RU2415869C2 - Dll4 antibodies and methods of application thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents an airra-DLL4-antibody, a coding polynucleotide, a vector, a host cell, a method for producing an antibody, compositions containing such antibodies, and methods of application thereof.

EFFECT: invention can be used effectively for treatment or prevention of pathological conditions associated with DLL4 expression or activity.

27 cl, 59 dwg, 3 tbl, 14 ex

Description

FIELD OF THE INVENTION

In General terms, the present invention relates to the field of molecular biology. More specifically, the present invention relates to anti-DLL4 antibodies and their use.

State of the art

Providing blood supply is a basic need for many physiological and pathological processes. For active tissue growth, such as embryos and tumors, adequate blood supply is needed. This blood supply is ensured by the production of pro-angiogenic factors that stimulate the formation of new blood vessels in a process called angiogenesis. Vascular formation is a complex but systematic biological process that includes all or many of the following stages: a) proliferation of endothelial cells (EC) from existing ECs or their differentiation from progenitor cells; b) EC migration and coalescence to form rope-like structures; c) tubulogenesis of vascular cords with the formation of vessels with a central lumen; d) proliferation of existing cords or vessels with the formation of secondary vessels; e) subsequent remodeling and the formation of new forms from the primary vascular plexus; and f) recruiting periendothelial cells to the endothelial vessels, which provides these vessels with supporting and modulatory functions, where the indicated cells are pericytes for small capillaries, smooth muscle cells for larger vessels, and myocardial cells in the heart. Hanahan, Science 277: 48-50 (1997); Hogan & Kolodziej, Nat. Rev. Genet. 3: 513-23 (2002); Lubarsky & Krasnow, Cell 112: 19-28 (2003).

It is now well known that angiogenesis is involved in the pathogenesis of various diseases. Such diseases are solid tumors and metastases, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular diseases such as proliferative retinopathies, such as diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, and immune tissue , rheumatoid arthritis and psoriasis. Folkman et al., J. Biol. Chem. 267: 10931-34 (1992); Klagsbrun et al., Annu. Rev. Physiol. 53: 217-39 (1991); and Garner A., “Vascular diseases,” In: Pathobiology of Ocular Disease. A Dynamic Approach, Garner A., Klintworth GK, eds., 2nd Edition (Marcel Dekker, NY, 1994), pp. 1625-1710.

In the case of tumor growth, angiogenesis obviously plays a decisive role in the transition from hyperplasia to neoplasia and in ensuring the supply of nutrients necessary for tumor growth and the formation of metastases. Folkman et al., Nature 339: 58 (1989). Neovascularization promotes the predominant growth of tumor cells and their acquisition of proliferative autonomy compared to normal cells. Tumor development usually begins with one aberrant cell, which can proliferate only to a size of several cubic millimeters, which is due to a certain distance from the available capillary bed, and such a cell may remain “dormant”, that is, it may not undergo further growth and dissemination during long period. Then, some tumor cells acquire an angiogenic phenotype with activation of endothelial cells, which, after proliferation and maturation, form new capillary blood vessels. These newly formed vessels not only contribute to the continued growth of the primary tumor, but also the dissemination and recolonization of metastatic tumor cells. In accordance with this, there is a correlation between the density of microvessels in tumor sections and the survival rate of patients with breast cancer and some other tumors. Weidner et al., N. Engl. J. Med. 324: 1-6 (1991); Horak et al., Lancet 340: 1120-24 (1992); Macchiarini et al., Lancet 340: 145-46 (1992). The exact mechanisms governing the switching of angiogenesis have not yet been established, although it is likely that neovascularization of the tumor mass is the result of a common ratio of many stimulants and inhibitors of angiogenesis (Folkman, Nat. Med. 1 (1): 27-31 (1995)).

The process of vascular development is strictly regulated. It is now known that a large number of molecules, mainly secreted factors produced by surrounding cells, regulate the differentiation, proliferation, migration and coalescence of the EU with the formation of rope-like structures. For example, vascular endothelial growth factor (VEGF) has been identified as a key factor involved in stimulating angiogenesis and in inducing vascular permeability. Ferrara et al., Endocr. Rev. 18: 4-25 (1997). The discovery of the fact that the loss of even one VEGF allele leads to embryo death indicates the indispensable role that this factor plays in the development and differentiation of the vascular system. In addition, VEGF has been shown to be a key mediator of neovascularization associated with the development of tumor and intraocular diseases. Ferrara et al., Endocr. Rev. see above. VEGF mRNA is overexpressed by most of the studied human tumors. 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).

In addition, VEGF concentrations in the intraocular fluid are significantly correlated with active proliferation of blood vessels in patients with diabetic retinopathy and other ischemia-associated retinopathy. Aiello et al., N. Engl. J. Med. 331: 1480-87 (1994). In addition, studies have been conducted that have identified VEGF localization in choroidal neovascular membranes in patients with AMD. Lopez et al., Invest. Ophthalmol. Vis Sci. 37: 855-68 (1996).

Neutralizing anti-VEGF 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); Borgstrum et al., Cancer Res. 56: 4032-39 (1996); Melnyk et al., Cancer Res. 56: 921-24 (1996)) also inhibit intraocular angiogenesis in models of ischemic retinal diseases (Adamis et al., Arch. Ophthalmol. 114: 66-71 (1996)). Therefore, monoclonal anti-VEGF antibodies or other inhibitors of the action of VEGF are promising candidates for their use in the treatment of tumor and various intraocular neovascular diseases. Such antibodies are described, for example, in EP 817648, published January 14, 1998; in applications WO 98/45331 and WO 98/45332 published on October 15, 1998. One of the anti-VEGF antibodies, bevacizumab, was approved by the Food and Drug Administration for the use of the drug in combination with chemotherapy for the treatment of specific cancers, a fragment of the anti-VEGF antibody, ranibizumab, has been approved by the FDA for the treatment of age-related (wet) macular degeneration. Both of these drugs are currently undergoing clinical trials.

It is obvious that the need to obtain agents that have clinical signs that are optimal for the development of therapeutic agents remains relevant. The invention described in this application satisfies this requirement and has other advantages.

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

Description of the invention

The present invention is based in part on the identification of various DLL4 binding agents (such as immunoconjugates, antibodies, and fragments thereof). DLL4 is an important and preferred therapeutic target, and the present invention relates to compositions and methods based on binding to DLL4. The DLL4 binding agents described herein according to the invention are important therapeutic and diagnostic agents that can be used to treat pathological conditions associated with the expression and / or activity of the Notch DLL4 receptor reaction cascade. Accordingly, the present invention relates to methods, compositions, kits, and industrial products associated with DLL4 binding.

The present invention relates to antibodies that bind (e.g., specifically bind) to DLL4.

In one aspect, the present invention provides an isolated anti-DLL4 antibody, wherein the full-length IgG antibody form specifically binds to human DLL4 with a binding affinity of about 1 nM or more or about 500 pM or more. As is well known to specialists, the affinity of binding of a ligand to its receptor can be determined using various assays and expressed in various quantitative quantities. Accordingly, in one embodiment of the invention, the binding affinity is expressed by Kd values and represents the natural binding affinity (for example, with minimal avidity effects). Typically and preferably, binding affinity is measured in vitro o in a cell-free or cell medium. To measure the binding affinity, any of the known assays can be used, including the assays described here, for example Biacore®, radioimmunoassay (RIA) and ELISA. In some embodiments of the invention, the isolated anti-DLL4 antibody binds to human and murine DLL4 with similar affinity, that is, the binding affinity of the human DLL4 exceeds the binding affinity of the murine DLL4 by no more than 100 times or less. In some embodiments of the invention, the binding affinity for human DLL4 exceeds the binding affinity for murine DLL4 by no more than 10 times or less. In some embodiments of the invention, the antibody specifically binds to murine DLL4 with a binding affinity of about 1 nM or more, or about 500 pM or more.

In one aspect, the present invention provides an isolated anti-DLL4 antibody, wherein the full-length IgG form of the antibody specifically binds to human DLL4 with k _{on of} about 2 × 10 ⁵ or more or about 1 × 10 ⁵ or more. As is well known to those skilled in the art, k _on binding of a ligand to its receptor can be determined using various assays and expressed in various quantitative quantities.

In one aspect, the present invention provides an isolated antibody that binds to the ligand binding region of DLL4. In some embodiments of the invention, the isolated antibody binds to a polypeptide comprising the extracellular domain of DLL4 or consisting of, or essentially consisting of, this domain. In some embodiments of the invention, the isolated antibody binds to a polypeptide comprising, consisting of, or essentially consisting of, amino acids 252-282, 1-252, 1-286, 1-324 and / or 219-286 of a human DLL4.

In one aspect, the present invention provides an isolated anti-DLL4 antibody that competes with the Notch receptor for binding to DLL4.

In one of its aspects, the present invention relates to an isolated anti-DLL4 antibody that inhibits, reduces and / or blocks the biological activity of DLL4.

In one aspect of the invention, an anti-DLL4 antibody of the invention includes:

(a) at least one, two, three, four, five, or six sequences of hypervariable regions (HVR) selected from the group consisting of:

(i) an HVR-L1 comprising the sequence A1-A11, wherein A1-A11 is RASQDVSTAVA (SEQ ID NO: 10);

(ii) an HVR-L2 containing the sequence B1-B7, where B1-B7 is SASFLYS (SEQ ID NO: 11);

(iii) an HVR-L3 containing the sequence C1-C9, wherein C1-C9 is QQSYNGPST (SEQ ID NO: 15);

(iv) an HVR-H1 comprising the sequence D1-D10, wherein D1-D10 is GFTFTDNWIS (SEQ ID NO: 1);

(v) an HVR-H2 containing the sequence E1-E18, where E1-E18 is GVINPNSGATEYADSVKG (SEQ ID NO: 5) and

(vi) an HVR-H3 comprising the sequence F1-F15, wherein F1-F15 is VYYCARDNFGGYFDY (SEQ ID NO: 9); and

(b) at least one HVR variant, wherein said HVR sequence variant has a modification of at least one residue of the sequence shown in SEQ ID NO: 1-18.

In one aspect of the invention, an anti-DLL4 antibody of the invention includes:

(a) at least one, two, three, four or five sequences of hypervariable regions (HVR) selected from the group consisting of:

(i) an HVR-L1 comprising the sequence A1-A11, wherein A1-A11 is RASQDVSTAVA (SEQ ID NO: 10);

(ii) an HVR-L2 containing the sequence B1-B7, where B1-B7 is SASFLYS (SEQ ID NO: 11);

(iii) an HVR-L3 containing the sequence C1-C9, wherein C1-C9 is QQSVNGPAT (SEQ ID NO: 14);

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

(v) an HVR-H2 containing the sequence E1-E18, where E1-E18 is GVINPNSGSTDYADSVKG (SEQ ID NO: 3);

(vi) an HVR-H3 comprising the sequence F1-F15, wherein F1-F15 is VYYCARDNFGGYFDY (SEQ ID NO: 9); and

(b) at least one HVR variant, wherein said HVR sequence variant has a modification of at least one residue of the sequence shown in SEQ ID NO: 1-18.

In one aspect of the invention, an anti-DLL4 antibody of the invention includes:

(a) at least one, two, three, four or five sequences of hypervariable regions (HVR) selected from the group consisting of:

(i) an HVR-L1 comprising the sequence A1-A11, wherein A1-A11 is RASQDVSTAVA (SEQ ID NO: 10);

(ii) an HVR-L2 containing the sequence B1-B7, where B1-B7 is SASFLYS (SEQ ID NO: 11);

(iii) an HVR-L3 containing the sequence C1-C9, wherein C1-C9 is QQSYTGTVT (SEQ ID NO: 18);

(iv) an HVR-H1 comprising the sequence D1-D10, wherein D1-D10 is GFTFTDNWIS (SEQ ID NO: 1);

(v) an HVR-H2 containing the sequence E1-E18, where E1-E18 is GYISPNSGFTYYADSVKG (SEQ ID NO: 8) and

(vi) an HVR-H3 comprising the sequence F1-F15, wherein F1-F15 is VYYCARDNFGGYFDY (SEQ ID NO: 9); and

(b) at least one HVR variant, wherein said HVR sequence variant has a modification of at least one residue of the sequence shown in SEQ ID NO: 1-18.

In one of its aspects, the present invention relates to an antibody comprising one, two, three, four, five or six HVRs, where each HVR comprises a sequence or consists of, or mainly consists of, a sequence selected from the group consisting of SEQ ID NO: 1-18, and where SEQ ID NO: 10 corresponds to HVR-L1, SEQ ID NO: 11 corresponds to HVR-L2, SEQ ID NO: 12, 13, 14, 15, 16, 17 or 18 correspond to HVR-L3, SEQ ID NO: 1 or 2 correspond to HVR-H1, SEQ ID NO: 3, 4, 5, 6, 7 or 8 correspond to HVR-H2, SEQ ID NO: 9 corresponds to HVR-H3. In one embodiment of the invention, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each of these regions includes, in that order, SEQ ID NO: 10, 11, 12, 1, 3, 9. In one embodiment of the invention, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, where each of these regions includes in this order SEQ ID NO: 10, 11, 13, 1, 4, 9. In one embodiment of the invention, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each of these areas includes, in that order, SEQ ID NO: 10, 11, 14, 2, 3, 9. In one embodiment, of the invention, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each of these regions includes, in that order, SEQ ID NO: 10, 11, 15, 1, 5, 9. In one embodiment of the invention, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each of these regions includes, in that order, SEQ ID NO: 10, 11, 16, 1, 6, 9. In one embodiment of the invention, the antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each of these areas includes in that order SEQ ID NO: 10, 11, 17, 1, 7, 9. In one embodiment of the invention, an antibody o according to the invention contains HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each of these areas includes in this order SEQ ID NO: 10, 11, 18, 1, 8 , 9.

Variants of the HVR in an antibody of the invention may have modifications in one or more (e.g., two, three, four, five or more) residues in the HVR.

In one embodiment of the invention, the HVR-L3 variant contains 1-6 (1, 2, 3, 4, 5, or 6) substitutions in any combination in the following positions: 91 (S or W), 92 (Y or F), 93 ( T, N or S), 94 (T or G), 95 (P, Q, A or T) and / or 96 (P, S, A or V).

In one embodiment of the invention, the HVR-H2 variant contains 1-4 (1, 2, 3 or 4) substitutions in any combination in the following positions: 50 (V, L or Y), 52 (N or S), 52a (P or S) or 53 (N, Q, T or I).

The letters in brackets after each position indicate a representative substitution (i.e., a representative replacement) of amino acids; and as one skilled in the art will recognize, the feasibility of replacing one amino acid with another amino acid in accordance with the invention described herein can be evaluated by routine methods known and / or described herein.

In one of its aspects, the present invention relates to an antibody comprising an HVR-H1 region containing the sequence of SEQ ID NO: 1 or 2. In one of its aspects, the present invention relates to an antibody comprising an HVR-H2 region containing a sequence of SEQ ID NO: 3, 4, 5, 6, 7 or 8. In one of its aspects, the present invention relates to an antibody comprising an HVR-H3 region comprising the sequence of SEQ ID NO: 9. In one embodiment, the present invention provides an antibody comprising an HVR-L1 region comprising the sequence of SEQ ID NO: 10. In one embodiment, the present invention provides an antibody comprising an HVR-L2 region comprising the sequence of SEQ ID NO: 11. In one embodiment, the present invention provides an antibody comprising an HVR-L3 region comprising the sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17, or 18.

In one of its aspects, the present invention relates to 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: 1;

(ii) an HVR-H2 sequence comprising the sequence of SEQ ID NO: 5;

(iii) an HVR-H3 sequence comprising the sequence of SEQ ID NO: 9.

