A large number of promoters recognized by a variety of potential host cells are well known. The promoter can be operably linked to the coding light or heavy chain by removing the selected promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Child DNA. Native promoter sequences and a variety of heterologous promoters can be used to direct amplification and/or expression of the targeted gene. In certain embodiments, a heterologous promoter is utilized because the heterologous promoter typically allows the expressed gene to be transcribed faster and has a higher yield than the native targeting polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoA promoter, beta-galactosidase and lactose promoter systems, tryptophan (Trp) promoter systems, and hybrid promoters (such as the tac or trc promoter). However, other promoters that function in bacteria, such as other known bacterial or bacteriophage promoters, are also suitable. Nucleotide sequences have been published, thereby enabling those skilled in the art to use ligators or adaptors to operate and encode cistrons that target light and heavy chains (Siebenlist et al., 1980). ) Cell 20: 269) Engage to supply any desired restriction sites.

In one aspect of the invention, each cistron in the recombinant vector comprises a secretory signal sequence component that directs transmembrane translocation of the expressed polypeptide. In general, the signal sequence can be a component of a vector, or it can be a portion of the targeted polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention should be those which are recognized and processed by the host cell (i.e., cleaved by signal peptidase). For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence is replaced, for example, by a prokaryotic signal sequence selected from the group consisting of alkaline phosphatase, penicillinase, Ipp or heat stable enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequence used in the two cistrons of the expression system is the STII signal sequence or a variant thereof.

In another aspect, the production of an immunoglobulin according to the present invention can occur in the cytoplasm of a host cell, and thus there is no need for a secretory signal sequence to be present in each cistron. In this regard, both the immunoglobulin light and heavy chains are expressed, folded, and assembled within the cytoplasm to form a functional immunoglobulin. Certain host strains (eg, E. coli trxB -strain) provide cytoplasmic conditions that readily form disulfide bonds, thereby allowing proper folding and assembly of the protein subunits represented. Proba and Pluckthun Gene , 159: 203 (1995).

Prokaryotic host cells suitable for expression of the antibodies of the invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., Escherichia coli), Bacilli (such as B. subtilis), Enterobacteria, Pseudomonas species. (eg P. aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella Genus (Shigella), Rhizobia, Vitreoscilla or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as a host of the invention. Examples of E. coli strains include the W3110 strain (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, DC: American Society for Microbiology, 1987), pages 1190-1219; ATCC Accession No. 27,325) and derivatives thereof, including Genotype W3110 Δ fhuA( Δ tonA)ptr3 lac Iq lacL8 Δ ompT Δ (nmpc-fepE) degP41 kanR 33D3 strain (U.S. Patent No. 5,639,635). Other strains and derivatives thereof (such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31, 608)) are also suitable. These examples are for purposes of illustration and not limitation. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8: 309-314 (1990). )in. It is often necessary to consider the replicability of the replicon in bacterial cells to select the appropriate bacteria. For example, when a replicon is supplied using a well-known plastid such as pBR322, pBR325, pACYC177 or pKN410, Escherichia coli, Serratia or Salmonella can be suitably used as a host. Generally, host cells should secrete very small amounts of proteolytic enzymes, and additional protease inhibitors may be required to be incorporated into the cell culture.

II. Transformation of the host cells to make antibody produced by the above-described expression vectors and cultured in the appropriately modified conventional nutrient medium to the gene for inducing promoters, selecting or transition to amplify the coding sequences.

Transformation means introducing DNA into a prokaryotic host such that the DNA can be replicated as an extrachromosomal element or by a chromosomal element. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment with calcium chloride is commonly used for bacterial cells containing basic cell wall barriers. Another method of transformation uses polyethylene glycol/DMSO. Yet another technique used is electroporation.

Prokaryotic cell lines used to produce the polypeptides of the invention are grown in a medium known in the art and suitable for culturing selected host cells. Examples of suitable media include luria broth (LB) supplemented with essential nutrient supplements. In certain embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively allow growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture medium for growth of cells expressing the ampicillin resistance gene.

Any necessary supplements other than carbon, nitrogen and inorganic phosphate sources, introduced alone or as a mixture with other supplements or matrices (such as a complex nitrogen source), may also be included. Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol. alcohol.

The prokaryotic host cell line is cultured at an appropriate temperature. For E. coli growth, for example, a preferred temperature is in the range of from about 20 ° C to about 39 ° C, more preferably from about 25 ° C to about 37 ° C, even more preferably about 30 ° C. Depending on the host organism, the pH of the medium can be any pH in the range of from about 5 to about 9. For E. coli, the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0.

If an inducible promoter is used in the expression vector of the present 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, transformed host cells are cultured in phosphate-restricted media for induction. Preferably, the phosphate-limiting medium is CRAP medium (see, for example, Simmons et al, J. Immunol. Methods (2002), 263: 133-147). As is known in the art, a variety of other elicitors can be used depending on the vector construct used.

In one embodiment, the expressed polypeptide of the invention is secreted into the host cell stroma and recovered from the host cell stroma. Protein recovery typically involves splitting the microorganisms typically by means of osmotic shock, ultrasonic treatment or dissolution. After dividing the cells, cell debris or whole cells can be removed by centrifugation or filtration. For example, the protein can then be purified by affinity resin chromatography. Alternatively, the protein can be transported to the medium and isolated therein. The cells can be removed from the culture and the culture supernatant filtered and concentrated to further purify the resulting protein. The expressed polypeptide can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

In one aspect of the invention, a large amount of antibody production is carried out by a fermentation method. A variety of large-scale feed batch fermentation procedures can be used to produce recombinant proteins. Large-scale fermentation has a capacity of at least 1000 liters, for example, a capacity of about 1,000 to 100,000 liters. These fermenters use an impeller agitator to distribute oxygen and nutrients, especially glucose (preferably carbon/energy sources). Small-scale fermentation usually means fermenting in a fermenter that has a volume of only about 100 liters and can range from about 1 liter to about 100 liters.

During fermentation, the induction of protein expression is typically initiated after the cells have been grown to the desired density (eg, OD550 is about 180 to 220), during which the cells are in the early stationary phase. As is known in the art and as described above, a variety of elicitors can be used depending on the vector construct used. Cells can be grown for a short period of time prior to induction. Although longer or shorter induction times can be used, the cells are typically induced for about 12 to 50 hours.

In order to improve the yield and quality of the polypeptide of the present invention, various fermentation conditions can be varied. For example, to improve the proper assembly and folding of the secreted antibody polypeptide, additional vectors overexpressing the accompanying protein can be used to co-transform with host prokaryotic cells such as DsbA (DsbA, DsbB, DsbC, DsbD and / or DsbG) or FkpA (peptidyl amidoxime cis-trans isomerase with concomitant activity). Concomitant proteins have been shown to promote proper folding and lysis of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J Bio Chem 274: 19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275: 17106-17113; Arie et al, (2001) Mol. Microbiol. 39:199-210.

To minimize the proteolysis of the heterologous proteins (especially for proteolytically sensitive), certain host strains lacking proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified to effect gene mutations encoding genes of known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof. Certain E. coli protease deficient strains are available and are described, for example, in Joly et al., (1998), supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2: 63-72 (1996).

In one embodiment, an E. coli strain lacking a proteolytic enzyme and exhibiting a plastid transformation of one or more accompanying proteins is used as a host cell in the expression system of the invention.

Iii. Antibody Purification Standard protein purification methods known in this method can be used. The following procedure is a suitable exemplary purification procedure: fractionation on an immunoaffinity column or ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on vermiculite or cation exchange resin (such as DEAE), chromatographic aggregation (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 the full length antibody product of the invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureas that binds to the antibody Fc region with high affinity. Lindmark et al. (1983) J. Immunol. Meth. 62: 1-13. The solid phase of the immobilized protein A is preferably a column comprising a glass or vermiculite surface, more preferably a controlled microporous glass column or a citrate column. In some applications, the tubing is coated with a reagent such as glycerin in an attempt to prevent non-specific attachment of contaminants.

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

b. Production of antibodies using eukaryotic host cells: Vector components typically include, but are not limited to, one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, a enhancer element, a promoter, and a transcription termination sequence.

(i) Signal Sequence Component The vector used in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The selected heterologous signal sequence is preferably recognized and processed by the host cell (i.e., cleaved by signal peptidase). In mammalian cell expression, mammalian signal sequences can be used as well as viral secretion leader sequences, such as the herpes simplex gD signal.

The DNA of the precursor domain is ligated to the DNA encoding the antibody in the reading frame.

(ii) Origin of replication Generally, no replication origin component is required in the mammalian expression vector. For example, since the SV40 origin contains an early promoter, it is usually only possible to use this starting point.

(iii) Selection of gene components The expression and selection vectors may contain a selection gene, also referred to as a selectable marker. A typical selection gene encodes a protein that satisfies the following conditions: (a) confers resistance to antibiotics and other toxins such as ampicillin, neomycin, methotrexate or tetracycline; (b) supplements if relevant Missing; or (c) critical nutrients that are not available from the complex medium.

An example of a selection procedure utilizes drugs to arrest the growth of host cells. The cells that have successfully transformed with the heterologous gene produce a protein that confers drug resistance and thus survives in the selection protocol. Examples of such dominant choices are the drugs neomycin, mycophenolic acid and hygromycin.

Another example of a selectable marker suitable for use in mammalian cells is one that is capable of recognizing cells capable of absorbing antibody nucleic acids, such as DHFR, thymidine kinase, metallothionein-I and -II (preferably, primate metals) Sulfur protein gene), adenosine deaminase, ornithine decarboxylase, and the like.

For example, cells transformed with a DHFR selection gene are first identified by culturing all of the 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 lacking DHFR activity (e.g., ATCC CRL-9096).

Alternatively, the encoded antibody can be selected by cell growth in a medium containing a selection agent for a selectable marker such as an aglycoside antibiotic such as kanamycin, neomycin or G418 (wild type) The DNA sequence of the DHFR protein) and another selectable marker (such as aminoglycoside 3'-phosphotransferase (APH)) are transformed or co-transformed with host cells (especially wild-type hosts containing endogenous DHFR). See U.S. Patent No. 4,965,199.

(iv) Promoter Components The expression and selection vectors typically contain a promoter that is recognized by the host organism and operably linked to the antibody polypeptide nucleic acid. Promoter sequences of eukaryotes are known. In fact, all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Another sequence found 70 to 80 bases upstream of the transcription initiation point of a plurality of genes is the CNCAAT region, wherein N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which can be a signal that adds a poly A tail to the 3' end of the coding sequence. All of these sequences are suitable for insertion into a eukaryotic expression vector.

For example, by a viral genome (such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, sarcoma virus) (avian sarcoma virus), cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40), from a heterologous mammalian promoter (eg, actin promoter) Or an immunoglobulin promoter, a promoter obtained from a heat shock promoter to control transcription of an antibody polypeptide by a vector in a mammalian host cell, with the proviso that the promoter is compatible with the host cell system.