In one of its aspects, the present invention relates to 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: 10;

(ii) an HVR-L2 sequence comprising the sequence of SEQ ID NO: 11;

(iii) an HVR-L3 sequence comprising the sequence of SEQ ID NO: 15.

In one of its aspects, the present invention relates to 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: 1;

(ii) an HVR-H2 sequence comprising the sequence of SEQ ID NO: 8;

(iii) an HVR-H3 sequence comprising the sequence of SEQ ID NO: 9.

In one of its aspects, the present invention relates to 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: 10;

(ii) an HVR-L2 sequence comprising the sequence of SEQ ID NO: 11;

(iii) an HVR-L3 sequence comprising the sequence of SEQ ID NO: 18.

The amino acid sequences of SEQ ID NOs: 1-18 are numbered for each individual HVR (i.e., H1, H2 or H3), as shown in Figures 1a and 1b, where the numbering corresponds to the Kabat numbering system described below.

In one of its aspects, the present invention relates to antibodies comprising heavy chain HVR sequences, as shown in figures 1a and 1b.

In one of its aspects, the present invention relates to antibodies comprising light chain HVR sequences, as shown in figures 1a and 1b.

Some embodiments of antibodies of the invention comprise a light chain variable domain of humanized antibody ^{4D5 (huMAb4D5-8) (HERCEPTIN ®,} Genentech, Inc., South San Francisco, CA, USA) ( also described in U.S. Patent № 6,407,213 and Lee et al., J . Mol. Biol. (2004), 340 (5): 1073-93)) presented below in SEQ ID NO: 52.

1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107 (SEQ ID NO: 52) (HVR residues underlined).

In one embodiment of the invention, the huMAb4D5-8 light chain variable domain sequence is modified at one or more positions 30, 66 and 91 (Asn, Arg and His are shown above in bold / italics, respectively). In one embodiment of the invention, the modified huMAb4D5-8 sequence contains Ser at position 30, Gly at position 66, and / or Ser at position 91. Accordingly, in one embodiment of the invention, the antibody of the invention comprises a light chain variable domain having the sequence represented by below in SEQ ID NO: 53:

1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107 (SEQ ID NO: 53) (HVR residues underlined).

Replaced residues in huMAb4D5-8 are indicated above in bold / italics.

Antibodies according to the invention may contain any suitable sequence of the frame domain in the variable region, provided that the activity of binding to DLL4 will mainly remain. So, for example, in some embodiments of the invention, the antibodies according to the invention contain a consensus sequence of the frame region of the human heavy chain of subgroup III. In one embodiment of these antibodies, the consensus sequence of the framework regions comprises a substitution at position 71, 73 and / or 78. In some embodiments of these antibodies, position A is present at position 71, T is present at position 73 and / or A. is present at position 78. embodiment said antibodies comprise the variable domain framework sequences of huMAb4D5-8 heavy chain ^{(HERCEPTIN ®, Genentech, Inc.,} South San Francisco, CA, USA) ( also described in U.S. patents 6,407,213 and 5,821,337 №№ and publications Lee et al. J. Mol. Biol. (2004), 340 (5): 1073-93). In one embodiment of the invention, these antibodies also include the consensus sequence of the frame region of the human κI light chain. In one embodiment of the invention, said antibodies comprise huMAb4D5-8 light chain HVR sequences (described in US Pat. Nos. 6,472,213 and 5,821,337). In one embodiment, these antibodies comprise variable domain light chain huMAb4D5-8 ^{(HERCEPTIN ®, Genentech, Inc.,} South San Francisco, CA, USA) ( also described in U.S. Patents 6,407,213 and 5,821,337 №№ and Lee et al., J. Mol. Biol. (2004), 340 (5): 1073-93).

In one embodiment of the invention, said antibody according to the invention comprises a heavy chain variable domain, wherein the frame sequence comprises the sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and / or 37, the HVR sequences H1, H2 and H3 are SEQ ID NO: 1, 5 and / or 9, respectively. In one embodiment of the invention, the antibody of the invention comprises a light chain variable domain, wherein the frame sequence comprises the sequence of SEQ ID NO: 38, 39, 40 and / or 41, the HVR sequences L1, L2 and L3 are SEQ ID NO: 10, 11 and / or 15, respectively.

In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain, wherein the frame sequence comprises the sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36 and / or 37, the HVR sequences H1, H2 and H3 are SEQ ID NO: 2, 3 and / or 9, respectively. In one embodiment of the invention, the antibody of the invention comprises a light chain variable domain, wherein the frame sequence comprises the sequence of SEQ ID NO: 38, 39, 40 and / or 41, the HVR sequences L1, L2, and L3 are SEQ ID NO: 10, 11, and / or 14, respectively.

In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain, wherein the frame sequence comprises the sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36 and / or 37, the HVR sequences H1, H2 and H3 are SEQ ID NO: 1, 8 and / or 9, respectively. In one embodiment of the invention, the antibody of the invention comprises a light chain variable domain, wherein the frame sequence comprises the sequence of SEQ ID NO: 38, 39, 40 and / or 41, the HVR sequences L1, L2, and L3 are SEQ ID NO: 10, 11, and / or 18, respectively.

In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain, wherein the frame sequence comprises the sequence of SEQ ID NO: 46, 47, 48 and / or 49, the HVR sequences H1, H2 and H3 are SEQ ID NO: 1, 5, and / or 9, respectively. In one embodiment of the invention, the antibody of the invention comprises a light chain variable domain, wherein the frame sequence comprises the sequence of SEQ ID NO: 42, 43, 44 and / or 45, the HVR sequences L1, L2 and L3 are SEQ ID NO: 10, 11 and / or 15, respectively.

In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain, wherein the frame sequence comprises the sequence SEQ ID NO: 46, 47, 48 and / or 49, the HVR sequences H1, H2 and H3 are SEQ ID NO: 2, 3, and / or 9, respectively. In one embodiment of the invention, the antibody of the invention comprises a light chain variable domain, wherein the frame sequence comprises the sequence of SEQ ID NO: 42, 43, 44 and / or 45, the HVR sequences L1, L2 and L3 are SEQ ID NO: 10, 11 and / or 14, respectively.

In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain, wherein the frame sequence comprises the sequence SEQ ID NO: 46, 47, 48 and / or 49, the HVR sequences H1, H2 and H3 are SEQ ID NO: 1, 8 and / or 9, respectively. In one embodiment of the invention, the antibody of the invention comprises a light chain variable domain, wherein the frame sequence comprises the sequence of SEQ ID NO: 42, 43, 44 and / or 45, the HVR sequences L1, L2 and L3 are SEQ ID NO: 10, 11 and / or 18, respectively.

In one embodiment of the invention, the antibody of the invention is affinity matured to achieve the desired binding affinity for the target. In one example, an affinity matured antibody of the invention comprises a substitution at one or more positions of amino acids H28, H30, H31, H32, H33, L91, L92, L93, L94, L95 and / or L96. In one example, an affinity matured antibody according to the invention contains one or more of the following substitutions: (a) in the heavy chain, V50L, V50Y, N52S, P52aS, N53Q, N53T, N53I, S56A, S56F, T57S, D58E, D58I, D58A, D58Y, or (b), in the light chain, S91W, Y92F, T93N, T93S, T94G, P95Q, P95A, P95T, P96S, P96A, P96V.

In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 54 . In one embodiment of the invention, the antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO: 55 . In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 54, and a light chain variable domain comprising the sequence of SEQ ID NO: 55.

In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 56 . In one embodiment of the invention, the antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO: 57 . In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 56, and a light chain variable domain comprising the sequence of SEQ ID NO: 57.

In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 58 . In one embodiment of the invention, the antibody of the invention comprises a light chain variable domain comprising the sequence of SEQ ID NO: 59 . In one embodiment of the invention, the antibody of the invention comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 58, and a light chain variable domain comprising the sequence of SEQ ID NO: 59.

In one of its aspects, the present invention relates to an antibody that competes with any of the aforementioned antibodies for binding to DLL4. In one of its aspects, the present invention relates to an antibody that binds to the same epitope on DLL4 that binds to any of the above antibodies.

As is known to those skilled in the art and as described in detail below, the amino acid positions / boundaries defining the hypervariable region of an antibody may vary depending on the surrounding amino acids and on various definitions known to those skilled in the art (described below). Some positions in the variable domain can be considered as hybrid hypervariable positions, where these positions, obviously, can exist in the hypervariable region in accordance with one set of signs, can be present outside the hypervariable region in accordance with another set of signs. One or more of these positions may also be located in elongated hypervariable regions (as detailed below).

In some embodiments of the invention, the specified antibody is a monoclonal antibody. In some embodiments of the invention, the specified antibody is a polyclonal antibody. In some embodiments of the invention, said antibody is selected from the group consisting of a chimeric antibody, an affinity matured antibody, a humanized antibody, and a human antibody. In some embodiments of the invention, said antibody is an antibody fragment. In some embodiments of the invention, said antibody is Fab, Fab ', Fab'-SH, F (ab') _2, or scFv.

In one embodiment of the invention, said antibody is a chimeric antibody, for example, an antibody comprising antigen binding sequences derived from a non-human donor and attached to a heterologous non-human, human or humanized sequence (e.g., frame and / or constant domain sequences). In one embodiment of the invention, said non-human donor is a mouse. In one embodiment of the invention, the antigen binding sequence is synthetic, for example, it can be obtained by mutagenesis (for example, by screening by phage display, etc.). In one embodiment of the invention, the chimeric antibody of the invention has murine V regions and a human C region. In one embodiment of the invention, the murine light chain V region is attached to the human kappa light chain. In one embodiment of the invention, the murine heavy chain V region is coupled to the human IgG1 C region.

The humanized antibodies of the invention are those having amino acid substitutions in FR and affinity matured variants with substitutions in the attached CDRs. Substituted amino acids in the CDR or FR are not limited to the amino acids present in the antibody donor or recipient. In other embodiments of the invention, the antibodies of the invention also comprise substitutions of amino acid residues in the Fc region that improve effector function, including improved CDC and / or ADCC function and enhanced cytolysis of B cells. Other antibodies of the invention are antibodies having specific substitutions that increase stability. In other embodiments of the invention, the antibodies of the invention comprise substitutions of amino acid residues in the Fc region that impair effector function, including attenuation of CDC and / or ADCC function and / or attenuation of cytolysis of B cells. In some embodiments of the invention, the antibodies of the invention are characterized by a reduced level of binding (e.g., lack of binding) to the C1q factor of the human complement system and / or to the human Fc receptor on natural killer cells (NK). In some embodiments of the invention, the antibodies of the invention are characterized by a reduced level of binding (e.g., lack of binding) to human FcγRI, FcγRIIA and / or FcγRIIIA. In some embodiments of the invention, the antibodies of the invention belong to the IgG class (e.g., IgG1 or IgG4) and contain at least one mutation in E233, L234, G236, D265, D270, N297, E318, K320, K322, A327, A330, P331 and / or P329 (numbering was carried out in accordance with the European numbering system (EU)). In some embodiments of the invention, these antibodies include a mutation of L234A / L235A or D265A / N297A.

In one of its aspects, the present invention relates to antibodies against DLL4 polypeptides containing any of the antigen binding sequences described herein, wherein said antibodies against DLL4 polypeptides specifically bind to DLL4.

Antibodies of the invention bind (e.g. specifically bind) to DLL4, in some embodiments of the invention, they can modulate one or more kinds of DLL4-associated effects, including, but not limited to, attenuation or blocking of Notch receptor activation, attenuation or blocking of a molecular signal, transmitted after Notch receptor signaling, disruption or blocking of Notch receptor binding to DLL4 and / or stimulation of endothelial cell proliferation and / or inhibition of endothelial differentiation ialnyh cells and / or inhibition of arterial differentiation, and / or inhibition of tumor vascular perfusion, and / or treatment and / or prevention of tumor development, klotochno proliferative disorder or cancer; and / or treating or preventing a disorder associated with the expression and / or activity of DLL4 and / or treating or preventing a disorder associated with the expression and / or activity of the Notch receptor. In some embodiments of the invention, the antibody of the invention specifically binds to DLL4. In some embodiments of the invention, the antibody specifically binds to the extracellular domain of DLL4 (ECD). In some embodiments of the invention, the antibody specifically binds to a polypeptide consisting or mainly consisting of the extracellular domain of DLL4. In some embodiments of the invention, the antibody specifically binds to DLL4 with a KD of about 1 nM or more, or about 500 pM or more. In some embodiments of the invention, the antibody specifically binds to human DLL4 with k _{on of} about 2 × 10 ⁵ or more, or about 1 × 10 ⁵ or more. In some embodiments of the invention, the antibody according to the invention weakens, inhibits and / or blocks the activity of DLL4 in vivo and / or in vitro o. In some embodiments, the antibody competes with a DLL4 ligand for binding to DLL4 (weakens and / or blocks the binding of Notch receptor to DLL4).

In one of its aspects, the present invention relates to compositions comprising one or more antibodies of the invention and a carrier. In one embodiment of the invention, said carrier is pharmaceutically acceptable.

In one of its aspects, the present invention relates to nucleic acids encoding an anti-DLL4 antibody of the invention.

In one of its aspects, the present invention relates to vectors containing a nucleic acid according to the invention.

In one aspect, the present invention relates to compositions comprising one or more nucleic acids of the invention and a carrier. In one embodiment of the invention, said carrier is pharmaceutically acceptable.

In one of its aspects, the present invention relates to host cells comprising a nucleic acid or a vector according to the invention. The specified vector can be any type of vector, for example, a recombinant vector, such as an expression vector. In this case, any host cells can be used. In one embodiment of the invention, the host cell is a prokaryotic cell, for example, E. coli . In one embodiment, the host cell is a eukaryotic cell, for example, a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

In one of its aspects, the present invention relates to methods for producing antibodies according to the invention. Thus, for example, the present invention relates to methods for producing an anti-DLL4 antibody (which, as defined herein, is a full-length antibody or antibody fragment) or an immunoconjugate, wherein said method comprises expressing in a suitable host cell a recombinant vector of the invention encoding the specified antibody (or fragment thereof), and the selection of the specified antibodies.

In one of its aspects, the present invention relates to an industrial product comprising a container and a composition contained in that container, wherein said composition comprises one or more anti-DLL4 antibodies of the invention. In one embodiment of the invention, said composition also comprises an antiangiogenic agent. In one embodiment of the invention, said anti-angiogenic agent is an anti-VEGF antibody, for example, bevacizumab. In one embodiment of the invention, said composition comprises a nucleic acid according to the invention. In one embodiment of the invention, said antibody-containing composition also comprises a carrier, which, in some embodiments of the invention, is pharmaceutically acceptable. In one embodiment of the invention, a second composition is present in said container, wherein said second composition comprises an anti-angiogenic agent. In one embodiment of the invention, said anti-angiogenic agent is an anti-VEGF antibody, for example, bevacizumab. In one embodiment of the invention, an industrial product according to the invention also includes instructions for administering said composition (s) (e.g., antibodies) to an individual (e.g., instructions for using any of the methods described herein).

In one of its aspects, the present invention relates to a kit comprising a first container comprising a composition comprising one or more anti-DLL4 antibodies of the invention, and a second container comprising a buffer. In one embodiment of the invention, said buffer is pharmaceutically acceptable. In one embodiment of the invention, the composition comprising the antibody also comprises a carrier, which, in some embodiments of the invention, is pharmaceutically acceptable. In one embodiment of the invention, said kit also includes instructions for administering said composition (eg, antibody) to an individual.

In one aspect, the present invention relates to the use of an anti-DLL4 antibody of the invention for the manufacture of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as cancer, tumor and / or cell proliferative disorder. In some embodiments of the invention, said disorder is a pathological condition associated with angiogenesis.

In one of its aspects, the present invention relates to the use of a nucleic acid according to the invention for the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as cancer, tumor and / or cell proliferative disorder. In some embodiments of the invention, said disorder is a pathological condition associated with angiogenesis.

In one of its aspects, the present invention relates to the use of an expression vector according to the invention for the manufacture of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as cancer, tumor and / or cell proliferative disorder. In some embodiments of the invention, said disorder is a pathological condition associated with angiogenesis.

In one aspect, the present invention relates to the use of a host cell according to the invention for the manufacture of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as cancer, tumor and / or cell proliferative disorder. In some embodiments of the invention, said disorder is a pathological condition associated with angiogenesis.

In one aspect, the present invention relates to the use of an industrial product according to the invention for the manufacture of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as cancer, tumor and / or cell proliferative disorder. In some embodiments of the invention, said disorder is a pathological condition associated with angiogenesis.

In one of its aspects, the present invention relates to the use of a kit according to the invention for the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as cancer, tumor and / or cell proliferative disorder. In some embodiments of the invention, said disorder is a pathological condition associated with angiogenesis.

The present invention relates to methods and compositions that can be used to treat pathological conditions associated with the expression and / or activity of DLL4, for example, with increased or decreased expression and / or activity, or undesired expression and / or activity of DLL4.

In one aspect, the present invention provides methods for treating or preventing a tumor, cancer, and / or cell proliferative disorder associated with increased expression and / or activity of DLL4, wherein said methods comprise administering an effective amount of an anti-DLL4 antibody to an individual in need such a treatment.

In one aspect, the present invention provides methods for attenuating, inhibiting, blocking, or preventing the growth of a tumor or cancer, wherein said methods comprise administering an effective amount of an anti-DLL4 antibody to an individual in need of such treatment.

In one of its aspects, the present invention relates to methods for treating a tumor, cancer and / or cell proliferative disorder, wherein said methods comprise administering an effective amount of an anti-DLL4 antibody to an individual in need of such treatment.

In one aspect, the present invention relates to methods for inhibiting angiogenesis, wherein said methods comprise administering an effective amount of an anti-DLL4 antibody to an individual in need of such treatment.

In one of its aspects, the present invention relates to methods for treating a pathological condition associated with impaired angiogenesis, wherein said methods comprise administering an effective amount of an anti-DLL4 antibody to an individual in need of such treatment. In some embodiments of the invention, said pathological condition associated with angiogenesis is a tumor, cancer, and / or cell proliferative disorder. In some embodiments of the invention, said pathological condition associated with angiogenesis is an intraocular neovascular disease.

The methods of the invention can be used to treat any relevant pathological condition. Representative disorders are described herein, and such disorders are cancers selected from the group consisting of small cell lung cancer, neuroblastoma, melanoma, breast carcinoma, stomach cancer, colon and colon cancer (CRC), and hepatocellular carcinoma, including metastases of these cancer diseases.

The methods of the invention may also include additional treatment steps. Thus, for example, in one embodiment of the invention, the method further comprises a step in which the target cell and / or target tissue (eg, cancer cell) is subjected to radiation therapy or chemotherapy, or treated with an antiangiogenic agent.

In another aspect, the present invention relates to methods for detecting DLL4, where these methods include detecting the complex "DLL4-anti-DLL4 antibody" in the sample. As used herein, the term “detection” includes qualitative and / or quantitative detection (at measurable levels) in the presence or absence of control.

In another aspect, the present invention relates to methods for diagnosing a disorder associated with the expression and / or activity of DLL4, where these methods include the detection of the complex "DLL4-anti-DLL4 antibody" in a biological sample taken from a patient suffering from the specified disorder or with suspicion to the specified disorder. In some embodiments of the invention, the expression of DLL4 is increased or abnormal. In some embodiments of the invention, said disorder is a tumor, cancer, and / or cell proliferative disorder.

In another aspect, the present invention relates to any of the anti-DLL4 antibodies described herein, wherein said anti-DLL4 antibody contains a detectable label.

In another aspect, the present invention relates to a complex consisting of any anti-DLL4 antibodies and DLL4 described herein. In some embodiments of the invention, said complex is present in vivo or in vitro o. In some embodiments of the invention, said complex comprises cancer cells. In some embodiments, the anti-DLL4 antibody is detectably labeled.

A brief description of the graphic material

Figure 1a, b: HVR loop sequences of the heavy chain and light chain of anti-DLL4 antibodies. The figures show the heavy chain HVR sequences, H1, H2 and H3, and the light chain HVR sequences, L1, L2 and L3. The sequences are numbered as follows: clone 152.26 (HVR-H1 represents SEQ ID NO: 1; HVR-H2 represents SEQ ID NO: 3; HVR-H3 represents SEQ ID NO: 9; HVR-L1 represents SEQ ID NO: 10; HVR-L2 represents SEQ ID NO: 11; HVR-L3 represents SEQ ID NO: 12); clone 152.26.6 (HVR-H1 represents SEQ ID NO: 1; HVR-H2 represents SEQ ID NO: 4; HVR-H3 represents SEQ ID NO: 9; HVR-L1 represents SEQ ID NO: 10; HVR -L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 13); clone 152.26.14 (HVR-H1 represents SEQ ID NO: 2; HVR-H2 represents SEQ ID NO: 3; HVR-H3 represents SEQ ID NO: 9; HVR-L1 represents SEQ ID NO: 10; HVR -L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 14); clone 152.26.20 (HVR-H1 represents SEQ ID NO: 1; HVR-H2 represents SEQ ID NO: 5; HVR-H3 represents SEQ ID NO: 9; HVR-L1 represents SEQ ID NO: 10; HVR -L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 15); clone 152.26.34 (HVR-H1 represents SEQ ID NO: 1; HVR-H2 represents SEQ ID NO: 6; HVR-H3 represents SEQ ID NO: 9; HVR-L1 represents SEQ ID NO: 10; HVR -L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 16); clone 152.26.40 (HVR-H1 represents SEQ ID NO: 1; HVR-H2 represents SEQ ID NO: 7; HVR-H3 represents SEQ ID NO: 9; HVR-L1 represents SEQ ID NO: 10; HVR -L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 17); and clone 152.26.82 (HVR-H1 is SEQ ID NO: 1; HVR-H2 is SEQ ID NO: 8; HVR-H3 is SEQ ID NO: 9; HVR-L1 is SEQ ID NO: 10; HVR-L2 is SEQ ID NO: 11; HVR-L3 is SEQ ID NO: 18).

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

Figures 2 and 3: representative human acceptor framework consensus sequences that are used to implement the present invention using the following sequence identifiers:

Consensus framework sequences of the variable region of the heavy chain (VH) (figa, b)

The consensus sequence of the human framework region VH subgroup I minus Kabat CDR (SEQ ID NO: 19)

The consensus sequence of the human framework region VH subgroup I minus the elongated hypervariable region (SEQ ID NO: 20-22)

The consensus sequence of the human framework region VH subgroup II minus Kabat CDR (SEQ ID NO: 23)

The consensus sequence of the human framework region VH subgroup I minus the elongated hypervariable region (SEQ ID NO: 24-26)

The consensus sequence of the human framework region VH subgroup II minus elongated

The consensus sequence of the human framework region VH subgroup III minus Kabat CDR (SEQ ID NO: 27)

The consensus sequence of the human framework region VH subgroup III minus the elongated hypervariable region (SEQ ID NO: 28-30)

The acceptor sequence of the human framework region VH minus Kabat CDR (SEQ ID NO: 31)

The acceptor sequence of the human framework region VH minus the elongated hypervariable region (SEQ ID NO: 32-33)

Kabat acceptor sequence 2 of the human framework region VH minus CDR (SEQ ID NO: 34)

The acceptor sequence 2 of the human framework region VH minus the elongated hypervariable region (SEQ ID NO: 35-37)

Consensus sequence of the frame region of the variable region of the light chain (VL) (figure 3)

Consensus sequence of the human framework region of the kappa VL chain of subgroup I (SEQ ID NO: 38)

Consensus sequence of the human framework region of the kappa VL subgroup II chain (SEQ ID NO: 39)

Consensus sequence of the human framework region of the kappa VL subgroup III chain (SEQ ID NO: 40)

Consensus sequence of the human framework region of the kappa chain VL subgroup IV (SEQ ID NO: 41)

Figure 4: The sequence of the frame region of the light and heavy chains of huMAb4D5-8. The numbers in the upper index in bold indicate the Kabat numbered amino acid positions.

Figure 5: Modified / altered sequences of the frame region of the light and heavy chains of huMAb4D5-8. The numbers in the upper index in bold indicate the amino acid positions numbered by Kabat.

Figure 6: Variable regions of the heavy chain and variable regions of the light chain of antibody clones 26.20, 26.14 and 26.82.

Figure 7: DLL4-mediated Notch signaling regulates EC proliferation. a-c, f: HUVEC proliferation assays in 3-dimensional fibrin gels. Anti-DLL4 antibody (YW26.82) or DBZ stimulate proliferation of HUVECs (a). Ki67 staining showed that anti-DLL4 antibody or DBZ induces HUVEC hyperproliferation (b). Anti-DLL4 antibody or DBZ increase HUVEC proliferation in the presence of SF-conditioned medium (c). d, h: Systemic delivery of anti-DLL4 antibodies induces massive EC accumulation in the neonatal retina. Images of the vascular system of the retina obtained with a confocal microscope with a small increase (top) and a large increase (bottom) (staining with isolectin). (d): Ki67 staining indicated an increase in EC proliferation in the neonatal retina treated with anti-DLL4 antibody (h). e: Notch activation by immobilized DLL4 resulted in inhibition of HUVEC proliferation. f: Anti-VEGF antibody inhibited the proliferation of HUVEC in the presence or absence of DBZ. g: Regulation of VEGFR2 by Notch. Quantitative PCR analysis for VEGFR2 expression in response to Notch blockade in a 3-dimensional fibrin gel HUVEC cell culture (7 days) by anti-DLL4 antibody or DBZ (left) or Notch activation in a 2-dimensional gel HUVEC cell culture (36 hours) under the influence of immobilized DLL4 (right). Anti-DLL4 antibody and DBZ were used at 5 μg / ml and 0.08 μM, respectively (a-c, e-g).

Figure 8: DLL4-mediated Notch signaling regulates EC differentiation. a: Cavity-like structures (white arrows) formed by HUVEC cells growing in fibrin gels were eliminated in the presence of an anti-DLL4 antibody or DBZ. Instead, these growths were densely packed with cells (black arrows). b: Regulation of TGFβ2 by Notch. Quantitative PCR analysis of TGFβ2 expression in response to Notch blockade in HUVEC cell culture in 3-dimensional fibrin gel (7 days) by anti-DLL4 antibody or DBZ (left) or Notch activation in HUVEC cell culture in 2-dimensional gel ( 36 hours) under the influence of immobilized DLL4 (right). c: Anti-DLL4 antibody blocks the development of arteries. Images of retina stained with alpha-actin smooth muscle (ASMA) and isolectin obtained for a newborn mouse using a confocal microscope. Newborn mice were treated as shown in Fig. 1d. d: Images of a stained ASMA and retinal isolectin obtained with a confocal microscope for an adult mouse. 8-week-old mice were treated with PBS or anti-DLL4 antibody (10 mg / kg, twice a week) for two weeks.

Figure 9: Selective blocking of DLL4 and / or VEGF disrupts tumor angiogenesis and inhibits tumor growth. a-f: Results for tumor models: HM7 (a), Colo205 (b), Calu6 (c), MDA-MB-435 (d), MV-522 (e) and WEHI3 (f). Presents the mean tumor volume with standard error. g-h: Histological analysis of tumor vessels. Immunohistochemical analysis for anti-CD31 antibodies performed on sections of EL4 tumors taken from control mice and mice treated with anti-DLL4 antibody and anti-VEGF antibody (g): Lectin perfusion and staining with anti-CD31 antibody in sections EL4 tumors (h). ip: Results for tumor models: SK-OV-3X1 (i), LL2 (j), EL4 (k), H1299 (l), SKMES-1 (m), MX-1 (n), SW620 (o ) and LS174T (p).

Figure 10: DLL4 / Notch does not play an important role in mouse small intestine homeostasis. Immunohistochemical studies of the small intestine taken from control mice (a, d, g, j) and mice treated with anti-DLL4 antibody (10 mg / kg, twice a week for 6 weeks) were carried out (b, e, h, k), and in mice treated with DBZ (30 μmol / kg daily for 5 days) (c, f, i, l). As shown by staining with H&E (a, b, c) and Alcian blue (d, e, f), DBZ causes the replacement of the TA cell population with goblet cells. This change was completely absent after treatment with anti-DLL4 antibody. Staining of Ki67 (g, h, i) and HES-1 (j, k, l) further confirmed that the anti-DLL4 antibody was unable to mimic the effect of DBZ.

Figure 11: Characterization of anti-DLL4 antibodies. a: Mapping of the epitope of the monoclonal anti-DLL4 antibody (Mab) YW26.82. Schematic representation of a set of DLL4 mutants expressed as C-terminal fusion proteins of alkaline phosphatase (ALP) in a human placenta. 293T cell conditioned media containing fusion proteins was tested on 96-well microtiter plates sensitized with purified anti-DLL4 antibody (YW26.82, 0.5 μg / ml). The associated DLL4.AP was detected using 1-Step PNPP (Pierce) as a substrate and absorbance (OD) was measured at 405 nm. b-d: Selective binding of YW26.82 to DLL4. Nunc MaxiSorp ™ 96-well plates were coated with purified recombinant proteins as described above (1 μg / ml). The binding of YW26.82 at the indicated concentrations was measured by ELISA analysis. Bound antibodies were detected using an anti-human antibody-HRP conjugate using TMB as a substrate and absorbance (OD) was measured at 405 nm. An anti-HER2 antibody and recombinant ErbB2-ECD (b) were used as an analytical control. FACS analysis of 293 cells temporarily transfected with a vector full-sized DLL4, Jag1 or DLL1. A significant level of binding of YW26.82 was detected only on DLL4-transfected cells (top panel). The expression of Jag1 and DLL1 was confirmed by binding of recombinant rat Notch1-Fc (rrNotch1-Fc, middle panel) and recombinant rat Notch2-Fc (rrNotch2-Fc, bottom panel), respectively. YW26.82, rrNotch1-Fc or rrNotch2-Fc (R & D system) was used at a concentration of 2 μg / ml, then a goat anti-human IgG antibody conjugated with PE was used (1: 500, Jackson ImmunoResearch) (c). Anti-DLL4 antibody blocked the binding of DLL4-AP, but not DLL1-AP, with rNotch1 deposited, with an IC50 of ~ 12 nM calculated (left panel). The anti-DLL4 antibody blocked the binding of DLL4-His, but not Jag1-His, to rNotch1, at an IC50 of ~ 8 nM (right panel) (d). e: Specific binding of YW26.82 to endogenously expressed DLL4. FACS analysis of HUVEC transfected with control or DLL4-specific siRNA. YW26.82 was used at 2 μg / ml, then a goat anti-human IgG antibody conjugated with PE (1: 500, Jackson ImmunoResearch) (e) was used.

Figure 12: Positive regulation of DLL4 through activation of Notch. HUVECs were stimulated with immobilized C-terminal His-tagged human DLL4 (amino acids 1-404) in the absence or presence of DBZ (0.08 μM). 36 hours after stimulation, endogenous expression of DLL4 was evaluated by FACS analysis using anti-DLL4 antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to anti-DLL4 antibodies that can be used, for example, to treat or prevent pathological conditions associated with the expression and / or activity of DLL4, for example, with increased expression and / or activity or with undesired and / or activity of DLL4. In some embodiments of the invention, the antibodies of the invention are used to treat a tumor, cancer and / or cell proliferative disorder. In some embodiments of the invention, the antibodies of the invention are used to treat a pathological condition associated with angiogenesis.