The early and late promoters of the SV40 virus are conveniently obtained, which are SV40 restriction fragments that also contain the SV40 viral origin of replication. An immediate early promoter of human cytomegalovirus, which is a HindIII E restriction fragment, is conveniently obtained. A system for expressing DNA in a mammalian host using bovine papillomavirus as a vector is disclosed in U.S. Patent No. 4,419,446. Modifications to this system are described in U.S. Patent No. 4,601,978. Alternatively, the long terminal repeat of Rous Sarcoma Virus can be used as a promoter.

(v) Enhancer element assembly The transcription of DNA encoding the antibody polypeptide of the present invention by higher eukaryotes can generally be increased by insertion of the enhancer sequence into the vector. A variety of enhancer sequences from mammalian genes (blood globulin, elastin, albumin, alpha-fetoprotein, and insulin) are known. However, we usually use enhancers from eukaryotic cells. Examples include the SV40 enhancer on the posterior side of the replication origin (100-270 bp), the cytomegalovirus early promoter enhancer, the polyomarous enhancer on the posterior side of the replication origin, and the adenovirus enhancer. See also Yaniv, Nature 297: 17-18 (1982) for enhanced elements for activation of eukaryotic promoters. The enhancer can be spliced to the 5' to 3' position of the sequence encoding the antibody polypeptide in the vector, but it is typically located 5' to the position of the promoter.

(vi) Transcription termination component in a eukaryotic host cell for the expression vector should usually contain termination of transcription and mRNA stabilizing sequences as are necessary. Such sequences are typically obtained from the 5' and sometimes the 3' positions of the untranslated region of eukaryotic or viral DNA or cDNA. The regions contain nucleotide segments transcribed as poly-adenylated fragments of the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors disclosed therein.

(vii) Selection and Transformation of Host Cells Host cells suitable for use in the selection or expression of DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. It has become a routine procedure to propagate vertebrate cells in cultures (tissue cultures). Examples of useful mammalian host cell lines are monkey kidney CV1 strain transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 selected for growth in suspension culture) Cells, 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. USA 77:4216 (1980)); mouse sertoli cell (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cell (CV1 ATCC CCL) 70); African green monkey kidney cell (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo ratl cell (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse breast tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals NYAcad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human liver tumor cell lines (Hep G2).

The host cell is transformed with the above-described expression or selection vector for antibody production and cultured in a suitably modified conventional nutrient medium for inducing a promoter, selecting a transformant or amplifying a gene encoding the desired sequence.

(viii) Culturing Host Cells Host cells for producing the antibodies of the present invention can be cultured in a variety of media. Such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM), Sigma The usual medium is suitable for culturing host cells. Furthermore, any of the media described in the following literature can be used as a medium for host cells: Ham et al, Meth. Enz . 58:44 (1979); Barnes et al, Anal. Biochem. 102:255 (1980); Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469; WO 90/03430; WO 87/00195; or US Patent Overview 30,985. Any of these media may optionally supplement 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 ^TM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or the like Energy efficiency. Any other necessary supplements that will be of a suitable concentration known to those skilled in the art may also be included. Culture conditions, such as temperature, pH, and the like, are previously used by the host cell selected for expression and will be apparent to those of ordinary skill.

(ix) Antibody purification When recombinant techniques are used, antibodies can be produced intracellularly or secreted directly into the culture medium. If the antibody is produced intracellularly as the first step, the host cell or the fragment of the dissolved fragment is usually removed, for example, by centrifugation or ultrafiltration. In the case of secretion of antibodies into the culture medium, a commercially available protein concentration filter (such as an Amicon or Millipore Pellicon ultrafiltration device) is typically first used to concentrate the supernatant from the performance systems. Protease inhibitors (such as PMSF) can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and the isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, J. Immunol. Meth . 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al, EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices may also be used. Mechanically stable matrices such as controlled microporous glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than agarose. Where the antibody comprises a CH3 domain, Bakerbond ABX ^TM resin (JTBaker, Phillipsburg, NJ) is suitable for purification. Depending on the antibody to be recovered, other techniques for protein purification, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, vermiculite chromatography, anion or cation exchange resins (such as polyaspartic amine), may also be used. the heparin SEPHAROSE ^TM chromatography on an acid column), aggregation chromatography, SDS-PAGE, and ammonium sulfate precipitation.

After any primary purification step, the mixture comprising the antibody of interest and the contaminant can be subjected to a dissolution buffer having a pH between about 2.5 and 4.5, preferably at a low salt concentration (e.g., about 0-0.25 M salt). Low pH hydrophobic interaction chromatography performed under).

Immunoconjugate

The invention also provides immunoconjugates (alternatively referred to as "antibody-drug conjugates" or "ADCs") comprising cytotoxic agents (such as chemotherapeutic agents), drugs, growth inhibitors, toxins (eg, bacteria, Any of the anti-OX40L antibodies described herein are bound by an enzymatically active toxin of fungal, plant or animal origin, or a fragment thereof, or a radioisotope (ie, a radioconjugate).

Antibody drug conjugates are used for the local delivery of cytotoxic agents or cytostatic agents (i.e., drugs used to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19: 605-614; Niculescu -Duvaz and Springer (1997) Adv. Dr. Del. Rev. 26: 151-172; U.S. Patent No. 4,975,278)) the use of which allows for the targeted delivery of a drug moiety to a tumor in which intracellular accumulation is performed, wherein Systemic administration of a combination agent can cause unacceptable toxic levels in normal cells as well as tumor cells intended to be eliminated (Baldwin et al., (1986) Lancet, pp. (March 15, 1986): 603-05; Thorpe, ( 1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, "in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506). Therefore, seek maximum utility and minimal toxicity. Multiple strains of antibodies and monoclonal antibodies have been reported for use in such strategies (Rowland et al. (1986) Cancer Immunol. Immunother., 21: 183-87). The drugs used in these methods include daunorubicin, 14-hydroxydaunorubicin, methotrexate and vindesine (Rowland et al. (1986), supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin; phytotoxins such as ricin; small molecule toxins such as geldanamycin (Mandler et al. (2000) Jour. of the Nat. Cancer Inst. 92 (19): 1573-1581; Mandler et al., (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al., (2002) Bioconjugate Chem. 13: 786-791); Medenzin (EP 1391213; Liu et al, (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623) and calicheamicin (Lode et al. (1998) Cancer Res. 58: 2928; Hinman et al. (1993) Cancer Res. 53: 3336-3342). These toxins can affect their cytotoxicity and cell growth inhibition by a mechanism including tubulin binding, DNA binding or topoisomerase inhibition. Certain cytotoxic drugs tend to be inactive or have lower activity when combined with large antibody or protein receptor ligands.

ZEVALIN (Ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope conjugate consisting of murine IgG1 against CD20 antigens found on the surface of normal and malignant B lymphocytes. Monoclonal antibodies and ¹¹¹ In or ⁹⁰ Y radioisotopes bound by a thiourea linker-chelator (Wiseman et al., (2000) Eur. Jour. Nucl. Med. 27(7): 766-77; Wiseman et al. (2002) Blood 99(12): 4336-42; Witzig et al., (2002) J. Clin. Oncol. 20(10): 2453-63; Witzig et al., (2002) J. Clin. Oncol. (15): 3262-69). Although ZEVALIN is active against B-cell non-Hodgkin's lymphoma (NHL), administration causes severe and long-term blood loss in most patients. MYLOTARG ^TM (gemtuzumab (gemtuzumab ozogamicin), Wyeth Pharmaceuticals) is a hu CD33 antibody by the antibody drug doxorubicin constitutes cnidarian connected to a conjugate, which was approved in 2000 for the treatment of acute myeloid leukemia injection (Drugs Of the Future (2000) 25(7): 686; U.S. Patent Nos. 4,970,198, 5,079,233, 5,558,089, 5,560,040, 5,693,762, 5,739,116, 5,767,285, 5,773,001. Cantuzumab mertansine (Immunogen, Inc.) is an antibody drug conjugate consisting of a huC242 antibody linked to the medenoxin drug moiety DM1 via a disulfide linker SPP, which is used in the phase II trial to treat cancers that exhibit CanAg , such as colon cancer, pancreatic cancer, stomach cancer and other cancers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.) is an antibody drug conjugate composed of an anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the Medincinol drug moiety DM1, which has been developed. Used for the potential treatment of prostate tumors. Aristatin peptide (auristatin E (AE)) and monomethyl auristatin (MMAE) (a synthetic analog of dolastatin) and chimeric monoclonal antibody cBR96 (for cancer) Luyi Y has specificity) and cAC10 (specific for CD30 in hematological malignancies) (Doronina et al. (2003) Nature Biotechnology 21(7): 778-784) combines and is used for therapeutic development.

Chemotherapeutic agents suitable for use in generating immunoconjugates are described herein (eg, above). The enzyme-active toxins and fragments thereof can be used, including diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin toxin A chain, and radix erythropoietin ( Modeccin) A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogen (mitogellin), limited sputum ( Restrictocin), phenomycin, enomycin, and trichothecene. See, for example, WO 93/21232, published October 28, 1993. A variety of radionuclides can be used to generate radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re. The combination of an antibody and a cytotoxic agent is prepared using a plurality of bifunctional protein coupling agents such as N-succinimide-3-(2-pyridyldithiol) propionate ( SPDP), iminothiolane pentane (IT), bismuth imidate (such as dimethyl dimethyl imidate hydrochloride), bifunctional derivative, active ester (such as diammonium suberate) Amines), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzylidene) hexamethylenediamine), bis-diazonium derivatives (such as bis-( P-diazonium benzylidene)-ethylenediamine), diisocyanate (such as toluene 2,6-diisocyanate) and bis-active fluoro compound (such as 1,5-difluoro-2,4-dinitrobenzene) . For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science, 238: 1098 (1987). Carbon 14-labeled 1-isothiocyanobenzyl-3-methyldiethylidamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding radioactive nucleotides to antibodies. See WO 94/11026.

Also contemplated herein are combinations of antibodies with one or more small molecule toxins such as echinomycin, maytansinol, dolastatin, auristatin, trichothecenes, and CC1065, as well as toxin activity. Derivatives of such toxins.

i. Maytansine and Medensinol In some embodiments, the immunoconjugate comprises an antibody (full length or fragment) of the invention that binds to one or more mayenoxin molecules.

Medensinol is a mitotic inhibitor that works by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produced maytansinol, such as maytansinol and C-3 maytansinol (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Patent Nos. 4,137,2,304, 248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268. No. 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 No. 4,371,533.

The Medensinol drug moiety is the most interesting part of the drug drug conjugate because it is: (i) relatively easy to obtain by fermentation or chemical modification, derivatization of the fermentation product; (ii) Subject to derivatization by a functional group suitable for binding to the antibody via a non-disulfide linker; (iii) stable in plasma; and (iv) effective against a variety of tumor cell lines.

An immunoconjugate comprising a merinocin, a method for its manufacture, and a therapeutic use thereof are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416, 064, and European Patent No. EP 0 425 235 B1, the disclosures of each of which are incorporated by reference. The manner of this is explicitly incorporated herein. Liu et al, Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996) describe immunoconjugates comprising Metadenol (referred to as DM1) linked to monoclonal antibody C242 against human colorectal cancer. The conjugate was found to be highly cytotoxic to the cultured colon cancer cells and it exhibited anti-tumor activity in an in vivo tumor growth assay. Chari et al, Cancer Research 52: 127-131 (1992) describe immunoconjugates in which the Mycensinocin binds to a murine antibody A7 that binds to an antigen of a human colon cancer cell line via a disulfide linker, or Another murine monoclonal antibody TA.1 binds to the HER-2/neu oncogene. The cytotoxicity of the TA.1-Medensin conjugate was tested in vitro in human breast cancer cell line SK-BR-3, which showed 3 x 10 ⁵ HER-2 surface antigen per cell. The drug conjugate achieves a degree of cytotoxicity similar to that of the free medenoxin drug, which can be increased by increasing the number of mayonnino molecules per antibody molecule. The A7-Medensinol conjugate exhibited low systemic cytotoxicity in mice.

The antibody-Medensinol conjugate can be prepared by chemically linking the antibody to the Mycensino molecule without significantly reducing the biological activity of the antibody or the Mycensino molecule. For example, see U.S. Patent No. 5,208,020, the disclosure of which is expressly incorporated herein by reference. On average, binding of 3-4 Mydenoxin molecules per antibody molecule has demonstrated an effect of enhancing the cytotoxicity of the targeted cells without adversely affecting antibody function or solubility, although even one molecule of toxin/antibody is expected to be enhanced. Cytotoxicity (better than using pure antibodies). Medensinol is well known in the art and can be synthesized by known techniques or isolated from natural sources. A suitable US Patent No. 5,208,020 and other patents and non-patent publications mentioned above are hereby incorporated by reference. Preferably, Medensinol is a maytansinol and a modified maytansinoid analog (such as various maytansinol) in the aromatic ring or at other positions of the maytansinoid molecule.

A variety of linkers for the manufacture of antibody-Medensinol conjugates are known in the art, including those disclosed in, for example, U.S. Patent No. 5,208,020; or EP Patent No. 0 425 235 B1; Chari et al. Cancer Research 52: 127-131 (1992); and U.S. Patent Application Serial No. 10/960,602, the disclosure of which is incorporated herein by reference. The antibody-Medensinol conjugate comprising the linker component SMCC can be prepared as disclosed in U.S. Patent Application Serial No. 10/960,602, issued toK. The linker includes a disulfide group, a thioester group, an acid labile group, a photolabile group, a peptidase labile group, or an esterase labile group, as disclosed in the above identified patents, Disulfide and thioester groups are preferred. Additional linkers have been described and illustrated herein.

The combination of an antibody and mayenoxin can be prepared using a variety of bifunctional protein coupling agents such as N-succinimide-3-(2-pyridyldithio)propionate. (SPDP), amber quinone imido-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), imidothioindole cyclopentane (IT), hydrazine Bifunctional derivatives of imidates (such as dimethyl dimethyl imidate hydrochloride), active esters (such as perylene dimerate), aldehydes (such as glutaraldehyde), double- An azido compound (such as bis-(p-azidobenzylidene) hexamethylenediamine), a bis-diazonium salt derivative (such as bis-(p-diazonium benzylidene)-ethylenediamine), Diisocyanate (such as toluene 2,6-diisocyanate) and bis-active fluorine compound (such as 1,5-difluoro-2,4-dinitrobenzene). A preferred coupling agent includes N-succinimide-3-(2-pyridyldithio)propionate (SPDP) for providing a disulfide bond (Carlsson et al., Biochem. J. 173:723). -737 (1978)) and 4-(2-pyridylthio)pentanoic acid N-succinimide (SPP).

Depending on the type of attachment, the linker can be attached to each position of the Mindensinol molecule. For example, ester linkages can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction can occur at the C-3 position with a hydroxyl group, at the C-14 position modified with a hydroxymethyl group, at the C-15 position modified with a hydroxy group, and at the C-20 position having a hydroxy group. In a preferred embodiment, the linkage is formed at the C-3 position of the maytansinol or maytansinol analog.

Ii. Auristatin and Dolastatin In some embodiments, the immunoconjugate comprises an antibody of the invention that binds to a peptide analog of a dolastatin or a rabbit toxin and a derivative of auristatin (U.S. Patent No. 5,563, 482, No. 5780588). Naptoxin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have resistance Cancer (US5663149) and antifungal activity (Pettit et al, (1998) Antimicrob. Agents Chemother. 42: 2961-2965). The dolastatin or auristatin drug moiety can be attached to the antibody via the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety (WO 02/088172).

An exemplary auristatin embodiment comprises a N-terminal auristatin drug moiety DE and DF linked to the N-terminus, disclosed in "Monomethylvaline Compounds Capable of Conjugation to Ligands", published November 5, 2004, US Serial Number 10/983,340, the disclosure of which is expressly incorporated herein by reference in its entirety.

Generally, a peptide-based drug moiety can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can, for example, be based on liquid phase synthesis methods well known in the art of peptide chemistry (see E. Schr Der and KL Bke, "The Peptides", Vol. 1, pp. 76-136, 1965, Academic Press). The auristatin/conne toxin drug moiety can be prepared according to the following literature: US 5,635,483; US 5,780,588; Pettit et al., (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13: 243-277; Pettit, GR et al, Synthesis, 1996, 719-725; and Pettit et al, (1996) J. Chem. Soc. Perkin Trans. 15: 859-863 . See also Doronina (2003) Nat Biotechnol 21(7): 778-784; "Monomethylvaline Compounds Capable of Conjugation to Ligands", published November 5, 2004, U.S. Serial No. 10/983,340, which is incorporated by reference in its entirety. The manner of incorporation herein (discloses, for example, linkers and methods for preparing monomethylproline compounds such as MMAE and MMAF in combination with a linker).

Iii. Gynostocin In other embodiments, the immunoconjugate comprises an antibody of the invention that binds to one or more echinomycin molecules. The echinomycin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of the echinomycin family conjugates, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296 (both assigned) American Cyanamid Company). Structural analogs of echinomycin that may be used include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-ethylindolyl-γ ₁ ^I , PSAG, and θ ^I ₁ (Hinman et al., Cancer Research 53: 3336-3342 (1993); Lode et al, Cancer Research 58: 2925-2928 (1998); and the above-mentioned U.S. patent to American Cyanamid). Another anti-tumor drug that can bind to antibodies is QFA, which is an anti-folate. Both echinomycin and QFA have intracellular sites of action and are not susceptible to crossing the plasma membrane. Thus, uptake of such agents via antibody-mediated internalization significantly enhances their cytotoxic effects.

Iv. Other cytotoxic agents Other anti-tumor agents that can bind to the antibodies of the invention include BCNU, streptozoticin, vincristine, and 5-fluorouracil (described in U.S. Patent Nos. 5,053,394, 5,770,710). A family of agents collectively referred to as the LL-E33288 complex, and esperamicin (U.S. Patent 5,877,296).

Enzyme-active toxins and fragments thereof which can be used include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, Acacia toxin A chain, and radix aurantiaxin A Chain, bacteriocin, tung tree protein, carnation protein, P. sinensis (PAPI, PAPII and PAP-S), bitter melon inhibitor, jatropha, crotonin, geranium inhibitor, paradise fruit protein, Mitogen, fentanin, phenolic mycomycin, INOmycin, and mycotoxins. See, for example, WO 93/21232, published October 28, 1993.

The invention further encompasses immunoconjugates formed between an antibody and a compound having nuclear cleavage activity (e.g., ribonuclease or endonuclease, such as deoxyribonuclease; DNase).

To selectively destroy a tumor, the antibody can comprise highly radioactive atoms. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu. When a conjugate is used for detection, it may comprise: a radioactive atom for scintigraphic studies, such as Tc ^99m or I ¹²³ ; or for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, Spin labeling of mri), such as iodine-123 and iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, cesium, manganese or iron.

Radioactive labels or other labels can be incorporated into the conjugate in a known manner. For example, the peptide can be biosynthesized, or the peptide can be synthesized by chemical amino acid synthesis using a suitable amino acid precursor (eg, involving the replacement of hydrogen with a fluorine-19 amino acid). A label such as Tc ^99m or I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ and In ^{111 can} be attached to the peptide via a cysteine residue. The 钇-90 can be attached via an amine acid residue. The IODOGEN method (Fraker et al., (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

Combinations of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents such as N-succinimide-3-(2-pyridyldithio)propionate ( SPDP), amber quinone imido-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), imidothioindole cyclopentane (IT), 醯亚Bifunctional derivatives of amine esters (such as dimethyl dimethyl imidate hydrochloride), active esters (such as dinonyl succinimide), aldehydes (such as glutaraldehyde), bis-azide Base compound (such as bis-(p-azidobenzylidene) hexamethylenediamine), bis-diazonium salt derivative (such as bis-(p-diazonium benzylidene)-ethylenediamine), diisocyanate (such as toluene 2,6-diisocyanate) and a double active fluorine compound (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described by Vitetta et al., Science, 238: 1098 (1987). Carbon 14-labeled 1-isothiocyanobenzyl-3-methyldiethylidamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding radioactive nucleotides to antibodies. See WO 94/11026. A linker can be a "cleavable linker" that promotes the release of cytotoxic drugs in a cell. For example, an acid labile linker, a peptidase sensitive linker, a photolabile linker, a dimethyl linker or a disulfide containing linker can be used (Chari et al., Cancer Research 52: 127-131). (1992); U.S. Patent No. 5,208,020).

The compounds of the invention expressly encompass, but are not limited to, ADCs prepared using the following crosslinker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfonic acid Base-EMCS, sulfonate-GMBS, sulfonate-KMUS, sulfonate-MBS, sulfonate-SIAB, sulfonate-SMCC and sulfonate-SMPB and SVSB (amber quinone imine-(4) - Vinyl hydrazide) benzoate), such crosslinker reagents are commercially available (for example, from Pierce Biotechnology, Inc., Rockford, IL., USA). See 2003-2004 Applications Handbook and Catalog, pp. 467-498.

v. Preparation of Antibody Drug Conjugates In the antibody drug conjugate (ADC) of the present invention, the antibody (Ab) binds to one or more drug moieties (D) via a linker (L), for example, about 1 to each antibody. About 20 drug parts. The ADC 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) reacting a nucleophilic group of an antibody with a divalent linker reagent. Forming Ab-L via a covalent bond, followed by reaction with drug moiety D; and (2) reacting a nucleophilic group of the drug moiety with a divalent linker reagent to form D-L via a covalent bond, followed by antibody Nucleophilic group reaction. Additional methods for preparing ADCs have been described herein.