In another aspect of the invention, the anti-DLL4 antibodies of the invention can be used as reagents for detecting and / or isolating DLL4, for example, for detecting DLL4 in various types of tissues and cells.

The present invention also relates to methods for producing anti-DLL4 antibodies and polynucleotides encoding anti-DLL4 antibodies.

General methods

The methods and procedures described or cited here are essentially well known to those skilled in the art and are generally used in accordance with standard techniques known to those skilled in the art, for example the widely used techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. Eds., (2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Definitions

An “isolated” antibody is an antibody that has been identified and separated from its natural environment components and / or isolated from such components. Impurity components of its natural environment are substances that can adversely affect the diagnostic or therapeutic use of this antibody, and such substances can be enzymes, hormones, and other protein or non-protein dissolved substances. In preferred embodiments of the invention, said antibody can be purified (1) by more than 95% by weight of the antibody, as can be determined by the Lauri method, most preferably by more than 99% by weight of the antibody, (2) to a level sufficient to produce N- terminal or internal amino acid sequence consisting of at least 15 residues using a sequencer with centrifuge cups, or (3) until homogeneity, assessed by SDS page-SDS under reducing or non-reducing conditions s using Coomassie blue or preferably, silver stain. An isolated antibody is an in situ antibody in recombinant cells because at least one component of the natural environment of the antibody is missing. A similarly isolated antibody is an antibody in a medium surrounding recombinant cells. However, usually isolated antibody can be obtained in 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 impurity nucleic acid molecule with which it is usually associated in a natural source of nucleic acid encoding an antibody. An isolated nucleic acid molecule has a shape or structure that is different from the shape or structure of a natural molecule. Therefore, the isolated nucleic acid molecules are different from the nucleic acid molecules present in natural cells. However, the isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that typically express an antibody, where, for example, the nucleic acid molecule is present on the chromosome at a position different from that of the nucleic acid in natural cells.

As used herein, the term “Kabat numbered variable domain residue” or “Kabat numbered amino acid position” and variants thereof, means a system used to number the heavy chain variable domains or light chain variable domains of the antibodies described in the Kabat Antibody Reference et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991). In accordance with this numbering system, the actual primary amino acid sequence may contain a smaller number or an additional number of amino acids corresponding to a shortened or elongated FR or CDR region of the variable domain. So, for example, the variable domain of the heavy chain may include the insertion of one amino acid (residue 52a in accordance with Kabat numbering) after residue 52 of H2, and residues (for example, residues 82a, 82b and 82c and the like in accordance with Kabat numbering), embedded after the remainder of 82 FR heavy chain. Kabat residues of a given antibody can be numbered after aligning its sequence in areas of homology with a “standard” Kabat sequence.

As used herein, the terms “substantially similar” or “substantially the same” mean a fairly high degree of similarity between two numerical values (usually between one value corresponding to an antibody of the invention and another value corresponding to a reference / reference antibody) namely, such a degree of similarity that would allow a person skilled in the art to consider the difference between these two values as insignificant or biologically and / or statistically insignificant with respect to their biological voystv defined by said values (e.g., Kd values). The differences between the two values are preferably less than about 50%, more preferably less than about 40%, even more preferably less than about 30%, even more preferably less than about 20%, most preferably less than about 10%, compared to the values reference / comparison antibody.

The term “binding affinity” essentially means the strength of the total non-covalent interactions of one binding site of a molecule (eg, an antibody) with its binding partner (eg, antigen). Unless otherwise specified, the term “binding affinity” as used herein means a natural binding affinity in which a 1: 1 interaction occurs between members of the binding pairs (eg, antibodies with antigen). The binding affinity of molecule X with its partner Y, in general terms, may be called the dissociation constant (Kd). Affinity may be determined by standard methods known to those skilled in the art, including those described herein. Low affinity antibodies usually bind to the antigen at a slower rate and tend to easily dissociate, whereas high affinity antibodies usually bind to the antigen at a faster rate and tend to remain bound for a longer period of time. Various methods for measuring binding affinity are known to those skilled in the art, and any of these methods may be used to implement the present invention. Specific representative embodiments of the invention are described below.

In one embodiment of the invention, “Kd” or “Kd value” according to the invention is determined by a binding assay for a radiolabeled antigen (RIA) using a Fab variant of the antibody of interest and its antigen, described below, where the analysis allows measurement the affinity of Fab binding to antigen in solution by balancing Fab with a minimum concentration of ¹²⁵ I-labeled antigen in the presence of a titration set of unlabeled antigens, followed by immobilization of the associated antigen on a tablet sensitized with an anti-Fab antibody (Chen et al. (1999) J. Mol. Biol. 293: 865-881). In order to create appropriate conditions for analysis, microtiter plates (Dynex) were sensitized overnight with 5 μg / ml binding anti-Fab antibodies (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (mass / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23 ° C). In a non-absorbent plate (Nunc # 269620), 100 pM or 26 pM ¹²⁵ I antigens are mixed with serial dilutions of the Fab of interest (e.g., according to the evaluation of the anti-VEGF antibody, Fab-12, Presta et al. (1997) Cancer Res. 57: 4593-4599). The Fabs of interest are then incubated overnight, however, to ensure equilibrium, incubation can be carried out over a longer period of time (e.g. 65 hours). After this, the mixture is transferred to a plate for immobilization and incubated at room temperature (for example, for one hour). Then the solution was removed and the plate was washed eight times with 0.1% Tween-20 in PBS. After drying the plates, 150 μl / well of scintillation fluid (MicroScint ^™ -20; Packard) was added, and the plates were counted on a Topcount gamma counter (Packard) for ten minutes. The concentrations of each Fab, which are 20% or less of the maximum binding, are selected for their use in competitive binding assays. According to another embodiment of the invention, Kd or Kd value is measured in a surface plasmon resonance analysis using a BIAcore ^™ -2000 or BIAcore ^™ -3000 (BIAcore, Inc., Piscataway, NJ) at 25 ° C. using CM5 chips immobilized thereon antigen for response units (RU) of ~ 10. Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the instructions of the suppliers. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg / ml (~ 0.2 μM), then injected at a flow rate of 5 μl / min until the value of the response unit (RU) of the bound protein of ~ 10 is reached. After antigen injection, 1M ethanolamine is administered to block unreacted groups. To measure the kinetics of the reaction, two serial serial dilutions of Fab (0.78 nM - 500 nM) are injected in PBS containing 0.05% Tween® 20 (PBST) at 25 ° C and at a flow rate of approximately 25 μl / min. The association rate (k _on ) and the dissociation rate (k _off ) are calculated using a simple Langmuir 1: 1 binding model (BIAcore Evaluation Software version 3.2) while constructing association and dissociation sensograms. The equilibrium dissociation constant (Kd) is calculated as the ratio k _off / k _on . See, for example, Chen, Y., et al. (1999) J. Mol. Biol. 293: 865-881. If the association rate exceeds 10 ⁶ M ^-1 · s ^-1 , as was determined above by the surface plasmon resonance method, this association rate can be determined by the fluorescence quenching method, which allows one to measure an increase or decrease in the fluorescence intensity (excitation = 295 nm; = 340 nm, bandwidth 16 nm) at 25 ° C for 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing antigen concentrations, as measured on a spectrometer such as a spectrophotometer, equipped with the first flow limiter (Aviv Instrumtnts), or a spectrophotometer series SLM-Aminco 8000 (ThermoSpectronic), equipped with a stirring cuvette containing red dye.

The “association rate” or “k _on ” according to the invention can also be determined by the surface plasmon resonance method described above using BIAcore ^™ -2000 or BIAcore ^™ -3000 (BIAcore, Inc., Piscataway, NJ) at 25 ° C. and using chips CM5 with antigen immobilized on them with a response unit (RU) value of ~ 10. Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the instructions of the suppliers. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg / ml (~ 0.2 μM), then injected at a flow rate of 5 μl / min until the value of the response unit (RU) of the bound protein of ~ 10 is reached. After antigen injection, 1M ethanolamine is administered to block unreacted groups. To measure the kinetics of the reaction, two-fold serial dilutions of Fab (0.78 nM-500 nM) are injected in PBS containing 0.05% Tween® 20 (PBST) at 25 ° C and at a flow rate of approximately 25 μl / min. The association rate (k _on ) and dissociation rate (k _off ) are calculated using a simple Langmuir 1: 1 binding model (BIAcore Evaluation Software version 3.2) while constructing association and dissociation sensorograms. The equilibrium dissociation constant (Kd) is calculated as the ratio k _off / k _on . See, for example, Chen, Y., et al. (1999) J. Mol. Biol. 293: 865-881. However, if the association rate exceeds 10 ⁶ M ^-1 · s ^-1 , as was determined above by analysis of the surface plasmon resonance method, then this association rate is preferably determined by the fluorescence quenching method, which allows one to measure an increase or decrease in the fluorescence intensity (excitation = 295 nm; radiation = 340 nm, passband 16 nm) at 25 ° C for 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen measured on a spectrometer such as with ektrofotometr provided with a flow limiter (Aviv Instrumetnts) or spectrophotometer series SLM-Aminco 8000 (ThermoSpectronic), equipped with a cuvette for mixing.

As used herein, the term “vector” means a nucleic acid molecule capable of transferring another nucleic acid to which it is attached. One type of vectors is a “plasmid", which is a circular double-stranded DNA loop with 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 to the viral genome. Some vectors are capable of autonomously replicating in the host cell into which they were introduced (for example, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (for example, non-episomal mammalian vectors) can integrate into the genome of the host cell after they are introduced into the specified host cell, as a result of which they can replicate along with the host genome. In addition, some vectors are able to regulate the expression of genes to which they were functionally attached. Such vectors are referred to herein as “recombinant expression vectors” (or simply “recombinant vectors”). In general terms, expression vectors commonly used in recombinant DNA techniques are often in the form of plasmids. As used herein, the terms “plasmid” and “vector” may be used interchangeably since plasmid is the most common form of vector.

As used herein, the terms “polynucleotide” or “nucleic acid” are used interchangeably to mean polymers of any length consisting of nucleotides, and such polymers are DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer via DNA or RNA polymerase, or through a synthesis reaction. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogues. If necessary, the modification of the nucleotide structure can be carried out before or after the assembly of the polymer. Nucleotide sequences may be interrupted by non-nucleotide components. The polynucleotide can be further modified after synthesis, for example by conjugating it to a label. Modifications of other types are, for example, “caps”; replacement of one or more natural nucleotides with their analogues; internucleotide modifications, such as, for example, modifications by introducing uncharged bonds (e.g. methylphosphonates, phosphotriethers, phosphoamidates, carbamates, etc.) and charged bonds (e.g. phosphorothioates, phosphorodithioates, etc.); modifications containing side groups, such as, for example, proteins (for example, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); modifications containing intercalating agents (for example, acridine, psoralen, etc.); modifications containing chelating agents (for example, metals, radioactive metals, boron, oxidizing metals, etc.); modifications containing alkylating agents; modifications containing modified bonds (for example, alpha-anomeric nucleic acids, etc.), also unmodified forms of polynucleotide (s). In addition, any hydroxyl groups typically present in sugars can be replaced, for example, with phosphonate groups and phosphate groups; protected by standard protective groups; or activated to form additional bonds with additional nucleotides; or they can be conjugated to solid or semi-solid carriers. 5'- and 3'-terminal OH can be phosphorylated or substituted by amines or organic “capping” groups consisting of 1-20 carbon atoms. Other hydroxyls may also be derivatized with standard protecting groups. Polynucleotides may also contain similar forms of sugars, such as ribose or deoxyribose, known to those skilled in the art, including, for example, 2'-O-methyl-ribose-2'-O-allylribose;2'-fluoro or 2'-azidoribose; carbocyclic analogues of sugars; alpha anomeric sugars; epimeric sugars such as arabinose, xylose or lyxose; pyranose sugar, furanose sugar; sedoheptulose; acyclic analogs and basic nucleoside analogs such as methylriboside. One or more phosphodiester bonds may be replaced by alternative linker groups. Such alternative linker groups include, but are not limited to, variants in which phosphate is replaced by P (O) S (“thioate”), P (S) S (“dithioate”), (O) NR ₂ (“amidate”), P (O) R, P (O) OR ', CO or CH ₂ (“formacetal”), where each of R or R' independently is H or substituted or unsubstituted alkyl (C _1-20 ), optionally containing an ether bond (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all bonds in the polynucleotide must be identical. The above description applies to all polynucleotides used herein, including RNA and DNA.

As used herein, the term “oligonucleotide” generally means short, mostly single-stranded, usually synthetic polynucleotides, the length of which is mainly, but not necessarily, less than about 200 nucleotides. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above description of polynucleotides can equally and fully relate to oligonucleotides.

The “percentage (%) amino acid sequence identity" of peptides or polypeptides is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after aligning these sequences and introducing “gaps” in them, if necessary, for achieving a maximum percentage of sequence identity, and any conservative substitutions are not considered as part of the identical sequence of the notes. Alignment carried out in order to determine the percent identity of amino acid sequences can be carried out by various methods known to those skilled in the art, for example using commonly available computer programs such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR). The person skilled in the art can independently determine the appropriate alignment parameters, including the use of any algorithms necessary to achieve optimal alignment along the entire length of the compared sequences. However, for the purposes of the present invention,% amino acid sequence identity is calculated using ALIGN-2 computer programs used for sequence comparison. The computer program ALIGN-2, designed to compare sequences, was developed by Genentech, Inc. and the specified source code was registered in the user documentation in the United States Copyright Office, Washington, D.C., 20559, under registration number U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is a publicly available program provided by Genentech, Inc., South San Francisco, California. The ALIGN-2 program must be designed for use on a UNIX operating system, preferably a digital UNIX V4.0D system. All sequence comparison parameters were set in the ALIGN-2 program and were not changed.

If the ALIGN-2 program is used to compare amino acid sequences, then the% identity of a given amino acid sequence A with a given amino acid sequence B (which could alternatively be called a given amino acid sequence A having or constituting a certain% identity with a given amino acid sequence B) is calculated as follows way:

100 X / Y,

where X represents the number of amino acid residues that were estimated to be completely consistent when aligning the sequences using the ALIGN-2 program, which compared sequences A and B, and where Y represents the total number of amino acid residues in sequence B. note that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then% identity of amino acid sequence A of amino acid sequence In atelnosti not equal the% amino acid sequence identity in amino acid sequence A.

Unless otherwise specified, the total% identity of the amino acid sequences used here was calculated as described in the previous paragraph using the ALIGN-2 computer program.

As used herein, the term “DLL4” (which is synonymous with the term “delta-like 4”) means, unless otherwise specified or from the context of the invention, any native or modified (natural or synthetic) DLL4 polypeptide. The term “native sequence”, in particular, encompasses natural truncated or secreted forms (eg, extracellular domain sequence), natural variants (eg, alternatively spliced forms), and natural allelic variants. The term "wild-type DLL4" basically means a polypeptide containing the amino acid sequence of the natural DLL4 protein. The term “wild-type DLL4 sequence” basically means the amino acid sequence present in native DLL4.

As used herein, the term "Notch receptor" (which is synonymous with the term "Notch") means, unless otherwise specified or as follows from the context of the invention, any native or modified (natural or synthetic) Notch receptor polypeptide. In humans, there are four Notch receptors (Notch1, Notch2, Notch3, and Notch4). As used herein, the term "Notch receptor" includes any one or all four human Notch receptors. The term “native sequence”, in particular, encompasses natural truncated or secreted forms (eg, extracellular domain sequence), natural variants (eg, alternatively spliced forms), and natural allelic variants. The term "wild-type Notch receptor" generally means a polypeptide containing the amino acid sequence of a natural Notch receptor protein. The term “wild-type Notch receptor sequence” generally means an amino acid sequence present in a Notch native receptor.