Ab-(L-D) _p I

A linker can be composed of one or more linker components. Exemplary linker components include 6-maleimido hexamethylene ("MC"), maleimide propyl ketone ("MP"), lysine- citrulline ("val- Cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), 4-(2-pyridylthio)pentanoic acid N-succinimide ("SPP"), 4-(N-maleimidomethyl)cyclohexane-1carboxylic acid N-succinimide ("SMCC") and (4-iodo-ethenyl)amine N-Amber succinimide ("SIAB"). Additional linker components are known in the art and some are described herein. See also "Monomethylvaline Compounds Capable of Conjugation to Ligands", published November 5, 2004, U.S. Serial No. 10/983,340, the disclosure of which is incorporated herein in its entirety.

In some embodiments, the linker can comprise an amino acid residue. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides or pentapeptides. Exemplary tripeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-proline-glycine (gly-val-cit) and glycine-glycine-glycine. Amino acid residues comprising an amino acid linker component include those naturally occurring, as well as a small number of amino acids and non-naturally occurring amino acid analogs such as citrulline. The amino acid linker component can be designed and optimized for selectivity to enzymatic cleavage by specific enzymes (e.g., tumor associated proteases, cathepsins B, C and D or fibrinolytic enzymes).

Nucleophilic groups on the antibody include, but are not limited to, (i) an N-terminal amine group; (ii) a side chain amine group, such as an amine acid; (iii) a side chain thiol group, such as cysteine And (iv) a saccharide hydroxyl or amine group in which the antibody is glycosylated. The amine group, thiol group and hydroxyl group are nucleophilic groups and are capable of reacting to form a covalent bond with the linker moiety and the electrophilic group of the linker reagent, including: (i) an active ester such as NHS Esters, HOBt esters, haloformates and acid halides; (ii) alkyl and benzyl halides such as haloacetamide; (iii) aldehydes, ketones, carboxyl groups and maleimine groups. Certain antibodies have a reducible intrachain disulfide, a cysteine bridge. The antibody can be reacted with a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Thus, in theory each cysteine bridge will form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody via reaction of the amine acid with 2-iminothiolane (Traut's reagent) to convert the amine to a thiol. By introducing one, two, three, four or more cysteine residues (for example, preparing a mutant antibody comprising one or more non-natural cysteine amino acid residues) A reactive thiol group is introduced into the antibody (or a fragment thereof).

The antibody drug conjugates of the invention can also be produced by modifying an antibody to introduce an electrophilic moiety that can react with a nucleophilic substituent on a linker reagent or drug. The sugar of the glycosylated antibody can be oxidized by, for example, a periodate oxidizing reagent to form an aldehyde or ketone group reactive with the linker reagent or the amine group of the drug moiety. The resulting imine Schiff base group can form a stable bond or can be reduced, for example, by a borohydride reagent to form a stable amine bond. In one embodiment, the reaction of the carbohydrate moiety of the glycosylated antibody with galactose oxidase or sodium metaperiodate provides a carbonyl (aldehyde and ketone) in the protein that reacts with a suitable group of the drug (Hermanson, Bioconjugate Techniques). In another embodiment, a protein containing an N-terminal serine acid or a threonine residue can be reacted with sodium metaperiodate to produce an aldehyde that replaces the first amino acid (Geoghegan and Stroh, (1992) Bioconjugate Chem. 3: 138-146; US5362852). Such aldehydes can be reacted with a drug moiety or a linker nucleophile.

Likewise, the nucleophilic groups of the drug moiety include, but are not limited to, amines, thiols, hydroxyls, hydrazines, hydrazines, hydrazines, thiosemicarbazones, bismuth carboxylates, and aryl sulfonium groups, which are capable of The reaction forms a covalent bond with the electrophilic group of the linker moiety and the linker reagent, and the electrophile comprises: (i) an active ester such as an NHS ester, a HOBt ester, a haloformate, and an acid halide; (ii) an alkyl group and a benzyl halide such as haloacetamide; (iii) an aldehyde, a ketone, a carboxyl group and a maleimine group.

Alternatively, fusion proteins comprising antibodies and cytotoxic agents can be made, for example, by recombinant techniques or peptide synthesis. The length of the DNA may comprise individual regions encoding two portions of the conjugate, the two portions of the conjugate being adjacent to each other or separated by regions encoding the linker peptide that do not impair the desired properties of the conjugate.

In yet another embodiment, the antibody can be combined with a "receptor" (such as streptavidin) for pretargeting the tumor, wherein the antibody-receptor conjugate is administered to the patient, followed by self-circulation using the scavenger The unbound conjugate is removed, and then a "coordinating group" (eg, avidin) that binds to a cytotoxic agent (eg, a radioactive nucleotide) is administered.

Medical formulation

Preparation of an aqueous solution by mixing an antibody of the desired purity with a physiologically acceptable optional carrier, excipient or stabilizer ( Remington's: The Science and Practice of Pharmacy , 20th Ed., ed. (2000)) A therapeutic formulation comprising an antibody of the invention in the form of a lyophilized or other dry formulation for storage. At the dosages and concentrations employed, acceptable carriers, excipients or stabilizers are non-toxic to the receptor and include buffers such as phosphates, citrates, histidines and other organic acids; antioxidants, including Ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexahydroxy quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl Alcohol; alkyl p-hydroxybenzoate, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol; low molecular weight (less than about 10 residues) polypeptide; protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acid, such as glycine, glutamic acid, day Aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol , trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (eg Zn) Protein complexes); and / or non-ionic surfactant, such as TWEEN ^TM, PLURONICS ^TM or polyethylene glycol (PEG).

Formulations herein may also contain more than one active compound as needed for the particular indication being treated, preferably those having complementary effects that do not adversely affect each other. These molecules are suitable for being present in the combination in an amount effective to achieve the intended purpose.

The active ingredient may also be entrapped in a gelled drug delivery system (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions (eg, by coacervation techniques or interfacial polymerization). The microcapsules (for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively). Such techniques are disclosed in Remington: The Science and Practice of Pharmacy, 20th Edition (2000).

Formulations to be administered in vivo must be sterile. This is easily accomplished by filtration through a sterile filter.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices containing solid hydrophobic polymers of the immunoglobulins of the invention, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), L- Copolymer of glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer (such as LUPRON DEPOT ^TM (lighted by lactic acid-glycolic acid copolymer and acetic acid) Injectable microspheres composed of pirarin) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable the release of molecules for more than 100 days, some hydrogels release proteins in a relatively short period of time. When encapsulated immunoglobulins remain in the body for extended periods of time, they can denature or agglomerate upon exposure to moisture at 37 °C, resulting in loss of biological activity and possible alteration of immunogenicity. Depending on the mechanism involved, a reasonable strategy can be designed with regard to stability. For example, if the agglutination mechanism is found to form an intermolecular S-S bond via a thio-disulfide interchange, the thiol residue can be modified, lyophilized from an acidic solution, moisture content controlled, and appropriate additives used. A specific polymer matrix composition is produced to achieve stability.

use

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

In one aspect, the invention provides methods for treating or preventing a tumor, cancer, and/or cell proliferative disorder associated with increased performance and/or activity of EphB4, the method comprising administering to a patient in need of such treatment An effective amount of an anti-EphB4 antibody.

In one aspect, the invention provides a method for reducing, inhibiting or preventing tumor or cancer growth, the method comprising administering to a patient in need of such treatment an effective amount of an anti-EphB4 antibody.

EphB4 is involved in motor axon guidance and neural ridge cell migration in embryonic development. Thus, the antibodies of the invention are also useful in the treatment (including prevention) of conditions involving pathology involving cell degeneration or dysfunction, such as treatment of various (chronic) neurodegenerative disorders and acute neuronal damage. Such neurodegenerative disorders include, but are not limited to, peripheral neuropathy; motor neuron disorders such as amyloidosis lateral sclerosis (ALS, Lug Grieg), Bell's palsy, and various disorders involving spinal muscular atrophy or paralysis Conditions; and other human neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's disease, Down's syndrome, neurological deafness, and Meniere's disease And acute neuronal damage caused by, for example, trauma or spinal cord injury.

The antibodies of the invention are also useful for inhibiting angiogenesis. In some embodiments, the site of angiogenesis is a tumor or cancer.

In one aspect, the invention provides a method for inhibiting angiogenesis comprising administering to a patient in need of such treatment an effective amount of an anti-EphB2 antibody.

In one aspect, the invention provides a method for treating a pathological condition associated with angiogenesis comprising administering to a patient in need of such treatment an effective amount of an anti-EphB2 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 disorder.

Furthermore, at least some of the antibodies of the invention may bind antigens from other species. Thus, the antibodies of the invention can be used in, for example, cell cultures containing antigens, in human patients, or in other mammalian patients having antigens that cross-react with antibodies of the invention (e.g., orangutans, baboons, simians, macaques, and Binding specific antigen activity in rhesus, pig or mouse). In one embodiment, the antibody can be used to inhibit antigenic activity by contacting the antibody of the invention with an antigen to inhibit antigenic activity. Preferably, the antigen is a human protein molecule.

In one embodiment, an antibody of the invention is for use in a method of binding an antigen in a patient suffering from a condition associated with increased antigenic expression and/or activity, the method comprising administering to the patient an antibody of the invention, thereby binding to the patient antigen. Preferably, the antigen is a human protein molecule and the patient is a human patient. Alternatively, the patient can be a mammal that exhibits an antigen that binds to an antibody of the invention. Also, the patient can be a mammal that has been introduced (e.g., by administering an antigen or by expressing an antigen-transforming gene) antigen. The antibodies of the invention can be administered to a human patient for therapeutic purposes. Furthermore, an antibody of the invention may be administered to a non-human mammal (eg, a primate, pig or mouse) that exhibits an antigen that cross-reacts with an immunoglobulin for veterinary purposes or as an animal of human disease. model. In the latter case, such animal models can be used to analyze the therapeutic utility of the antibodies of the invention (e.g., test dosing amount and time course).

The antibodies of the invention are useful for treating, inhibiting, delaying the progression of a disease, disorder or condition associated with the performance and/or activity of one or more antigenic molecules, preventing/delaying the recurrence, amelioration or prevention of such diseases, disorders or conditions. , illness or condition.

Exemplary conditions include cancer, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma. , including gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrium or Uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, genital cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma, brain and head and neck cancer and related metastasis . In some embodiments, the cancer line is selected from the group consisting of small cell lung cancer, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma.

In certain embodiments, an immunoconjugate comprising an antibody that binds to one or more cytotoxic agents is administered to a patient. In some embodiments, the immunoconjugate and/or the antigen bound thereto are internalized by the cell, causing an increase in the therapeutic utility of the immunoconjugate to kill the targeted cell to which it binds. In one embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the targeted cell. In one embodiment, the cytotoxic agent targets or interferes with microtubule polymerization. Examples of such cytotoxic agents include any of the chemotherapeutic agents noted herein (e.g., maytansinol, auristatin, dolastatin or calicheamicin), radioisotopes or ribonucleases or DNA endonucleases.

The antibodies of the invention may be used in therapy 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 inhibitors. If the antibody of the invention inhibits tumor growth, it may in particular be desirable to combine it with one or more other therapeutic agents that also inhibit tumor growth. Additionally or alternatively, the patient may receive combined radiation therapy (e.g., external radiation or treatment with a radiolabeled agent such as an antibody). The combination therapies noted above include combination administration (in which two or more agents are included in the same or separate formulations) and administration alone, and administration of the antibody of the present invention may be administered adjuvant therapy in the case of administration alone. Occurs before and / or after.

Combination Therapy As indicated above, the present invention provides combination therapies in which an anti-EphB4 antibody is administered with another therapy. For example, an anti-EphB4 antibody is used in combination with an anti-cancer therapeutic or anti-angiogenic therapeutic to treat various neoplastic or non-neoplastic conditions. In one embodiment, the neoplastic or non-neoplastic condition is characterized by a pathological condition associated with abnormal or poor angiogenesis. The anti-EphB4 antibody can be administered in the same composition or as a separate composition, either continuously or in combination with another therapeutic agent effective to achieve the same. Additionally or alternatively, a variety of EphB4 inhibitors can be administered.

Administration of the anti-EphB4 antibody (for example) as a single composition or as two or more distinct compositions can be accomplished simultaneously using the same or different routes of administration. Alternatively or additionally, the administration may be completed in succession in any order. In certain embodiments, there may be a time interval ranging from a few minutes to a few days to weeks to months between administrations of two or more compositions. For example, an anticancer agent can be administered first, followed by an EphB4 inhibitor. However, it is also contemplated to administer the drug simultaneously or first to the anti-EphB4 antibody.

The effective amount of the therapeutic agent administered in combination with the anti-EphB4 antibody will depend on the judgment of the physician or veterinarian. Dosing is administered and adjusted to achieve the maximum treatment of the condition to be treated. In addition, the dosage will depend on such factors as the type of therapeutic agent to be used and the particular patient to be treated. Suitable doses of the anticancer agent are those currently used and may be lowered by the combined action (synergy) of the anticancer agent and the anti-EphB4 antibody. In certain embodiments, the combination of inhibitors enhances the efficacy of a single inhibitor. The term "boosting" refers to an improvement in the efficacy of a therapeutic agent that is used in a conventional or approved dose. See also the section entitled Pharmaceutical Compositions herein.

In general, anti-EphB4 antibodies and anticancer agents are suitable for the same or similar diseases to block or reduce pathological conditions such as tumor growth or cancer cell growth. In one embodiment, the anticancer agent is an anti-angiogenic agent.

Anti-angiogenic therapies for cancer are aimed at inhibiting cancer treatment strategies that provide nutrients to support tumor vascular development required for tumor growth. Since angiogenesis is involved in both primary tumor growth and metastasis, the anti-angiogenic therapy provided by the present invention can inhibit tumor neoplastic growth at a primary site and prevent tumor metastasis at a secondary site, thus allowing Other therapeutic agents attack the tumor.

Many anti-angiogenic agents have been identified and are known in the art, including those anti-angiogenic agents listed herein, such as those listed in the definitions section and, for example, Carmeliet and Jain, Nature 407:249-257 ( 2000); Ferrara et al, Nature Reviews: Drug Discovery, 3: 391-400 (2004); and Sato Int. J. Clin. Oncol., 8: 200-206 (2003) listed for their anti-angiogenesis Agent. See also U.S. Patent Application No. US 20030055006. In one embodiment, a fragment that includes, but is not limited to, a soluble VEGF receptor (eg, VEGFR-1, VEGFR-2, VEGFR-3, neuropilin (eg, NRP1, NRP2)) can be blocked VEGF or VEGFR, aptamers that neutralize anti-VEGFR antibodies, low molecular weight inhibitors of VEGFR tyrosine kinase (RTK), antisense strategies for VEGF, ribonuclease against VEGF or VEGF receptors, antagonist variants of VEGF An anti-EPHB4 antibody is used in combination with an anti-VEGF neutralizing antibody (or fragment) thereof and/or another VEGF antagonist or VEGF receptor antagonist. In addition or in addition to the VEGF antagonist and other agents, two or more angiogenesis inhibitors may be administered to the patient as appropriate. In one embodiment, one or more additional therapeutic agents, such as an anticancer agent, can be administered in combination with an anti-EphB4 antibody, a VEGF antagonist, and an anti-angiogenic agent.

In certain aspects of the invention, other therapeutic agents that can be used in combination with anti-EphB4 antibodies for tumor therapy include other cancer therapies (eg, surgery, radiation therapy (eg, involving irradiation or administration of radioactive materials), chemotherapy, Anticancer agents or combinations thereof as listed herein and known in the art). Additionally or alternatively, two or more antibodies that bind to the same antigen or two or more different antigens disclosed herein can be co-administered to the patient. Sometimes it may be beneficial to also administer one or more cytokines to the patient.

Chemotherapeutic Agents In certain aspects, the present invention provides an effective amount of an EphB4 antagonist and/or angiogenesis inhibitor and one or more chemotherapeutic agents administered to a patient susceptible to or diagnosed with cancer. A method of blocking or reducing tumor growth or cancer cell growth. A variety of chemotherapeutic agents can be used in the combination therapies of the invention. In this document, the "Definitions" section provides an illustrative and non-limiting list of chemotherapeutic agents covered.

It will be understood by those skilled in the art that the appropriate dosage of the chemotherapeutic agent will generally be about the doses that have been used in the clinical therapies in which the chemotherapeutic agents are 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 dose for the individual patient.

The invention also provides methods and compositions for inhibiting or preventing the growth of recurrent tumors or the growth of recurrent cancer cells. Recurrent tumor growth or recurrence cancer cell lines are used to describe where the patient experiences one or more currently available therapies (eg, cancer therapy, eg, chemotherapy, radiation therapy, surgery, hormone therapy, and/or biological therapy/immunotherapy, anti-VEGF) Antibody therapy, especially for standard treatment regimens for specific cancers, or treatment with it is not clinically sufficient to treat patients or patients no longer benefit from the therapy, thus making such patients in need of additional effective therapies. As used herein, the phrase may also refer to a condition of a "non-reactive/refractory" patient, for example, a patient who is responsive to therapy but has side effects, a patient who develops resistance, and a patient who does not respond to therapy. Patients who do not respond satisfactorily to therapy. In various embodiments, the cancer is a recurring tumor growth or recurring cancer cell growth in which the number of cancer cells has not decreased significantly or has increased or the tumor size has not decreased significantly or has increased or the size or number of cancer cells is no longer further reduced. Use of any of the "relapsed" or "refractory" or "non-reactive" meanings accepted in the art in the context, by any method known in the art for determining the effectiveness of cancer cell treatment The cancer cells can be determined in vivo or in vitro for recurrence of tumor growth or recurrence of cancer cell growth. Anti-VEGF treated resistant tumors are examples of recurrent tumor growth.

The present invention provides a method of blocking or reducing the growth of recurrent tumors or recurring cancer cells in a patient by administering one or more anti-EphB4 antibodies to block or reduce the growth of recurrent tumors or relapsed cancer cells in a patient. In certain embodiments, the antagonist can be administered after the cancer therapeutic. In certain embodiments, an anti-EphB4 antibody is administered concurrently with a cancer therapy. Additionally or alternatively, the anti-EphB4 antibody therapy alternates with another cancer therapy, which can be performed in any order. The invention also encompasses methods of administering one or more inhibitory antibodies to prevent cancer onset or relapse in a patient who is predisposed to having cancer. In general, patients have received or are receiving cancer therapy. In one embodiment, the cancer therapy is treatment with an anti-angiogenic agent (eg, a VEGF antagonist). Anti-angiogenic agents include those known in the art as anti-angiogenic agents and their anti-angiogenic agents found in the definitions herein. In one embodiment, the anti-angiogenic agent is an anti-VEGF neutralizing antibody or fragment (eg, humanized A4.6.1, AVASTIN) (Genentech, South San Francisco, CA), Y0317, M4, G6, B20, 2C3, etc.). For example, see U.S. Patent Nos. 6,582,959, 6,884,879, 6, 703, 020, WO 98/45332, WO 96/30046, WO 94/10202, EP 0 666 868 B1, U.S. Patent Application Nos. 20030206899, 20030190317, 20030203409, and 20050112126; Popkov et al., Journal of Immunological Methods 288: 149-164 (2004); and WO 2005012359. Additional agents can be administered in combination with VEGF antagonists and anti-EphB4 antibodies to block or reduce recurrence of tumor growth or recurrence of cancer cell growth, for example, see the section entitled Combination Therapy herein.

Recurrent Tumor Growth The present invention also provides methods and compositions for inhibiting or preventing recurring tumor growth or recurring cancer cell growth. Recurrent tumor growth or recurrence cancer cell lines are used to describe a patient undergoing one or more currently available therapies (eg, cancer therapy, eg, chemotherapy, radiation therapy, surgery, hormone therapy, and/or biological therapy/immunotherapy, anti-VEGF antibodies) Therapies, especially for standard treatments for specific cancers, or treatments thereof are not clinically sufficient to treat patients or patients no longer benefit from the therapy, thus making such patients in need of additional effective therapies. As used herein, the phrase may also refer to a condition of a "non-reactive/refractory" patient, for example, a patient who is responsive to therapy but has side effects, a patient who develops resistance, and a patient who does not respond to therapy. Patients who do not respond satisfactorily to therapy. In various embodiments, the cancer is a recurring tumor growth or recurring cancer cell growth in which the number of cancer cells has not decreased significantly or has increased or the tumor size has not decreased significantly or has increased or the size or number of cancer cells is no longer further reduced. Use of any of the "relapsed" or "refractory" or "non-reactive" meanings accepted in the art in the context, by any method known in the art for determining the effectiveness of cancer cell treatment The cancer cells can be determined in vivo or in vitro for recurrence of tumor growth or recurrence of cancer cell growth. Anti-VEGF treated resistant tumors are examples of recurrent tumor growth.

The present invention provides a method of blocking or reducing the growth of recurrent tumors or recurring cancer cells in a patient by administering one or more anti-EphB4 antibodies to block or reduce the growth of recurrent tumors or relapsed cancer cells in a patient. In certain embodiments, the antagonist can be administered after the cancer therapeutic. In certain embodiments, an anti-EphB4 antibody is administered concurrently with a cancer therapy. Additionally or alternatively, the anti-EphB4 antibody therapy alternates with another cancer therapy, which can be performed in any order. The invention also encompasses methods of administering one or more inhibitory antibodies to prevent cancer onset or relapse in a patient who is predisposed to having cancer. In general, patients have received or are receiving cancer therapy. In one embodiment, the cancer therapy is treatment with an anti-angiogenic agent (eg, a VEGF antagonist). Anti-angiogenic agents include those known in the art as anti-angiogenic agents and their anti-angiogenic agents found in the definitions herein. In one embodiment, the anti-angiogenic agent is an anti-VEGF neutralizing antibody or fragment (eg, humanized A4.6.1, AVASTIN) (Genentech, South San Francisco, CA), Y0317, M4, G6, B20, 2C3, etc.). For example, see U.S. Patent Nos. 6,582,959, 6,884,879, 6, 703, 020, WO 98/45332, WO 96/30046, WO 94/10202, EP 0 666 868 B1, U.S. Patent Application Nos. 20030206899, 20030190317, 20030203409, and 20050112126; Popkov et al., Journal of Immunological Methods 288: 149-164 (2004); and WO 2005012359. Additional agents can be administered in combination with VEGF antagonists and anti-EphB4 antibodies to block or reduce recurrence of tumor growth or recurrence of cancer cell growth, for example, see the section entitled Combination Therapy herein.