As used herein, the terms “antibody” and “immunoglobulin” are used synonymously in the broadest sense and include monoclonal antibodies (eg, full-size or intact monoclonal antibodies), polyclonal antibodies, polyvalent antibodies, multispecific antibodies (eg, bispecific antibodies, provided that they possess the desired biological activity) may also include some antibody fragments (described in more detail below). The antibody may be human, humanized and / or affinity matured.

The term “variable” refers to certain parts of the variable domains that have significant differences in the sequences of different antibodies and are involved in the binding of each specific antibody to its specific antigen, also determine the specificity of each specific antibody with respect to its specific antigen. However, variability is not evenly distributed across all variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions (HVRs) present in the variable domains of both light and heavy chains. The most highly conserved parts of variable domains are called framework regions (FR). Each variable domain of the native heavy and light chains contains four FRs having mainly a β-folded configuration and connected by three CDRs that form loops connecting, in some cases, forming part of the β-folded structure. The hypervariable regions (CDRs) in each chain are held in close proximity to each other by FR, and, together with the CDRs of the other chain, are involved in the formation of the antigen-binding site of antibodies (see Kabat et al. Sequences of Proteins of Immunological Interest , Fifth Edition, National Institutes of Health, Bethesda, MD (1991)). The constant domains do not directly participate in the binding of the antibody to the antigen, but have various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity.

Hydrolysis of antibodies by papain leads to the production of two identical antigen binding fragments, called Fab fragments, each of which has one antigen binding site, and another Fc fragment, the designation of which indicates its ability to rapidly crystallize. Pepsin treatment leads to the formation of an F (ab ') ₂ fragment, which has two antigen-binding sites and has the ability to cross-bind to the antigen.

"Fv" is a minimal antibody fragment containing a full-sized antigen recognition site and an antigen binding site of the antibody. In double-stranded Fv, this region consists of a dimer, which includes one variable domain of the heavy chain and one variable domain of the light chain, rigidly connected by a non-covalent bond. In a single-chain Fv, one variable domain of the heavy chain and one variable domain of the light chain can be covalently linked by a flexible peptide linker, as a result of which the light and heavy chains can be associated with each other to form a “dimeric” structure similar to the structure of a double-chain Fv. It has a configuration in which three CDRs of each variable domain interact with each other and define an antigen-binding site on the surface of the VH-VL dimer. In total, six CDRs confer antigen binding specificity to this antibody. However, even one variable domain (or half of the Fv containing only three CDRs specific for the antigen) may be able to recognize the antigen and bind to this antigen, although it may have a lower affinity than the full binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab'fragments differ from Fab fragments by several residues attached at the carboxy-end of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is Fab 'in which the cysteine (e) residue (s) of the constant domains have a free thiol group. By their nature, F (ab ') ₂ fragments of an antibody are produced as pairs of Fab'fragments that are linked together by hinged cysteine residues. Other chemical bonds of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) originating from vertebrates of any kind can be assigned to one of two completely different types of chains, called kappa (k) and lambda (λ), based on the amino acid sequences of their constant domains.

Immunoglobulins, depending on the amino acid sequence of the constant domain of their heavy chains, can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of them can also be divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to immunoglobulins of various classes are denoted by α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional configurations of immunoglobulins of various classes are well known in the art.

“Antibody fragments” contain only a portion of an intact antibody, wherein said portion, if present in the intact antibody, preferably retains at least one function, preferably most of the functions, or all functions normally found in that part of a natural intact antibody. Examples of antibody fragments are Fab, Fab'-, F (ab ') ₂ - and Fv fragments; diantibodies; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment of the invention, the antibody fragment contains the antigen-binding site of the intact antibody, therefore, it retains its ability to bind to the antigen. In another embodiment, an antibody fragment, for example, a fragment containing an Fc region, if present in an intact antibody, retains at least one of the biological functions typically found in the Fc region of an intact antibody, such as binding to FcRn, the ability to modulate time antibody half-life, ADCC function and complement binding. In one embodiment of the invention, the antibody fragment is a monovalent antibody that has an in vivo half-life substantially similar to the half-life of an intact antibody. So, for example, such an antibody fragment may contain an antigen binding region attached to the Fc sequence and capable of imparting stability to the said fragment in vivo .

As used herein, the terms “hypervariable region”, “HVR” or “HV” mean regions of the variable domain of an antibody that are hypervariable in this sequence and / or form loops of a specific structure. In general terms, these antibodies contain six hypervariable regions; three areas in VH (H1, H2, H3) and three areas in VL (L1, L2, L3). This application uses a number of hypervariable regions, which is included in the scope of the present invention. Kabat hypervariable regions (complementarity determining regions (CDRs)) have a high degree of sequence variability and are most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda , MD. (1991)). Instead of defining hypervariable regions by Kabat, Chotia proposed defining hypervariable regions by localization of structural loops (Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987)). The definition of the hypervariable AbM regions is a compromise between the Kebat “CDR” and the Chotia “structural loops”, and such definitions are used in Oxford Molecular's AbM antibody modeling software. “Contact” hypervariable regions are determined based on an analysis of the existing complex crystalline structures. The remains of each of these hypervariable regions are given below:

The loop Kabatu AbМ By Chotia Contact areas L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35V H26-H35V H26-H32 N30-N35V (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Cotia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

The term “hypervariable regions” may include “elongated hypervariable regions”, namely: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in VL; and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. The remains of the variable domains for each of these definitions are numbered according to Kebat et al., See above.

“Frame” or “FR” residues are those of the variable domains, with the exception of those of the hypervariable regions defined above.

As used herein, the term “monoclonal antibody” means an antibody derived from a population of substantially homogeneous antibodies, that is, the individual antibodies within such a population are identical and / or bind to the same (same) epitope (s) ), with the exception of the possible variants that can be formed during the production of a monoclonal antibody, where these variants are mainly present in small quantities. Such a monoclonal antibody is typically an antibody containing a target binding polypeptide sequence, wherein said target binding polypeptide sequence is prepared by a method that comprises selecting one target binding polypeptide sequence from a plurality of polypeptide sequences. So, for example, such a selection method can be a selection of a unique clone from a variety of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be noted that the selected target-binding sequence can also be modified, for example, to increase the affinity of the target, to humanize the sequence that binds to the target, to improve its production in cell culture, to reduce its immunogenicity in vivo , to create a multispecific antibody and the like, and that said antibody comprising a modified target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which are usually different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against one determinant on an antigen. Monoclonal antibody preparations, in addition to their specificity, have other advantages, that is, they usually do not contain other impurity immunoglobulins. The term "monoclonal" defines the nature of the antibody, obtained mainly from a homogeneous population of antibodies, but does not mean that this antibody must be produced by any specific method. For example, the monoclonal antibodies used in accordance with the present invention can be obtained by various methods, including, for example, the hybridoma method (e.g., Kohler et al., Nature , 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier, NY, 1981)), recombinant DNA methods (see , for example, US patent No. 4816567), phage presentation technologies (see, for example, Clackson et al., Nature , 352: 624-628 (1991); Marks et al., J. Mol. Biol. , 222: 581- 597 (1991); Sidhu et al., J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al., J. Mol. Biol . 340 (5): 1073-1093 (2004) ; Fellouse, Proc. Nat. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al. J. Immunol. Methods 284 (1-2): 119-132 (2004), and methods for producing human or humanized antibodies in animals that have parts or all of the loci of the human immunoglobulin or genes encoding the sequences of the human immunoglobulin (see, for example, WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature , 362: 255-258 (1993); Bruggemann et al., Year in Immuno. 7:33 (1993); U.S. Patent Nos. 5,545,806; 5,569,825; 5,591,669 (all from GenPharm); 5,545,807; WO 1997/17852; U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5633425 and 5661016; Marks et al., Bio / Technology , 10: 779-783 (1992); Lonberg et al., Nature , 368: 856-859 (1994); Morrison, Nature , 368: 812-813 (1994); Fishwild et al., Nature Biotechnology , 14: 845-851 (1996); Neuberger, Nature Biotechnology , 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

“Humanized” forms of non-human antibodies (eg, murine antibodies) are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (a recipient antibody) in which residues derived from the hypervariable region of a given recipient antibody are replaced by residues derived from a hypervariable region of a non-human antibody (donor antibody), such as a mouse antibody, a rat antibody , a rabbit or non-human primate antibody, wherein said antibodies have the desired specificity, affinity and binding ability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues not found in the recipient antibody or in the donor antibody. These modifications are introduced to improve the properties of the antibodies. In general terms, a humanized antibody may comprise substantially all or at least one, typically two variable domains in which all or almost all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or almost all of the FRs are FR with human immunoglobulin sequence. A humanized antibody also contains, but not necessarily, at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin region. For a more detailed description, see Jones et al. Nature 321: 522-525 (1986); Riechmann et al. Nature 332: 323-329 (1998) and Presta Curr. Op. Struct. Biol ., 2: 593-596 (1992). See also the following review articles and references cited there: 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).

"Chimeric antibodies" (immunoglobulins) have a part of the heavy and / or light chain that is identical or homologous to the corresponding sequences of antibodies originating from a particular species or belonging to a particular class or subclass, the remaining chain (s) are identical (s) or homologous ( s) the corresponding sequences of antibodies derived from another species or belonging to another class or subclass of antibodies, also from fragments of such antibodies, provided that they have the desired biological activity (US patent No. 4816567; Morrison et al, Proc Natl Acad Sci USA 81:..... 6851-6855 (1984)). The humanized antibody used herein is a subsequence of chimeric antibodies.

A “single chain Fv fragment” or “scFv fragment” of an antibody includes the VH and VL domains of an antibody, wherein said domains are present in a single polypeptide chain. Typically, the scFv polypeptide also contains between the VH and VL domains a polypeptide linker that provides the formation of scFv with the structure necessary for binding to the antigen. A description of scFv can be found in Pluckthun's The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg & Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "antigen" means a predefined antigen with which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other natural or synthetic compound. A preferred target antigen is a polypeptide.

The term “diantibodies” means small antibody fragments with two antigen-binding sites, where these fragments include the heavy chain variable domain (VH) linked to the light chain variable domain (VL) in the same polypeptide chain (VH-VL). If the linker is too short to form a pair between two domains on the same chain, then the domains are forced to mate with the complementary domains of the other chain and form two antigen-binding sites. Diantibodies are described in more detail, for example, in EP 404097; in 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 an antibody produced in humans and / or obtained by any of the methods for constructing human antibodies described herein. Such a definition of a human antibody, in particular, excludes a humanized antibody containing non-human antigen binding residues.

An "affinity matured" antibody is an antibody having one or more modifications in one or more CDRs, which allows to increase the affinity of the antibody for the antigen, as compared to the parent antibody that does not have such (their) modification (s). Preferred affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are prepared by known methods. Marks et al. ( Bio / Technology 10: 779-783 (1992) describe affinity maturation resulting from permutation of the VH and VL domains. Nonspecific mutagenesis of CDRs and / or framework residues is described in Barbas et al., Proc. Natl. 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).

The term "effector functions" of antibodies means biological activities inherent in the Fc region (Fc region of a native sequence or Fc region of a variant of an amino acid sequence) of an antibody and varying depending on the isotype of the antibody. Examples of antibody effector functions are C1q binding and complement dependent cytotoxicity; binding to the Fc receptor; antibody dependent cell mediated cytotoxicity (ADCC); phagocytosis; negative regulation of cell surface receptors (e.g., B-cell receptor); and B cell activation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” means a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (eg, natural killer cells (NK), neutrophils and macrophages ) allows these cytotoxic effector cells to specifically bind to antigen-containing target cells and cause the death of target cells under the action of cytotoxins. These antibodies “arm” cytotoxic cells and are absolutely necessary for such cytolysis. Primary cells mediating ADCC, i.e., NK cells, express FcγRIII only, while monocytes express FcγRI, FcγRII and FcγRIII. The data obtained for the expression of FcR on hematopoietic cells are summarized in Table 3 on page 464 of Ravetch & Kinet Annu. Rev. Immunol. (1991) 9: 457-92. In order to evaluate the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as the assay described in US Pat. Nos. 5,500,362 or 5,821,337, or Presta, in US Pat. No. 6,737,056. Mononuclear effector cells suitable for such assays are peripheral blood cells (MCPC) and natural killer cells (NK). Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo , for example, in an animal model, such as the model described by Clynes et al., Proc. Natl. Acad. Sci., USA 95: 652-656 (1998).

“Human effector cells” are white blood cells that express one or more FcRs and perform effector functions. Preferably, such cells express at least FcγRIII and perform an ADCC effector function. Examples of human leukocytes mediating ADCC are peripheral blood mononuclear cells (MCPCs), natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils, with MCPC and NK being preferred. Effector cells can be isolated from a natural source, for example from blood.

The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody. Preferred FcR is human sequence native FcR. In addition, a preferred FcR is FcR, which binds to an IgG antibody (gamma receptor), and receptors of subclasses FcγRI, FcγRII and FcγRIII, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors are FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains in its cytoplasmic domain an tyrosine-based immunoreceptor motif (ITAM). The inhibitory receptor FcγRIIB contains in its cytoplasmic domain an tyrosine-based immunoreceptor inhibitory motif (ITIM). (see review by M. Daeron, Annu. Rev. Immunol ., 15: 203-234 (1997)). FcRs are described in Ravetch & Kinet, Annu. Rev. Immunol . 9: 457-92 (1991); Capel et al., Immunomethods, 4: 25-34 (1994) and de Haas et al., J. Lab. Clin. Med ., 126: 330-41 (1995). As used herein, the term “FcR” also covers other FcRs, including FcRs, which will be identified in the future. The term also includes a neonatal receptor, FcRn, which is responsible for the transmission of maternal IgG to the fetus (Guyer et al., J. Immunol ., 117: 587 (1976) and Kim et al., J. Immunol ., 24: 249 (1994)), and regulates immunoglobulin homeostasis. WO 00/42072 (Presta) describes antibody variants with increased or decreased levels of binding to FcR. The contents of this patent publication are specifically incorporated into this description by reference. See also Shields et al. J. Biol. Chem. 9 (2): 6591-6604 (2001).

Methods for measuring the level of binding to FcRn are known to those skilled in the art (see, for example, Ghetie 1997, Hinton, 2004). In vivo binding to human FcRn and the half-life of polypeptides having high binding affinity for human FcRn in serum can be evaluated, for example, in transgenic mice or in transfected human cell lines expressing human FcRn, or in primates that have been introduced with variants Fc polypeptides.

“Complement dependent cytotoxicity” or “CDC” means the lysis of target cells in the presence of complement. The classical complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (corresponding subclass) that bind to their cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described by Gazzano-Santoro et al., J. Immunol. Methods, 202: 163 (1996).

Polypeptide variants having modified amino acid sequences of the Fc region and having increased or decreased ability to bind to C1q are described in US patent No. 6194551B1 and in WO 99/51642. The contents of these patent publications are specifically incorporated into this description by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “Fc region containing polypeptide” means a polypeptide, such as an antibody or immunoadhesin (as defined below), which contains an Fc region. The C-terminal lysine (residue 447 according to the European Numbering System EU) of the Fc region can be removed, for example, by purification of the polypeptide or by recombinant construction of a nucleic acid encoding the polypeptide. Accordingly, a composition comprising a polypeptide having an Fc region of the invention may comprise polypeptides with a K447 residue; polypeptides where all K447 have been removed; or a mixture of polypeptides containing and not containing residue K447.

A “blocking” antibody or “antagonist antibody” is an antibody that inhibits or reduces the biological activity of the antigen with which it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

As used herein, the term “agonist antibody” means an antibody that mimics at least one of the functional activities of a polypeptide of interest.