The antibodies (and therapeutic adjuvants) of the invention may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and if necessary for topical treatment, may be administered intralesionally . Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Furthermore, antibodies are also suitable for administration by pulse infusion, in particular administration of antibodies in decreasing doses. Partially depending on the short-term or long-term nature of the administration, it can be administered by any suitable route (for example, by injection, such as intravenous or subcutaneous injection).

The antibody compositions of the invention should be formulated, administered and administered in a manner consistent with good pharmaceutical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiology of the condition, the delivery site of the agent, the method of administration, the time course of administration, and the medical practitioner's known Other factors. The antibody is not required but may be formulated with one or more agents currently used to prevent or treat the condition in question, as appropriate. The effective amount of such other agents will depend on the amount of the antibody of the invention present in the formulation, the type of disorder or treatment, and other factors discussed above. Such agents are usually administered in the same dosages and routes of administration as described above, or from about 1 to 99% of the doses hitherto used.

For the prevention or treatment of a disease, the appropriate dose of the antibody of the invention (when used alone or in combination with other agents such as chemotherapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and duration of the disease, whether or not Administration of antibodies, prior therapies, clinical history of the patient, response to antibodies, and judgment of the attending physician for prophylactic or therapeutic purposes. The antibody is suitable for administration to a patient once or via a series of treatments. Depending on the type and severity of the disease, antibodies from about 1 μg/kg to 15 mg/kg (eg, 0.1 mg/kg to 10 mg/kg) are, for example, administered by one or more separate doses or by continuous The initial candidate dose is administered to the patient upon infusion. A typical daily dose may range from about 1 μg/kg to 100 mg/kg or higher, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment will usually be continued until the desired inhibitory effect on the symptoms of the disease occurs. An exemplary dosage of one of the antibodies will range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to a patient. Such doses can be administered intermittently, such as weekly or every three weeks (e.g., such that the patient receives about two doses to about twenty doses (e.g., about six doses) of antibody). A higher initial loading dose can be administered followed by one or more lower doses. An exemplary dosing regimen comprises administering an antibody at an initial loading dose of about 4 mg/kg followed by administration of a weekly maintenance dose of about 2 mg/kg of antibody. However, other dosage regimens are also available. The advancement of this therapy is that it is easy to monitor by conventional techniques and assays.

The anti-EphB4 antibodies of the invention can be used in assays for detecting EphB4 expression in a particular cell or tissue (e.g., in a diagnostic or prognostic assay) wherein the antibody is labeled and/or immobilized on an insoluble substrate as described below.

In another aspect, the invention provides a method for detecting EphB4, the method comprising detecting an EphB4-anti-EphB4 antibody complex in a sample. The term "detecting" as used herein includes qualitative and/or quantitative detection (measurement content) with or without reference to a control.

In another aspect, the invention provides a method for diagnosing a condition associated with EphB4 expression and/or activity, the method comprising detecting EphB4- in a biological sample from a patient having or suspected of having the condition Anti-EphB4 antibody complex. In some embodiments, EphB4 exhibits increased performance or abnormal (bad) performance. In some embodiments, the condition is a tumor, cancer, and/or a cell proliferative disorder.

In another aspect, the invention provides any of the anti-EphB4 antibodies described herein, wherein the anti-EphB4 antibody comprises a detectable label.

In another aspect, the invention provides a complex of any of the anti-EphB4 antibodies and EphB4 described herein. In some embodiments, the complex is in vivo or in vitro. In some embodiments, the complex comprises cancer cells. In some embodiments, the anti-EphB4 anti-system is detectably labeled.

Anti-EphB4 antibodies can be used to detect EphB4 in any of several well known detection assays. For example, a sample is obtained from a desired source, the sample is mixed with an anti-EphB4 antibody to form an antibody/EphB4 complex with any EphB4 present in the mixture, and any antibody/EphB4 complex present in the mixture is detected. In this way, EphB4 of biological samples can be assayed. Biological samples for assays can be prepared by methods known in the art for use in a particular sample. The method of mixing the sample with the antibody and the method of detecting the antibody/EphB4 complex are selected depending on the type of assay used. Such assays include immunohistochemistry, competitive and sandwich assays, and stereosuppression assays.

EphB4 assays used one or more of the following reagents: labeled EphB4 analog, immobilized EphB4 analog, labeled anti-EphB4 antibody, immobilized anti-EphB4 antibody, and stereoconjugate. Labeled reagents are also known as "tracers."

The label used is any detectable functional group that does not interfere with the binding of EphB4 to an anti-EphB4 antibody. Various labels for immunoassays are known, examples include directly detectable moieties such as chromophores, chemiluminescent agents, and radioactive labels, as well as moieties such as enzymes that must be reacted or derivatized for detection. Examples of such labels include: radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I, fluorophores (such as rare earth metal chelators or luciferins and derivatives thereof), rhodamine and Its derivatives, dansyl, ketone, luciferase (such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456)), luciferin, 2,3-dihydrogen Pyridazinone, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, sugar oxidase (eg glucose oxidase, galactose oxidase and glucose) -6-phosphate dehydrogenase), heterocyclic oxidase (such as uricase and porphyrin oxidation) coupled with an enzyme that oxidizes a dye precursor (such as HRP, lactoperoxidase or microperoxidase) with hydrogen peroxide Enzymes), biotin/avidin, spin labeling, phage labeling, stable free radicals and the like.

These labels can be covalently bound to a protein or polypeptide using conventional methods. For example, a coupling agent such as dialdehyde, carbodiimide, dimaleimide, bis-imide, bis-diazoniumbenzidine, and the like can be used to label the above-mentioned fluorescent light, Chemiluminescent and enzymatically labeled antibodies. See, for example, U.S. Patent Nos. 3,940,475 (fluorescence assay) and 3,645,090 (enzyme); 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. Binding of the marker (including the enzyme) to the antibody is a standard procedure for those skilled in the art of immunoassay. See, for example, Methods in Enzymology, JJ Langone and H. Van Vunakis, eds., Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166, O'Sullivan et al., "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay".

Some assay methods require a curing reagent. Curing requires isolation of any anti-EphB4 antibody from any EphB4 remaining in solution. Typically, the anti-EphB4 antibody or EphB4 analog is insoluble (eg, by adsorption to a water insoluble matrix or surface (Bennich et al., US 3,720,760) by covalent covalent coupling (eg, using pentane) prior to the assay procedure. The dialdehyde crosslinks)) or by subsequent insolubilization of the anti-EphB4 antibody or EphB4 analog (for example by immunoprecipitation).

Immunohistochemistry and staining protocols can be used to examine the performance of the protein in the sample. Immunohistochemical staining of tissue sections has been shown to be a reliable method for analyzing or detecting the presence of proteins in a sample. Immunohistochemistry ("IHC") techniques typically detect and visualize cellular antigens in situ using antibodies based on chromogenic or fluorescent methods. For sample preparation, tissue or cell samples from mammals (usually human patients) can be used. Examples of samples include, but are not limited to, cancer cells such as colon, breast, prostate, ovary, lung, stomach, pancreas, lymphoma, and leukemia cancer cells. Samples may be obtained by various procedures known in the art including, but not limited to, surgical resection, inhalation, or biopsy. The tissue can be fresh or frozen. In one embodiment, the sample is fixed and embedded in paraffin or the like. Tissue samples can be fixed (i.e., preserved) by conventional methods. Those of ordinary skill in the art will appreciate that the choice of fixative will be determined by histological staining or analysis of the sample. It is also generally understood by those skilled in the art that the fixed length will depend on the size of the tissue sample and the fixative used.

IHC can be performed in combination with additional techniques such as morphological staining and/or fluorescence in situ hybridization. Two general methods of IHC are available; direct and indirect assays. The binding of the antibody to a targeting antigen (e.g., EphB4) is determined directly according to the first assay. This direct assay uses a labeling reagent such as a fluorescent label or an enzymatically labeled primary antibody that can be visualized without further antibody interaction. In a typical indirect assay, an unbound primary antibody binds to an antigen and the labeled secondary antibody then binds to the primary antibody. If the secondary antibody binds to the enzymatic label, a chromogenic or fluorescent matrix is added to provide antigenic visualization. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody.

Primary and/or secondary antibodies for immunohistochemistry should generally be labeled with a detectable moiety. Various markers can be used, which can generally be classified into the following categories: In addition to the sample preparation procedures discussed above, tissue sections may need to be further processed before, during, or after IHC. For example, an epitope-determining method can be performed, such as heating a tissue sample in a citrate buffer (see, for example, Leong et al, Appl. Immunohistochem. 4(3): 201 (1996)).

After an optional blocking step, the tissue sections are exposed to the primary antibody for a sufficient period of time under appropriate conditions such that the primary antibody binds to the targeted protein antigen in the tissue sample. Suitable conditions for achieving this can be determined by routine experimentation. The extent to which the antibody binds to the sample should be determined by using any of the detectable labels discussed above. Preferably, the label is an enzymatic label (e.g., HRPO) that catalyzes the chemical alteration of a chromonic substrate such as a 3,3'-diaminobenzidine chromophore. The enzymatic label preferably binds to an antibody that specifically binds to the primary antibody (eg, the primary antibody is a rabbit polyclonal antibody and the secondary antibody is a goat anti-rabbit antibody).

The specimen thus prepared can be mounted and covered with a cover slip. Next, the slide analysis is determined, for example, using a microscope, and staining intensity standards conventionally used in the art can be used. The staining intensity criteria can be analyzed as follows:

A staining pattern score of about 2+ or higher in the IHC assay is usually diagnostic and/or prognostic. In some embodiments, a staining pattern of about 1+ or higher is scored as diagnostic and/or prognostic. In other embodiments, a staining pattern of about 3 or higher is scored as diagnostic and/or prognostic. It will be appreciated that when IHC is used to examine cells and/or tissues from tumor or colon adenomas, staining is typically determined or analyzed in tumor cells and/or tissues (as compared to the matrix or surrounding tissue that may be present in the sample).

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

The competitive assay relies on the ability of the tracer EphB4 analog to compete with the test sample EphB4 for a limited number of anti-EphB4 antibody-antigen binding sites. The anti-EphB4 antibody is typically insoluble before or after competition, followed by isolation from the unconjugated tracer and EphB4 to the anti-EphB4 antibody tracer and EphB4. This separation is achieved by decantation (wherein the binding partner is not dissolved in advance) or by centrifugation (where the binding partner is precipitated after a competitive reaction). The amount of test sample EphB4 is inversely proportional to the amount of bound tracer as measured by the amount of labeled material. A dose response curve using a known amount of EphB4 was prepared and compared to the test results to quantitatively determine the amount of EphB4 present in the test sample. When an enzyme is used as a detectable label, such assays are referred to as ELISA systems.

Another competitive assay called the "homologous" assay does not require phase separation. Therefore, a combination of the enzyme and EphB4 was prepared and used such that when the anti-EphB4 antibody binds to EphB4, the presence of the anti-EphB4 antibody changes the enzyme activity. In this case, EphB4 or an immunologically active fragment thereof is bound to an enzyme such as a peroxidase by a bifunctional organic bridge. The conjugate is selected for use with an anti-EphB4 antibody such that binding to the anti-EphB4 antibody inhibits or enhances the enzymatic activity of the label. This method is itself widely practiced under the name EMIT.

Stereoconjugates are used in steric methods for homologous assays. These conjugates are synthesized by covalently linking a low molecular weight hapten to a small EphB4 fragment such that the antibody against the hapten is substantially incapable of binding to the conjugate simultaneously with the anti-EphB4 antibody. In this assay, EphB4 present in the test sample will bind to the anti-EphB4 antibody, thereby allowing the anti-hapten to bind to the conjugate, resulting in a change in the characteristics of the conjugate hapten, such as when the hapten is a fluorophore. Light changes.

Sandwich assays are especially useful for assaying EphB4 or anti-EphB4 antibodies. In a continuous sandwich assay, the immobilized anti-EphB4 antibody is used to adsorb the test sample EphB4, the test sample is removed by washing, and the bound EphB4 is used to adsorb the secondary, labeled anti-EphB4 antibody, and then self-residual The bound material is separated in the tracer. The amount of bound tracer is directly proportional to the test sample EphB4. In the "simultaneous" sandwich assay, the test sample was not separated prior to the addition of labeled anti-EphB4. A continuous sandwich assay using an anti-EphB4 monoclonal antibody as one antibody and multiple anti-EphB4 antibodies as another antibody can be used to test EphB4 in a sample.

The foregoing is only an exemplary detection test for EphB4. Other methods of determining EphB4 using anti-EphB4 antibodies, which will be developed today or thereafter, are included in their scope, including the bioassays described herein.

Manufacturing goods

In another aspect of the invention, an article of manufacture comprising a material useful for treating, preventing, and/or diagnosing a condition described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition, alone or in combination with other compositions effective to treat, prevent, and/or diagnose the condition, and may have a sterile access port (eg, the container may be an intravenous solution bag or have a stopper pierceable by a hypodermic needle) The vial). At least one active agent in the composition is an antibody of the invention. The marker or package insert indicates that the composition is used to treat a condition selected, such as cancer. Further, the article of manufacture may comprise: (a) a first container comprising the composition, wherein the composition comprises an antibody of the invention; and (b) a second container comprising the composition, wherein the composition comprises another cytotoxicity Agent. In this embodiment of the invention, the article of manufacture may additionally comprise a package insert that should indicate that the first and second compositions are useful for treating a particular condition, such as cancer. Additionally or alternatively, the article of manufacture may additionally comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer Ringer's solution and dextrose solution. It may additionally include other materials that are 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 will be appreciated that various other embodiments may be implemented, given the general description provided above.

Instance Example 1: Production of anti-EphB4 antibody

Phage display was used and EphB4-His protein was used to select phage to generate anti-EphB4 antibodies. A variety of methods for generating phage display libraries are known in the art, and antibodies of interest can be obtained from such libraries. One method of producing an antibody of interest is the use of a phage antibody library as described in Lee et al, J. Mol. Biol. (2004), 340(5): 1073-93. Phage display was used to screen the selected pure lines using phage ELISA relative to EphB4-His protein. The unique pure line was selected for further characterization by phage competition ELISA and blocking assay to determine if the phage antibody line could block the EphB4-ephrinB2 interaction. Pure line 30.35 is preferably performed and selected for further analysis. To improve the affinity of pure lineage 30.35, phage display libraries were generated in the YW30.35 background, each targeting a selected HVR for soft or hard randomization. Selected pure lines were screened by phage ELISA and then visualized as Fab proteins and their affinity was determined using Biacore. The selected pure lines were recombined into full length IgG and their affinity was determined using Biacore. The sequence of the parent pure line 30.35 and the affinity matured line is shown in Figure 1.

Example 2: Characterization of anti-EphB4 antibodies

To determine the binding affinity of an anti-EphB4 Mab, using the surface plasmon resonance ^{(SRP) BIAcore TM -3000 (BIAcore} , Inc., Piscataway, NJ) to obtain the measurements. Briefly, according to the supplier's instructions, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide ( NHS) Activated carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc.). Anti-EphB4 antibody was diluted to 5 μg/ml with 10 mM sodium acetate (pH 4.8), followed by injection at a flow rate of 5 microliters per minute to achieve approximately 500 reaction units (RU) of conjugated antibody. Next, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, human or murine EphB4-His molecules (0.7 nM to 500 nM) were injected in two-fold serial dilutions in PBS with 0.05% Tween 20 at 25 μl/min flow rate at 25 °C. . The association rate ( _kon ) and dissociation rate ( _koff ) were calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software, version 3.2). The equilibrium dissociation constant (Kd) was calculated as k _off / k _on the ratio. The results of this test are shown in Figure 3. "NA" indicates that the measurement was not performed.

The cross-reactivity of the anti-EphB4 antibody line 30.35 was tested relative to other EphB receptors in different assays. Briefly, COS7 cells were transiently transfected with plastids expressing full length human EphB1, human EphB2, human EphB4 or human EphB6. 24 hours after transfection, cells were subjected to FACS analysis to detect possible binding by anti-EphB4 antibodies. The anti-EphB4 pure line 30.35 does not cross-react with human EphB1, human EphB2, human EphB3 or human EphB6.

Example 3: EphB4 receptor signaling blocked by anti-EphB4 antibodies in cell-based assays

To demonstrate the ability of anti-EphB4 antibodies to block membrane-bound interactions between 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 EphB4 and low EphrinB2, and the ability of anti-EphB4 antibodies to inhibit EphB4 activation was tested.

3T3 cells overexpressing human EphrinB2 were prepared as follows: Human full length EphrinB2 was colonized in pcDNA5/FRT vector (Invitrogen) according to the manufacturer's manual and subsequently used to generate stable cell lines with 3T3.Flp cells (Invitrogen).

3T3 cells overexpressing human EphrinB2 were overlaid on HUVEC cells for 15 or 30 minutes in the presence or absence of anti-EphB4 antibodies. EphB4 protein was immunoprecipitated, followed by anti-phospho- tyrosine antibody-(antibody 4G10; Upstate), using western dot method to detect the presence or absence of receptor tyrosine phosphorylation, thereby analyzing EphB4 receptor activation. Briefly, cells were lysed with RIPA buffer. Cell lysates were sorted by centrifugation and anti-EphB4 antibody 35.2D8 was added at 5 μg per sample. After incubation for two hours at 4 ° C, the immune complex was resolved using Protein A Sepharose. EphB4 phosphorylation was analyzed by Western blotting using an anti-phosphonium tyrosine antibody 4G10 (Upstate) at a concentration of 1 μg/ml.

The results of this test are shown in Figure 7. Overlay of 3T3 cells on HUVEC cells causes a dramatic tyrosine phosphorylation of EphB4 (bands 4 and 5). HUVECs were pre-incubated with anti-EphB4 antibody (pure line 35.2D8, 5 μg/ml) for 30 minutes to effectively eliminate EphB4 tyrosine phosphorylation induced by overlying 3T3-EphrinB2 cells (ribbon 6). In contrast, HUVEC cells treated with anti-EphB4 alone did not cause EphB4 activation in HUVEC (ribbons 2 and 3), and untreated HUVEC cells did not demonstrate EphB4 activation. These results establish that anti-EphB4 antibodies block ligand-receptor interactions in direct cell-cell contact content.

Although the foregoing invention has been described in some detail by way of illustration and example, the description and examples should not be construed as limiting the scope of the invention.

Figure 1: Heavy and light chain HVR loop sequences of anti-EphB4 antibodies. The figure shows heavy chain HVR sequences H1, H2 and H3 and light chain HVR sequences L1, L2 and L3. The sequence number is as follows: pure line 30.35 (HVR-H1 is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 3; HVR-H3 is SEQ ID NO: 7; HVR-L1 is SEQ ID NO: 9; HVR- L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 13); pure line 30.35.1D2 (HVR-H1 is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 3; HVR-H3 is SEQ ID NO:8; HVR-L1 is SEQ ID NO: 10; HVR-L2 is SEQ ID NO: 12; HVR-L3 is SEQ ID NO: 14); pure line 30.35.2D8 (HVR-H1 is SEQ ID NO: 2; HVR-H2 is SEQ ID NO: 4; HVR-H3 is SEQ ID NO: 7; HVR-L1 is SEQ ID NO: 9; HVR-L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 15) Pure line 30.35.2D12 (HVR-H1 is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 5; HVR-H3 is SEQ ID NO: 7; HVR-L1 is SEQ ID NO: 9; HVR-L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 16); and pure line 30.35.2D13 (HVR-H1 is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 6; HVR-H3 is SEQ ID NO: :7; HVR-L1 is SEQ ID NO:9; HVR-L2 is SEQ ID NO:11; HVR-L3 is SEQ ID NO:17).

The amino acid positions are numbered according to the Kabat numbering system as described below.

2A, 2B and 3 depict exemplary receptor human consensus framework sequences for practicing the invention, wherein the sequence identifiers are as follows: variable heavy chain (VH) consensus framework (Fig. 2) human VH subgroup I consensus framework minus Kabat CDR (SEQ ID NO: 19) human VH subgroup I consensus framework minus prolonged hypervariable regions (SEQ ID NO: 20-22) human VH subpopulation II consensus framework minus Kabat CDRs (SEQ ID NO: 23) human VH subgroup II consensus framework minus prolonged hypervariable regions (SEQ ID NO: 24-26) human VH subpopulation III consensus framework minus Kabat CDRs (SEQ ID NO: 27) human VH subpopulation III consensus framework minus prolonged Variable region (SEQ ID NO: 28-30) human VH receptor framework minus Kabat CDR (SEQ ID NO: 31) human VH receptor framework minus prolonged hypervariable region (SEQ ID NO: 32-33) human VH receptor The framework of the body 2 minus the Kabat CDR (SEQ ID NO: 34) human VH receptor 2 framework minus the prolonged hypervariable region (SEQ ID NO: 35-37)

Variable Light Chain (VL) Consistent Framework (Figure 3) Human VL Subgroup I Consistent Framework (SEQ ID NO: 38) Human VL Subgroup II Consistent Framework (SEQ ID NO: 39) Human VL Subgroup III Consistent Framework (SEQ ID NO: 40) Human VL Subgroup IV Consistent Framework (SEQ ID NO: 41)

Figure 4 depicts the framework regions of the huMAb4D5-8 light and heavy chains. The superscript/bold numbers indicate the position of the amino acid according to Kabat.