An “acceptor human framework region” used for the purposes of the present invention is a framework region comprising the amino acid sequence of a VL or VH framework region derived from a framework region of a human immunoglobulin or from a consensus framework region of a human immunoglobulin. The human acceptor framework region "originating from" the framework region of the human immunoglobulin or from the consensus framework region of the human immunoglobulin may include the same amino acid sequence, or it may contain an amino acid sequence with existing modifications. If amino acid modifications are already present, then it is preferable that their number be no more than 5, preferably 4 or less or 3 or less. If amino acid modifications are already present in VH, then it is preferable that such modifications are present only in three, two or in one of the positions 71H, 73H and 78H; for example, amino acid residues at such positions may be 71A, 73T and / or 78A. In one embodiment of the invention, the sequence of the acceptor human VL framework region is identical to the sequence of the VL framework region of the human immunoglobulin or the consensus sequence of the human immunoglobulin framework region.

A “consensus sequence of a human framework region” is a framework region that has the most common amino acid residue in a set of human immunoglobulin VL or VH framework sequences. Generally speaking, human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences. Typically, a subgroup of such sequences is a subgroup described by Kebat et al. In one embodiment of the invention, the subgroup for VL is the Kappa I subgroup described by Kebat and others. In one embodiment of the invention, the subgroup for VH is subgroup III described by Kebat et al.

"Consensus sequence of the VH framework region of subgroup III" includes a consensus sequence derived from amino acid sequences in the variable region of the heavy chain of subgroup III described by Kebat et al. In one embodiment of the invention, the consensus amino acid sequence of the framework region of VH subgroup III contains at least some or all the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 42) -H1-WVRQAPGKGLEWV (SEQ ID NO: 43) -H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 44) -H3-WGG.

A “consensus sequence of the VL subgroup I framework region” includes a consensus sequence derived from amino acid sequences in the variable region of the kappa subgroup I light chain described by Kabat et al. In one embodiment of the invention, the consensus amino acid sequence of the VL subgroup I framework region contains at least a portion or all of the following sequences:

DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 46) -L1-WYQQKPGKAPKLLIY (SEQ ID NO: 47) -L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 48) -L3-FEQQTK

The term “disorder” or “disease” means any condition that is susceptible to treatment with a substance / molecule or method of the invention. Such disorders are chronic and acute disorders or diseases, including pathological conditions that create a predisposition to a given disorder in a given mammal. Non-limiting examples of the disorders described herein to be treated are malignant and benign tumors: carcinoma, blastoma, and sarcoma.

The terms “cell proliferative disorder” and “proliferative disorder” mean disorders associated with a certain degree of abnormal cell proliferation. In one embodiment of the invention, said cell proliferative disorder is cancer.

As used herein, the term “tumor” means all neoplastic cells undergoing growth and proliferation, whether they are benign or malignant, and all precancerous and cancerous cells and tissues. As used herein, the terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive.

The terms “cancer” and “cancerous” as used herein refer to the physiological state of mammals, which is usually characterized by unregulated cell growth / proliferation. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers are squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, abdominal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, cancer of the bladder, hepatoma, breast cancer, colon cancer, cancer of the rectum and colon, endometrial or uterine carcinoma, salivary carcinoma, kidney cancer, liver cancer, pre prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, stomach cancer, melanoma and various types of head and neck cancer. Violation of angiogenesis can lead to the development of many disorders that can be treated with the compositions and methods of the invention. Such disorders are non-neoplastic and neoplastic conditions. Neoplastic disorders are, but are not limited to, the conditions described above. Non-neoplastic conditions include, but are not limited to, unwanted or aberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriasis plaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabetic and other proliferative retinopathy, neuropathic retinopathy, retinopathy and neuropathy glaucoma, senile macular degeneration, diabetic macular edema, corneal neovascularization, corneal neovascularization, corneal rejection corneal graft, neovascularization of the retina / choroid, neovascularization of the angle of the eyeball (redness of the eye), neovascular disease of the eyes, vascular restenosis, arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma, thyroid gland hyperplasia, including disease, associated with corneal transplantation of the eye and other tissues; chronic inflammation, pneumonia, acute lung injury / adult respiratory distress syndrome (RDSV), sepsis, primary pulmonary hypertension, malignant exudates in the lung area, cerebral edema (e.g. associated with stroke / closed injury / head injury), synovitis, pannus formation in RA, ossifying myositis, bone hypertrophy, osteoarthritis (OA), untreatable ascites, polycystic ovary, endometriosis, diseases associated with fluid sequestration in the third space (polo hollow organs) (pancreatitis, tunnel syndrome, burns, bowel disease), uterine fibroids, premature birth, chronic inflammation such as IBD (Crohn’s disease and ulcerative colitis), renal allograft rejection, inflammatory bowel disease, nephrotic syndrome, unwanted or aberrant tissue mass (non-cancerous tissue) growth, hemophilic arthropathy, hypertrophic scars, hair growth suppression, Osler-Weber syndrome, pyogenic granuloma, retrolental fibroplasia, scleroderma, trachoma, vask lar adhesion (fusion vessels), synovitis, dermatitis, preeclampsia, ascites, pericardial effusions region (e.g., associated with pericarditis) and pleural effusions.

As used herein, the term “treatment” means clinical intervention in order to alter the natural processes occurring in the body of an individual and in the cells being treated, and such treatment can be prophylactic, or it can be carried out in the process of developing clinical pathology. The desired effects of treatment are the prevention of relapse or a decrease in the frequency of relapses, the alleviation of symptoms, the reduction of any direct or indirect pathological consequences of the disease, the prevention of metastases, the reduction in the degree of progression of the disease, the weakening or reduction in the degree of the pathological condition, and the remission or improvement in prognosis. In some embodiments of the invention, the antibodies of the invention are used to slow the progression of a disease or disorder.

The term “individual” means a vertebrate, preferably a mammal, most preferably a human. The term “mammals” includes, but is not limited to, farm animals (such as cows), animals participating in sports, pets (such as cats, dogs and horses), primates, mice and rats.

“Mammals” to be treated are any animals classified as mammals, including humans, domestic and farm animals, also animals kept in zoos, animals involved in sports or pets, such as dogs, horses, cats, cows and etc. Preferred mammals are humans.

The term “effective amount” means an amount that is effective at certain doses and for a certain period of time necessary to achieve the desired therapeutic or prophylactic result.

The “therapeutically effective amount” of a substance / molecule according to the invention, an agonist or antagonist may vary depending on factors such as the pathological condition, age, gender and weight of the individual, as well as the ability of the substance / molecule, agonist or antagonist to produce the desired response in the individual. A therapeutically effective amount is also the amount upon administration of which the therapeutically beneficial effects of said substance / molecule, agonist or antagonist prevail over any toxic or side effects thereof. The term “prophylactically effective amount” means an amount that is effective at certain doses and for a certain period of time necessary to achieve the desired preventive result. Since the prophylactic dose is administered to individuals before the onset of the disease or at an early stage of the development of the disease, usually, but not necessarily, such a prophylactically effective amount should be less than a therapeutically effective amount.

As used herein, the term “cytotoxic agent” means a substance that inhibits or prevents the function of cells and / or causes destruction of cells. This term includes radioactive isotopes (e.g. At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu radioactive isotopes), chemotherapeutic agents, e.g. methotrexate, adriamycin, vinca alkaloids ( vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and their fragments, such as nucleolytic enzymes, antibiotics and toxins, such as small molecule toxins, or enzymatically active bacterial toxins, fungal, vegetable or animal origin, including fragments and / or variants thereof, and various antitumor or anticancer agents described below. Other cytotoxic agents are described below. An antitumor agent causes the destruction of tumor cells.

A “chemotherapeutic agent” is a chemical compound used to treat cancer. Examples of chemotherapeutic agents are alkylating agents such as thiotepa and cyclophosphamide cytoxan®; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboxwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylol melamine; acetogenins (and in particular, bullatacin and bullatacin); delta-9-tetrahydrocannabinol (dronabinol, marinol®); beta lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue of topotecan (gikamtin®), CPT-11 (irinotecan, camptosar®), acetyl camptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callistatin; CC-1065 (including its synthetic analogues, such as adoselesin, carzelesin and biselezin); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pankratistatin; sarcodiktiin; spongistatin; nitrogen analogues of mustard gas, such as chlorambucil, chlorofazine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembikhine, phenesterol, prednimustine, trophosphamide, uracil analog of mustard gas; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediin antibiotics (e.g. calicheamicin, in particular calicheamicin gamma II and calicheamicin omega II (see, for example, Agnew, Chem. Intl. Ed. Engl. , 33: 183-186 (1994)); dynamin, including dynemycin A; esperamycin, also neocarzinostatin chromophore and related chromophores, such as chromoprotein enediin antibiotics), aclacinomisins, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, carcinomycinin, carcinomycininazin, 5-oxo-L-norleucine, doxirubicin ad iamycin® (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycin, ritomicinomycinomycinomycin, pomidicinomycinomycinomycinomycetomyrinomyrinomycinomycinomyrolithrominomyrinomycinomycinomycinomyrolithrominomyrinomycinomycinomyrolithrominomyrinomycinomycinomycinomyrolithinomycinomycinomycinomyrolithinomycinomycinomyrolithinomycinomycinomycinomyrolithinomycinomycinomyrolithinomyrinomycinomycinomycinomyrolithinomyrinomycinomycinomycinomycinomycinomycinomyrolithinomycinomycinomycinomycinomycinomycinomycinomyretinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomyr? , tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimerexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxyfluuridine, enocytabine, phloxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostan, testolactone; antiadrenergic agents such as aminoglutethimide, mitotan, trilostane; a folic acid substitute such as frolinic acid; Aceglaton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisanthrene; edatraxate; defofamine; demecolcin; diaziquon; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytazine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pyrarubicin; losoxantone; 2-ethyl hydrazide; procarbiazcin; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spiro germanium; tenuazonic acid; triaziquon; 2,2 ', 2 ”trichlorotriethylamine; trichotecenes (and in particular, T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine (eldizin®, fildesin®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacitosin; arabinoside (“Ara-C”); thiotepa; taxoids, for example, paclitaxel Taxol® (Bristol-Myers Squibb Oncology, Princeton, NJ); non-cremophoric Abraxan ^TM , albumin-based composition of paclitaxel nanoparticles (American Pharmaceuticals Partners, Schaumberg, Illinois) and docetaxel Taxotere® (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (Gemzar®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (Velban®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (Oncovin®); oxaliplatin; leucovorin; vinorelbine (Navelbin®); novantron; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMF); retinoids such as retinoic acid; capecitabine (Xeloda®); pharmaceutically acceptable salts, oxides or derivatives of all of the above compounds, also combinations of two or more of the above compounds, such as CHOP, where the abbreviation is used in combination therapy and means cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, where the abbreviation is used in combination therapy and means oxaliplatin (Eloxatin®), 5-FU and leukovin.

This definition also includes anti-hormonal agents that regulate, reduce, block or inhibit the action of hormones that can stimulate the growth of a cancerous tumor, and these agents are often used for systemic or general treatment. These may include hormones themselves. Examples of such agents are antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen Nolvadex®), raloxifene Evista®, droloxifene, 4-hydroxy tamoxifen, trioxifen, keoxifen, LY117018, onapristone toremone; antiprogesterones; estrogen receptor inhibitors (ERD); agents that suppress or block ovarian function, for example, luteinizing hormone releasing hormone (LHRH) agonists such as Lupron® and Eligard® leuprolide acetate, goserelin acetate, buzerelin acetate and trypterelin; other antiandrogens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the aromatase enzyme and regulate the production of estrogen in the adrenal cortex, such as, for example, 4 (5) -imidazoles, aminoglutetimide, megestrol acetate Megaz®, exemestane Aromazin®, formestane, fadrozole, Vorozol Rivizor®, letrozole Anastrozole Arimidex®. In addition, this definition of chemotherapeutic agents includes bisphosphonates such as clodronate (e.g. Bonefos® or Ostak®), ethidronate Didrocal®, NE-58095, zoledronic acid / zoledronate Zometa®, alendronate Fozamax®, pamidronate Aredia®, tiludronate Skelid® or risedronate Actonel®, also troxacitabine (nucleoside analogue of 1,3-dioxolancytosine); antisense oligonucleotides, in particular oligonucleotides that inhibit gene expression of signaling pathways involved in unwanted cell proliferation, such as, for example, PKC alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as Teratopa® vaccine and vaccines used in gene therapy, for example Allovectin® vaccine, Leivectin® vaccine and Vaxid® vaccine; topoisomerase 1 inhibitor Lurtotecan®; rmRH Abarelix®; lapatinib ditosylate (a tyrosine kinase inhibitor consisting of two small molecules Erb-2 and EGFR, also known as GW 572016) and pharmaceutically acceptable salts, oxides or derivatives of all of the above compounds.

The term “growth inhibitory agent” as used herein means a compound or composition that inhibits cell growth (such as cells expressing DLL4), either in vitro or in vivo . Thus, an agent that inhibits cell growth can be an agent that significantly reduces the percentage of cells (such as cells expressing DLL4) in phase S. Examples of agents that inhibit cell growth are agents that block the cell cycle (in a phase other than phase S), such as phase G1 and phase M blocking agents. Classical phase M blocking agents are vinca alkaloids (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etop ozide and bleomycin. G1 phase blocking agents that also block S phase transitions are, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn & Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes and antineoplastic drugs”, Murakami et al. (WB Saunders: Philadelphia, 1995), in particular, on page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), obtained from European yew, is a semi-synthetic analogue of paclitaxel (Taxola®, Bristol-Myers Squibb). Paclitaxel and docetaxel induce the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which leads to inhibition of cell mitosis.

“Doxorubicin” is an anthracycline antibiotic. Doxorubicin has the full chemical name (8S-cis) -10 - [(3-amino-2,3,6-trideoxy-α-L-dioxo-hexapyranosyl) oxy] -7,8,9,10-tetrahydro-6, 8,11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5,12-naphthacenedione.

The term “antitumor composition” means a composition that can be used to treat cancer and containing at least one active therapeutic agent, for example, “anti-cancer agent”. Examples of therapeutic agents (anti-cancer agents, also referred to herein as “anti-tumor agents”) include, but are not limited to, for example, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents , toxins and other cancer treatments, for example, a neutralizing anti-VEGF antibody, a VEGF antagonist, an anti-HER-2 antibody, an anti-CD20 antibody, an epidermal receptor antagonist growth factor (EGFR) (e.g. tyrosine kinase inhibitor), HER1 / EGFR inhibitor, erlotinib, COX-2 inhibitor (e.g. celecoxib), interferons, cytokines, antagonists (e.g. neutralizing antibodies) that bind to one or more ErbB2 receptors, ErbB3, ErbB4 or VEGF, inhibitors of platelet growth factor tyrosine kinase receptors (PDGF) and / or stem cell factor (SCF) (e.g. imatinib mesylate (Gleevec ® Novartis)), TRAIL / Apo2L, and other biologically active agents and organic chemicals etc.

As used herein, the term “prodrug” means a precursor or derivative of a pharmaceutically active substance that, compared to the parent drug, is less cytotoxic to tumor cells and is capable of enzymatically activating or transforming into a more active mature 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., (Ed.), 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 drugs; glycosylated prodrugs; β-lactam-containing prodrugs; prodrugs containing optionally substituted phenoxyacetamide; or prodrugs containing, but not necessarily, substituted phenylacetamide; 5-fluorocytosine and other 5-fluoruridine prodrugs that can be converted into a more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized to produce a prodrug form for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.

The term “anti-angiogenic agent” or “angiogenesis inhibitor” means a low molecular weight substance, a polynucleotide (including, for example, an inhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, a recombinant protein, an antibody or its conjugates, or fusion proteins that are directly or indirectly inhibit angiogenesis, vasculogenesis, or unwanted vascular permeability. So, for example, an anti-angiogenic agent is an antibody or other antagonist against an angiogenic agent as defined above, for example, anti-VEGF antibodies, antibodies against VEGF receptors, fragments of soluble VEGF receptors or small molecules that block VEGF receptor signaling (e.g. PTK787 / ZK2284, SU6668, SUTENT® / SU11248 (sunitinib malate), AMG706 or the molecules described, for example, in International Patent Application WO 2004/113304). Antiangiogenic agents are also natural angiogenesis inhibitors, for example, anti-statin, 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) (for example, Table 3, which lists antiangiogenic agents for treating malignant melanoma); Ferrara & Alitalo, Nature Medicine 5 (12): 1359-1364 (1999); Tonini et al., Oncogene , 22: 6549-6556 (2003) (e.g., Table 2, which lists antiangiogenic factors); and Sato Int. J. Clin. Oncol. , 8: 200-206 (2003) (for example, table 1, which lists antiangiogenic agents used in clinical trials).

Compositions according to the invention and methods for their preparation

The present invention relates to compositions, including pharmaceutical compositions containing an anti-DLL4 antibody, and to polynucleotides containing sequences encoding an anti-DLL4 antibody. The compositions used herein comprise one or more antibodies that bind to DLL4 and / or one or more polynucleotides containing sequences encoding one or more antibodies that bind to DLL4. Such compositions may also contain suitable carriers, such as pharmaceutically acceptable excipients, including buffers well known in the art.

The present invention also encompasses variants of isolated antibodies and polynucleotides. The present invention also encompasses variants of substantially pure antibodies and polynucleotides.

Anti-DLL4 antibodies of the invention are preferably monoclonal antibodies. Fab, Fab'-, Fab'-SH- and F (ab ') ₂ fragments of the anti-DLL4 antibodies described herein are also within the scope of the present invention. These antibody fragments can be obtained by conventional methods, such as enzymatic hydrolysis, or they can be produced by recombinant DNA methods. These antibody fragments may be chimeric or humanized. These fragments can be used for diagnostic or therapeutic purposes, described below.

Monoclonal antibodies are obtained from a population of mainly homogeneous antibodies, that is, the individual antibodies that make up such a population are identical, with the exception of possible natural mutations, which may be present in small amounts. Thus, the term "monoclonal" defines the nature of the antibody, which is not a mixture of discrete antibodies.

The monoclonal anti-DLL4 antibodies of the invention can be prepared by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or they can be prepared by recombinant DNA methods (US Pat. No. 4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized to produce lymphocytes in it that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Antibodies against DLL4 are usually produced in animals after repeated subcutaneous (sc) or intraperitoneal (ip) injections of DLL4 and adjuvant. DLL4 can be obtained by well-known methods, some of which are described in more detail below. So, for example, recombinant production of DLL4 is described below. In one embodiment of the invention, animals are immunized with a DLL4 derivative that contains the extracellular domain (ECD) DLL4 attached to the Fc portion of the immunoglobulin heavy chain. In a preferred embodiment, the animals are immunized with a DLL4-IgG1 fusion protein. Animals are usually immunized with monophosphoryl lipid A (MPL) / trehalose dicrinomycolate (TDM) to elicit a response against immunogenic conjugates or DLL4 derivatives (Ribi Immunochem. Research, Inc., Hamilton, MT), and the resulting solution is injected transdermally into multiple sites. After two weeks, the animals are given a loading dose. After another 7-14 days, the animals were bled and the serum was analyzed for anti-DLL4 antibody titers. The animals are given a loading dose until the titer reaches a "plateau" (that is, a constant level).

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

The hybridoma cells thus obtained are seeded and cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of non-fused parental myeloma cells. For example, if the parent myeloma cells do not contain the hypoxanthine guanine phosphoribosyltransferase enzyme (HGPRT or HPRT), then the culture medium used to produce the hybridomas usually includes hypoxanthine, aminopterin and thymidine (NAT medium), that is, substances that prevent the growth of HGPRT -deficient cells.

Preferred myeloma cells are cells that are able to undergo efficient fusion, maintain stable production of high levels of antibodies by selected antibody-producing cells, and are sensitive to a medium, such as NAT medium. Of these cells, the preferred myeloma cell lines are murine myeloma lines, such as cell lines derived from murine tumor cells MORS-21 and MPC-11, available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP- cells. 2 or X63-Ag8-653, available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma cell lines and mouse-human heteromyeloma cell lines are also used to produce human monoclonal antibodies (Kozbor J. Immunol., 133: 3001 (1984) & Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51- 63 (Marcel Dekker, Inc., New York, 1987)).

The culture medium for the growth of hybridoma cells is analyzed for the production of monoclonal antibodies against DLL4. The specificity of binding of monoclonal antibodies produced by hybridoma cells is preferably determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of a monoclonal antibody can, for example, be determined using the Scatchard assay described by Munson et al., Anal. Biochem . 107: 220 (1980).

After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, clones can be subcloned by limiting dilution procedures and cultured using standard methods (Goding, Monoclonal Antibodies: Principles and Practice , pp. 59-103 (Academic Press, 1986)). Suitable culture media designed to achieve this goal are, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals.

Monoclonal antibodies secreted by subclones are appropriately isolated from culture medium, ascites fluid, or serum by standard immunoglobulin purification procedures, such as, for example, protein chromatography on A-Sepharose, chromatography on hydroxyapatites, gel electrophoresis, dialysis or affinity chromatography.

Anti-DLL4 antibodies according to the invention can be obtained using combinatorial libraries for screening for clones of synthetic antibodies with the desired activity or activities. In principle, clones of synthetic antibodies are selected by screening phage libraries containing a phage that represents various fragments of the antibody variable region (Fv) attached to the phage coat protein. Such phage libraries are subjected to panning by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed on the antigen and thereby separate them from non-binding clones in the library. Binding clones are then eluted from the antigen, after which the number of these clones can be increased by conducting additional antigen adsorption / elution cycles. Any of the anti-DLL4 antibodies of the invention can be prepared using a suitable antigen screening procedure for selection for a phage clone of interest, and then constructing a full-sized anti-DLL4 antibody clone using Fv sequences derived from the phage clone of interest and suitable sequences constant region (Fc) described by Kabat et al. , Sequences of Proteins of Immunological Interest , Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

The antigen binding domain of an antibody is formed from two variable (V) regions of approximately 110 amino acids, one in the light chain (VL), the other in the heavy chain (VH), and three hypervariable loops or complementarity determining in these variable regions region (CDR). Variable domains can be functionally represented on the phage either as single-chain Fv (scFv) fragments in which VH and VL are covalently linked to each other via a short flexible peptide, or as Fab fragments in which each of these variable domains is attached to a constant domain and undergoes non-covalent interaction, as described by Winter et al. Ann. Rev. Immunol. 12: 433-455 (1994). As used herein, scFv-encoding phage clones and Fab-encoding phage clones are generally referred to as “phage Fv clones” or “Fv clones”.

The VH and VL gene sets can be separately cloned by polymerase chain reaction (PCR) and subjected to non-specific recombination in phage libraries, then they can be analyzed for antigen-binding clones as described in Winter et al. Ann. Rev. Immunol. 12: 433-455 (1994). Libraries from immunized sources make it possible to obtain antibodies with high affinity for the immunogen, and thus do not require the construction of hybridomas. Alternatively, a wild-type human antibody kit can be cloned to produce a single source of human antibodies against non-autoantigens, as well as a wide range of autoantigens, without resorting to any immunization as described by Griffiths et al. EMBO J, 12: 725-734 (1993). Finally, the source libraries can also be synthesized by cloning unranged segments of the V gene from stem cells and using PCR primers containing a randomized sequence encoding highly variable CDR3 regions in order to achieve in vitro rearrangement as described by Hoogenboom & Winter, J. Mol. Biol. 227: 381-388 (1992).

Filamentous phage are used to represent antibody fragments by attaching them to the minor envelope protein pIII. Antibody fragments can be represented as single-chain Fv fragments in which the VH and VL domains are joined on the same polypeptide chain through a flexible polypeptide spacer, for example, as described by Marks et al. , J. Mol. Biol., 222: 581-597 (1991), or in the form of Fab fragments in which one chain is attached to pIII, the other is secreted into the periplasm of the bacterial host cell, where the structure of the Fab envelope protein is assembled, which is displayed on the surface of the phage by replacement some of the wild-type envelope proteins, for example, as described in Hoogenboom et al. , Nucl. Acids Res. 19: 4133-4137 (1991).

Typically, nucleic acids containing genes encoding antibody fragments are derived from immune cells taken from humans or animals. If it is necessary to obtain a library consisting mainly of clones of anti-DLL4 antibodies, the individual will be immunized with DLL4 to produce a humoral response, then spleen cells and / or circulating B cells that do not belong to the peripheral blood lymphocyte population (LPC) are isolated ) In a preferred embodiment of the invention, a gene library of fragments of a human antibody containing predominantly anti-DLL4 antibody clones is obtained by generating an anti-DLL4 antibody response in transgenic mice carrying an array of functional human immunoglobulin genes (and not containing a functional endogenous antibody producing system ), where such DLL4 immunization will lead to the formation of b cells producing human antibodies against DLL4. The generation of transgenic mice producing a human antibody is described below.

Additional enrichment of the cell population with reactive anti-DLL4 antibodies can be achieved using a suitable screening procedure to isolate B cells expressing a DLL4-specific membrane-bound antibody, for example, by cell separation using DLL4 affinity chromatography or cell adsorption on DLL4 labeled fluorochrome, and subsequent cell sorting with activation of fluorescence (FACS).

Alternatively, the use of spleen cells and / or B cells or other lipoproteins taken from an unimmunized donor provides a better representation of a possible antibody repertoire, and also allows the construction of an antibody library in animals of any kind (humans or other animals) in which DLL4 is not antigenic . To create libraries that include the construction of antibody genes in vitro , stem cells are taken from an individual and nucleic acids that encode un-arranged segments of antibody genes are isolated. Immune cells of interest can be isolated from animals of various species, such as humans, mice, rats, lagomorphs, wolves, dogs, cats, pigs, cows, horses, birds, and the like.

Nucleic acid encoding the antibody variable region gene segments (including VH and VL segments) are isolated from the cells of interest and amplified. In the case of using libraries of rearranged VH and VL genes, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) using primers corresponding to the 5 ′ and 3 ′ ends of the rearranged VH and VL genes, described in Orlandi et al. , Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), and thereby obtaining various sets of V genes for expression. V genes can be amplified from cDNA and genomic DNA using reverse primers at the 5 ′ end of the exon encoding the mature V domain and direct primers derived from the J segment as described in Orlandi et al. (1989) and Ward et al., Nature , 341: 544-546 (1989). However, for amplification from cDNA, reverse primers can also be obtained based on leader exon, as described in Jones et al. , Biotechnol. 9: 88-89 (1991), direct primers can be introduced into the constant region as described in Sastry et al., Proc. Natl. Acad. Sci. (USA) 86: 5728-5732 (1989). To maximize complementarity, degenerate primers can be prepared as described in Orlandi et al. (1989) or Sastry et al. (1989). Preferably, library diversity is maximized using PCR primers targeting each family of V genes in order to amplify all available VH and VL arrangements present in an immune cell nucleic acid sample, for example, by the method described in Marks et al., J. Mol . Biol. 222: 581-597 (1991), or by the method described in Orum et al., Nucleic Acids Res. 21: 4491-4498 (1993). For cloning amplified DNA into expression vectors, rare restriction sites may be introduced into the PCR primer as a label at one end, as described by Orlandi et al. (1989), or such an introduction can be carried out using PCR amplification using labeled primers, as described by Clackson et al., Nature, 352: 624-628 (1991).

Sets of synthetically rearranged V genes can be obtained in vitro from V gene segments. Most segments of human VH genes were cloned and sequenced (as reported in Tomlinson et al. , J. Mol. Biol., 227: 776-798 (1992)), then mapped (as reported in Matsuda et al. , Nature Genet ., 3: 88-94 (1993); and these cloned segments (including all major conformations of the H1 and H2 loops) can be used to obtain different sets of VH genes using PCR primers encoding H3 loops having different sequences and different lengths , as described in Hoogenboom and Winter, J. Mol Biol, 227:... 381-388 (1992) may also be prepared by n burs VH, having all the sequence diversity is centered in a long H3 loop of a single length as described in Barbas et al, Proc Natl Acad Sci USA, 89:...... 4457-4461 (1992) have been cloned and sequenced human segments Vκ and Vλ (as reported in Williams & Winter, Eur. J. Immunol ., 23: 1456-1461 (1993)) and such segments can be used to make synthetic light chain kits. The sets of synthesized V genes obtained on the basis of the folded structure of the VH and VL series, and L3 and H3 of different lengths will encode antibodies with a significantly varying structure. After amplification of the V gene coding DNA, the germline V gene segments can be rearranged in vitro by the methods described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Antibody fragment sets can be constructed by combining the VH and VL gene sets in several ways. Each set can be created in different vectors, and these vectors are subjected to in vitro recombination, for example, as described in the publication of Hogrefe et al. Gene , 128: 119-126 (1993), or in vivo by combinatorial infection, for example, with the loxP system described in Waterhouse et al. , Nucl. Acids Res. 21: 2265-2266 (1993). The in vivo recombination method uses the double-stranded nature of the Fab fragments to remove the restriction on library size and transformation efficiency of E. coli . The original sets of VH and VL are cloned separately: one into the phagemid, the other into the phage vector. Then the two libraries are combined by infection of the bacterium containing the phagemid, phage, so that each cell contains different combinations, the size of the library was limited only by the number of cells present (approximately 10 ¹² clones). Both vectors contain in vivo recombination signals, as a result of which the VH and VL genes undergo recombination to form one replicon and are together packaged in phage virions. These huge libraries represent a large number of different antibodies with high affinity (K _d ^-1 approximately 10 ^-8 M).

Alternatively, such kits can be sequentially cloned into the same vector, for example, as described in Barbas et al. , Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or they can be assembled using PCR, then cloned, for example, as described in the publication of Clackson et al. , Nature , 352: 624-628 (1991). PCR assembly can also be carried out to attach VH and VL DNA to DNA encoding a flexible peptide spacer to form single chain Fv (scFv) sets. In another method, “PCR assembly in cells” is used to combine VH and VL genes in lymphocytes by PCR followed by cloning of sets of linked genes, as described in Embleton et al. , Nucl. Acids Res. 20: 3831-3837 (1992).

Antibodies produced by the source libraries (natural or synthetic) may have low affinity (K _d ^-1 from about 10 ⁶ to 10 ⁷ M ^-1 ), however, affinity maturation can also be modeled in vitro by constructing and re-selecting from secondary libraries, as described in Winter et al. (1994), see above. For example, a mutation can be introduced accidentally in vitro using a polymerase reaction with a probability of error (reported in Leung et al. , Technique, 1: 11-15 (1989)) in the method described by Hawkins et al. , J. Mol. Biol., 226: 889-896 (1992) or the method described by Gram et al. , Proc. Natl. Acad. Sci USA 89: 3576-3580 (1992). In addition, affinity maturation can be accomplished by randomly mutating one or more CDRs, for example, by PCR using primers carrying a randomized sequence spanning CDR of interest in selected Fv clones, and by screening for clones with higher affinity. WO 9607754 (published March 14, 1996) describes a method for inducing mutagenesis in the hypervariable region of an immunoglobulin light chain to produce a library of light chain genes. Another effective method is the recombination of VH or VL domains selected by phage representation with sets of natural variants of the V domain isolated from non-immunized donors, and screening for higher affinity carried out in several rounds of chain permutation, as described in Marks et al . Biotechnol. 10: 779-783 (1992). This method allows the production of antibodies and antibody fragments with affinities in the range of 10 ^-9 M.