Figure 5 depicts the modified/variant framework region sequences of the huMAb4D5-8 light and heavy chains. The superscript/bold numbers indicate the position of the amino acid according to Kabat.

Figure 6 depicts the heavy chain variable region and the light chain variable region of the antibody pure lines 30.35, 30.35.1D2, 30.35.2D8, 30.35.2D12 and 20.25.2D13.

Figure 7: EphB4 receptor signaling blocked by anti-EphB4 monoclonal antibody in a cell-based assay.

<110> US-based Nandek <120> anti-EPHB4 antibody and method of using the same <130> P2300R1 <140> 0961-00340 <141> 2007-01-04 <150> US 60/756,889 <151> 2006-01-05 <150> US 60/760,892 <151> 2006-01-20 <160> 62 <210> 1 <211> 10 <212> PRT <213> Artificial sequence <220><223> HVR-H1 <400> 1 <210> 2 <211> 10 <212> PRT <213> Artificial sequence <220><223> HVR-H1 <400> 2 <210> 3 <211> 18 <212> PRT <213> Artificial sequence <220><223> HVR-H2 <400> 3 <210> 4 <211> 18 <212> PRT <213> Artificial sequence <220><223> HVR-H2 <400> 4 <210> 5 <211> 18 <212> PRT <213> Manual sequence <220><223> HVR-H2 <400> 5 <210> 6 <211> 18 <212> PRT <213> Artificial sequence <220><223> HVR-H2 <400> 6 <210> 7 <211> 17 <212> PRT <213> Manual sequence <220><223> HVR-H3 <400> 7 <210> 8 <211> 17 <212> PRT <213> Manual sequence <220><223> HVR-H3 <400> 8 <210> 9 <211> 11 <212> PRT <213> Manual sequence <220><223> HVR-L1 <400> 9 <210> 10 <211> 11 <212> PRT <213> Manual sequence <220><223> HVR-L1 <400> 10 <210> 11 <211> 7 <212> PRT <213> Manual sequence <220><223> HVR-L2 <400> 11 <210> 12 <211> 7 <212> PRT <213> Manual sequence <220><223> HVR-L2 <400> 12 <210> 13 <211> 9 <212> PRT <213> Manual sequence <220><223> HVR-L3 <400> 13 <210> 14 <211> 9 <212> PRT <213> Manual sequence <220><223> HVR-L3 <400> 14 <210> 15 <211> 9 <212> PRT <213> Manual sequence <220><223> HVR-L3 <400> 15 <210> 16 <211> 9 <212> PRT <213> Manual sequence <220><223> HVR-L3 <400> 16 <210> 17 <211> 9 <212> PRT <213> Manual sequence <220><223> HVR-L3 <400> 17 <210> 18 <211> 107 <212> PRT <213> Artificial sequence <220><223> Light chain variable domain of huMab4D5-8 <400> 18 <210> 19 <211> 87 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 19 <210> 20 <211> 81 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 20 <210> 21 <211> 80 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 21 <210> 22 <211> 79 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 22 <210> 23 <211> 87 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 23 <210> 24 <211> 81 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 24 <210> 25 <211> 80 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 25 <210> 26 <211> 79 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 26 <210> 27 <211> 87 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 27 <210> 28 <211> 81 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 28 <210> 29 <211> 80 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 29 <210> 30 <211> 79 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 30 <210> 31 <211> 87 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 31 <210> 32 <211> 81 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 32 <210> 33 <211> 80 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 33 <210> 34 <211> 87 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 34 <210> 35 <211> 81 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 35 <210> 36 <211> 80 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 36 <210> 37 <211> 79 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 37 <210> 38 <211> 80 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 38 <210> 39 <211> 80 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 39 <210> 40 <211> 80 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 40 <210> 41 <211> 80 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 41 <210> 42 <211> 23 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 42 <210> 43 <211> 15 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 43 <210> 44 <211> 32 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 44 <210> 45 <211> 10 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 45 <210> 46 <211> 25 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 46 <210> 47 <211> 13 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 47 <210> 48 <211> 30 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 48 <210> 49 <211> 11 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 49 <210> 50 <211> 32 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 50 <210> 51 <211> 30 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 51 <210> 52 <211> 107 <212>PRT<213> Artificial sequence <220><223> Sequence is synthesized <400> 52 <210> 53 <211> 128 <212> PRT <213> Artificial sequence <220><223> Heavy chain variable region of Mab30.35 <400> 53 <210> 54 <211> 108 <212> PRT <213> Artificial sequence <220><223> Light chain variable region of Mab30.35 <400> 54 <210> 55 <211> 128 <212> PRT <213> Artificial sequence <220><223> Heavy chain variable region of Mab30.35.1D2 <400> 55 <210> 56 <211> 108 <212> PRT <213> Artificial sequence <220><223> Light chain variable region of Mab30.35.1D2 <400> 56 <210> 57 <211> 128 <212> PRT <213> Artificial sequence <220><223> Heavy chain variable region of Mab30.35.2D8 <400> 57 <210> 58 <211> 108 <212> PRT <213> Artificial sequence <220><223> Light chain variable region of Mab30.35.2D8 <400> 58 <210> 59 <211> 128 <212> PRT <213> Artificial sequence <220><223> Heavy chain variable region of Mab30.35.2D12 <400> 59 <210> 60 <211> 108 <212> PRT <213> Artificial sequence <220><223> Light chain variable region of Mab30.35.2D12 <400> 60 <210> 61 <211> 128 <212> PRT <213> Artificial sequence <220><223> Heavy chain variable region of Mab30.35.2D13 <400> 61 <210> 62 <211> 108 <212> PRT <213> Artificial sequence <220><223> Light chain variable region of Mab30.35.2D13 <400> 62

Claims (40)

An isolated anti-EphB4 antibody comprising six HVRs, wherein SEQ ID NO: 9 or 10 corresponds to HVR-L1, SEQ ID NO: 11 or 12 corresponds to HVR-L2, SEQ ID NO: 13, 14, 15 16 or 17 corresponds to HVR-L3, SEQ ID NO: 1 or 2 corresponds to HVR-H1, SEQ ID NO: 3, 4, 5 or 6 corresponds to HVR-H2 and SEQ ID NO: 7 or 8 corresponds to HVR -H3. The antibody of claim 1, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each of which comprises SEQ ID NO: 9, 11, 13, 1 , 3 and 7. The antibody of claim 1, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each of which comprises SEQ ID NO: 10, 12, 14, 1 in sequence. , 3 and 8. The antibody of claim 1, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each of which comprises SEQ ID NO: 9, 11, 15, 2 in sequence. 4 and 7. The antibody of claim 1, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each of which comprises SEQ ID NO: 9, 11, 16, 1 , 5 and 7. The antibody of claim 1, wherein the antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each of which comprises SEQ ID NO: 9, 11, 17, 1 , 6 and 7. The antibody of any one of claims 1 to 6, wherein at least a portion of the framework sequence is a human consensus framework sequence. The antibody of any one of claims 1 to 6, wherein the antibody comprises a human kappa subgroup consensus framework sequence. The antibody of any one of claims 1 to 6, wherein the antibody comprises a heavy chain human subgroup III consensus framework sequence. The antibody of claim 9, wherein the antibody comprises a substitution at one or more of positions 71, 73 or 78 within the heavy chain human subgroup III consensus framework sequence, wherein the substitution at position 71 is R71A, the position 73 Substituted as N73T, and the substitution at position 78 is N78A. The antibody of claim 10, wherein the substitution is one or more of R71A, N73T or N78A. A polynucleotide encoding the antibody of any one of claims 1 to 11. A vector comprising the polynucleotide of claim 12. The vector of claim 13, wherein the vector is a performance vector. A host cell comprising the vector of claim 13 or 14. The host cell of claim 15, wherein the host cell is a prokaryotic cell. The host cell of claim 15, wherein the host cell is a eukaryotic cell. The host cell of claim 15, wherein the host cell is a mammalian cell. A method for making an anti-EphB4 antibody and/or immunoconjugate, the method comprising: (a) expressing a vector of claim 14 in a suitable host cell; and (b) recovering the antibody. The method of claim 19, wherein the host cell is a prokaryotic cell. The method of claim 19, wherein the host cell is a eukaryotic cell. A method for detecting EphB4, which is included in a biological sample The EphB4-anti-EphB4 antibody complex is detected, wherein the anti-EphB4 antibody is the antibody of any one of claims 1 to 11. A method for diagnosing a condition associated with EphB4 expression, the method comprising detecting an EphB4-anti-EphB4 antibody complex in a biological sample from a patient having or suspected of having the condition, wherein the anti-EphB4 antibody is derived from The antibody of any one of items 1 to 11. The method of any one of claims 22 to 23, wherein the anti-EphB4 anti-system is detectably labeled. A composition comprising the anti-EphB4 antibody of any one of claims 1 to 11. A composition comprising the polynucleotide of claim 12. The composition of claim 25 or 26, wherein the composition additionally comprises a carrier. Use of an anti-EphB4 antibody according to any one of claims 1 to 11 for the preparation of a medicament for inhibiting angiogenesis in a patient in need of inhibition of angiogenesis. The use of claim 28, wherein the agent is used in combination with an anti-angiogenic agent. The use of claim 29, wherein the anti-angiogenic agent is administered before or after administration of the anti-EphB4 antibody. The use of claim 29, wherein the anti-angiogenic agent is administered concurrently with the anti-EphB4 antibody. The use of any one of claims 29 to 31, wherein the anti-angiogenic agent is an antagonist of vascular endothelial growth factor (VEGF). The use of claim 32, wherein the VEGF antagonist is an anti-VEGF antibody. The use of claim 33, wherein the anti-VEGF antibody is bevacizumab. The use of any one of claims 28 to 31, wherein the agent is used in combination with a chemotherapeutic agent. Use of an anti-EphB4 antibody according to any one of claims 1 to 11 for the preparation of a medicament for the therapeutic and/or prophylactic treatment of a condition, wherein the condition is a cancer, tumor or cell proliferative disorder. Use of an anti-EphB4 antibody according to any one of claims 1 to 11 for the preparation of a medicament for the therapeutic and/or prophylactic treatment of a condition, wherein the condition is a neuropathy or a neurodegenerative disease. Use of an anti-EphB4 antibody according to any one of claims 1 to 11 for the preparation of a medicament for the therapeutic and/or prophylactic treatment of a condition, wherein the condition is a pathological condition associated with angiogenesis. The use of claim 38, wherein the pathological condition associated with angiogenesis is a tumor, a cancer, and/or a cell proliferative disorder. The use of claim 38, wherein the pathological condition associated with angiogenesis is intraocular neovascular disease.