Nucleic acid sequences and amino acid sequences of DLL4 are known to those skilled in the art. The nucleic acid sequence encoding DLL4 can be constructed using the amino acid sequence of the desired region of DLL4. Alternatively, the cDNA sequence (or fragments thereof) is deposited in GenBank under reg. No. NM_019074. DLL4 is a transmembrane protein. The extracellular region contains 8 EGF-like repeats, also the DSL domain, which is conserved for all Notch ligands and necessary for binding to the receptor. The predicted protein also contains a transmembrane region and a cytoplasmic tail, which does not contain any catalytic motifs. The human DLL4 protein is a protein of 685 amino acids and contains the following areas: signal peptide (amino acids 1-25); MNNL (amino acids 26-92); DSL (amino acids 155-217); EGF-like region (amino acids 221-251); EGF-like region (amino acids 252-282); EGF-like region (amino acids 284-322); EGF-like region (amino acids 324-360); EGF-like region (amino acids 366-400); EGF-like region (amino acids 402-438); EGF-like region (amino acids 440-476); EGF-like region (amino acids 480-518); transmembrane region (amino acids 529-551); cytoplasmic domain (amino acids 552-685). Human DLL4 has registration number NM_019074, mouse DLL4 has registration number NM_019454.

DNAs encoding DLL4 can be obtained by various methods known to those skilled in the art. Such methods include, but are not limited to, chemical synthesis carried out by any of the methods described in Engels et al. , Agnew. Chem. Int. Ed. Engl. 28: 716-734 (1989), such as methods using triester, phosphite, phosphoramidite and H-phosphonate. In one embodiment of the invention, codons that are preferred for expression in host cells are used to construct the DLL4 coding DNA. Alternatively, DNA encoding DLL4 may be isolated from a genomic library or cDNA library.

After constructing the DNA molecule encoding DLL4, this DNA molecule is functionally attached to the expression control sequence in an expression vector, such as a plasmid, where the regulatory sequence is recognized by a host cell transformed by this vector. In general terms, plasmid vectors contain replication sequences and regulatory sequences derived from species compatible with the host cell. Such a vector typically contains a replication site, as well as sequences encoding proteins that allow phenotypic selection in transformed cells. Suitable vectors for expression in prokaryotic and eukaryotic host cells are known in the art, and some of them are described in more detail below. In this case, eukaryotic organisms such as yeast or cells derived from multicellular organisms such as mammals can be used.

DNA encoding DLL4 may optionally be operably linked to a secretory leader sequence, which may result in secretion of the expression product by the host cell in the culture medium. Examples of secretory leader sequences are stII, ecotin, lamB, HD (glutamate dehydrogenase) of the herpes virus, lpp, alkaline phosphatase, invertase and alpha factor. The leader sequence of protein A consisting of 36 amino acids can also be used in the present invention (Abrahmsen et al. , EMBO J. , 4: 3901 (1985)).

Host cells are transfected, preferably transformed with the above expression or cloning vectors according to the invention and cultured in suitable culture media, modified, if necessary, to induce promoters, select transformants or amplify genes encoding the desired sequences.

The term "transfection" means the absorption of the expression vector by the host cell, regardless of whether it expresses any coding sequences or not. Various transfection methods are known to those of ordinary skill in the art, and such methods are, for example, CaPO ₄ precipitation and electroporation. Successful transfection is usually determined by any sign indicating the presence of the indicated vector in the host cell. Transfection methods are well known in the art, and some of them are described in more detail in this application.

The term "transformation" means the introduction of DNA into the body so that it can replicate as an extrachromosomal element or as a chromosomal component. Depending on the host cell used, the transformation is carried out using standard methods suitable for such cells. Methods of transformation are well known to those skilled in the art and some of them are described in more detail in this application.

Prokaryotic host cells used to produce DLL4 can be cultured mainly as described in Sambrook et al. , see above.

Mammalian host cells used to produce DLL4 can be cultured in various media that are well known in the art, and some of which are described in this application.

The host cells described in this application include cells in an in vitro culture, also cells of an animal host.

The cleaning of DLL4 can be accomplished by well-known methods, some of which are described in this application.

Purified DLL4 can be bound to an appropriate matrix, such as agarose gel spheres, acrylamide spheres, glass beads, cellulose, various acrylic copolymers, hydroxyl methacrylate gels, polyacrylic and polymethacrylic acid copolymers, nylon, neutral and ionic carriers, etc., in their use in affinity chromatography for the separation of clones represented on phages. Attachment of the DLL4 protein to the matrix can be accomplished by any of the methods described in Methods in Enzymology , vol. 44 (1976). Methods commonly used to attach protein ligands to polysaccharide matrices, such as agarose, dextran, or cellulose, include activating the carrier with cyanogen halides and then binding the primary aliphatic or aromatic amines of the peptide ligands to the activated matrix.

Alternatively, DLL4 can be used to coat the wells of adsorption plates and expressed on host cells attached to adsorption plates, or it can be used in cell sorting or conjugated with biotin to bind to streptavidin coated spheres, or it can be used in any other a well-known method for panning phage view libraries.

Samples of phage libraries are contacted with immobilized DLL4 under conditions suitable for binding at least a portion of the phage particles to an adsorbent. Typically, appropriate pH, ionic strength, temperature, and the like are chosen to simulate physiological conditions. The phages associated with the solid phase are washed, then eluted with acid, for example, as described in Barbas et al. , Proc. Natl. Acad. Sci USA , 88: 7978-7982 (1991), or alkali, for example, as described in Marks et al. , J. Mol. Biol., 222: 581-597 (1991), or by competitive binding to the DLL4 antigen, for example, in accordance with a procedure similar to that performed in the competitive antigen binding method, as described in Clackson et al. , Nature , 352: 624-628 (1991). Phages can be subjected to 20-1000-fold enrichment in one round of selection. In addition, enriched phages can be grown in bacterial culture and subjected to additional rounds of selection.

The effectiveness of the selection depends on many factors, including the kinetics of dissociation during washing, and on the ability of multiple antibody fragments to simultaneously bind to the antigen on the same phage. Antibodies with fast dissociation kinetics (and weak binding affinity) can be fixed due to short-term washes, the use of multivalent phage representation and high density coating of antigen on the solid phase. Such a high density not only stabilizes the phage due to multivalent interactions, but also favors the re-binding of the phage, which is dissociated. The selection of antibodies with weak dissociation kinetics (and with high binding affinity) can be improved through longer washes and the use of the monovalent phage representation described in Bass et al. , Proteins , 8: 309-314 (1990) and in WO 92/09690, also due to the low density of antigen coating, as described in Marks et al. Biotechnol. 10: 779-783 (1992).

Selection can be made between phage antibodies with different affinities, even with a slightly different affinity for DLL4. However, a random mutation of a selected antibody (for example, introduced during the implementation of some of the methods for affinity maturation described above) is likely to lead to the formation of many mutants, most of which will bind to the antigen, some of them with even greater affinity. With a limited level of DLL4, a rare phage with high affinity can withstand this competition. To preserve all mutants with higher affinity, phages can be incubated with an excess of biotinylated DLL4, but this biotinylated DLL4 must have a lower molar concentration than the molar target affinity constant for DLL4. Then, phages with high binding affinity can be captured by paramagnetic spheres coated with streptavidin. Such an “equilibrium capture” makes it possible to select antibodies according to their binding affinity with sensitivity, which allows mutant clones with only twice as high affinity to be isolated from a large excess of phages with lower affinity. The conditions used for washing the phages associated with the solid phase can also be changed to reveal their differences based on the kinetics of dissociation.

Clones of anti-DLL4 antibodies can be selected for their activity. In one embodiment, the present invention provides anti-DLL4 antibodies that block the binding of a Notch receptor (such as Notch1, Notch2, Notch3 and / or Notch4) to DLL4, but do not block the binding of a Notch receptor to a second protein. Fv clones corresponding to such anti-DLL4 antibodies can be selected by: (1) isolating the anti-DLL4 antibody clones from the phage library as described above, and, optionally, amplifying the isolated phage clone population by culturing such a population in a suitable a bacterial host; (2) selecting DLL4 and the second protein against which it is necessary to produce blocking and non-blocking activity, respectively; (3) adsorption of phage clones of anti-DLL4 antibodies on immobilized DLL4; (4) using an excess of the second protein to elute any unwanted clones that recognize DLL4-binding determinants that overlap with the binding determinants of the second protein or share binding determinants with it; and (5) eluting clones that remain adsorbed after stage (4). Clones with the desired blocking / non-blocking properties may also be, but not necessarily, enriched by one or more of the repeated selection procedures described in this application.

DNA encoding the hybridoma-produced monoclonal antibodies or Fv clones of the phage representation of the invention can be easily isolated and sequenced according to standard procedures (for example, using oligonucleotide primers that are able to specifically amplify the coding regions of the heavy and light chains of interest from the hybridoma or phage DNA template). After isolation, this DNA can be inserted into expression vectors, which are then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells, which otherwise do not produce immunoglobulin protein, as a result, the desired monoclonal antibodies are synthesized in these recombinant host cells. A discussion of recombinant expression of antibody-coding DNA in bacteria can be found in the review articles by Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding constant regions of the heavy chain and / or light chain (for example, the corresponding DNA sequences can be obtained as described in Kabat et al. , See above) with the formation of clones encoding full-sized heavy and / or light chains or fragments thereof. It should be noted that for this purpose, constant regions of any isotype can be used, including constant regions of IgG, IgM, IgA, IgD and IgE, and such constant regions can be obtained from humans or animals of any species. The definition of the term “chimeric” and “hybrid” antibody as used herein includes an Fv clone derived from the DNA of the variable domain of an animal of one species (eg, human), which was then attached to the DNA of a constant region of an animal of another species to form the coding sequence (s) (s) a “hybrid” of a full-sized heavy chain and / or light chain. In a preferred embodiment of the invention, an Fv clone derived from human variable domain DNA is coupled to the human constant region DNA to form the coding sequence (s) for all human full-sized heavy and / or light chains and / or fragments thereof.

DNA encoding an anti-DLL4 antibody derived from a hybridoma according to the invention can also be modified, for example, by replacing homologous mouse sequences derived from a hybridoma clone with a coding sequence for human constant domains of the heavy and light chains (for example, as in the method described by Morrison et al. , Proc. Natl. Acad. Sci. USA , 81: 6851-6855 (1984)). DNA encoding an antibody derived from a hybridoma or Fv clone, or a fragment thereof, can also be modified by covalently linking an immunoglobulin coding sequence to a full-length sequence encoding a non-immunoglobulin polypeptide, or a part thereof. In this way, “chimeric” or “hybrid” antibodies can be obtained having binding specificities for antibodies derived from an Fv clone or hybridoma clone.

Antibody Fragments

The present invention encompasses antibody fragments. In some cases, it is preferable to use fragments of antibodies, not full-sized antibodies. The smaller the size of the fragments, the faster their clearance and the higher the likelihood of improved delivery to solid tumors.

Various methods have been developed to obtain antibody fragments. Traditionally, these fragments are formed by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science , 229: 81 (1985)) . However, these fragments can be produced directly by recombinant host cells. Fab, Fv, and scFv fragments of antibodies can be expressed in and secreted from E. coli cells, which facilitates the production of these fragments in large quantities. Antibody fragments can be isolated from the phage antibody libraries discussed above. Alternatively, Fab'-SH fragments can be directly isolated 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 a culture of a recombinant host cell. Fab and F (ab ') ₂ fragments with an increased in vivo half-life containing residues of an epitope that binds to the rescue receptor are described in US Pat. No. 5,869,046. Other methods for preparing antibody fragments are known to those skilled in the art. In other embodiments, the selected antibody is a single chain Fv fragment (scFv). See WO 93/16185, US Pat. Nos. 5,571,894 and 5,587,458. Fv and sFv belong only to species that contain intact combination sites lacking constant regions, and therefore may be suitable to reduce levels of nonspecific binding when used in vivo . Hybrid scFv proteins can be designed to produce an effector protein hybrid at the amino or carboxy terminus of scFv. See Antibody Engineering, ed. Borrebaeck, see above. An antibody fragment may also be a “linear antibody,” for example, the antibody described in US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methods of humanizing non-human antibodies are known to those skilled in the art. Thus, for example, a humanized antibody may have one or more amino acid residues introduced into it, originating from a non-human source. These non-human amino acid residues are often referred to as “import” residues, which are usually taken from the “import” variable domain. Humanization can be carried out mainly by the method of Winter (Winter) and collaborators (Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988 ) Science 239: 1534-1536), by replacing the sequences of the hypervariable region with the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567), in which a substantially smaller portion than the variable domain of an intact human antibody is replaced by a corresponding sequence derived from a non-human antibody. In fact, humanized antibodies are typically human antibodies in which some residues of the hypervariable region and, possibly, some residues of the framework region (FR) are replaced by residues derived from similar regions of rodent antibodies.

To reduce antigenicity when creating humanized antibodies, it is very important to choose the variable domains of both the light and heavy chains of a human antibody. In accordance with the so-called fitting method, the sequence of the variable domain of a rodent antibody is screened throughout the library of known variable domain sequences of a human antibody. The human sequence, which is most similar to the rodent sequence, is then taken as the human framework region for creating a humanized antibody (Sims et al. (1993) J. Immunol . 151: 2296; Chothia et al. (1987), J. Mol. Biol . 196: 901). Another method uses a specific framework region, derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework region 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).

It is also important that antibodies can be humanized while maintaining high affinity for this antigen and other favorable biological properties. In accordance with one of the methods, to achieve this goal, humanized antibodies can be obtained by analyzing the source sequences and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are publicly available and are well known in the art. Computer programs exist that illustrate and represent probable three-dimensional conformational structures of selected immunoglobulin candidate sequences. The study of these ideas allows us to analyze the likely role of these residues in the functioning of the candidate immunoglobulin sequences, that is, residues that affect the ability of the candidate immunoglobulin to bind to its antigen. Thus, FR residues can be selected from recipient and “import” sequences and combined, as a result of which the desired antibody can be obtained with the desired properties, such as increased affinity for the target antigen (s) (s). In general terms, residues of the hypervariable region directly affect antigen binding, and are mainly involved in such binding.

Human antibodies

Human anti-DLL4 antibodies of the invention can be constructed by combining the sequence (s) of the variable domain of the Fv clone selected from libraries of phage presentation originating from humans with the known sequence (s) of the human constant domain, as described above. Alternatively, the human monoclonal anti-DLL4 antibodies of the invention can be prepared by the hybridoma method. Human myeloma cell lines and mouse-human heteromyeloma cell lines used to produce human monoclonal antibodies are described, for example, in Kozbor J. Immunol publications . 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).

Currently, transgenic animals (e.g., mice) can be obtained which, after immunization, will be able to produce a complete repertoire of human antibodies without producing endogenous immunoglobulin. For example, it was reported that a homozygous deletion of the gene of the junction region in the heavy chain (J _H ) of an antibody in chimeric and mutant germ line mice leads to complete inhibition of the production of endogenous antibodies. Transferring a set of human germline immunoglobulin genes to such mutant germline mice can lead to the production of human antibodies after antigen stimulation. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci ., USA, 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggerman et al., Year in Immunol , 7:33 (1993).

To obtain human antibodies from non-human animals, such as rodents, where the human antibody has the same affinity and specificity for the original non-human antibody, a gene swapping method can also be used. According to this method, also called “epitope imprinting,” the variable region of the heavy or light chain of a non-human antibody fragment obtained by the phage presentation methods described above is replaced with a set of human V-domain genes to produce a population of non-human chain chimeras / human scFv or Fab chain. " Selection using antigen allows the selection of “non-human chain / scFv or Fab human” chimeras, where the indicated human chain restores the antigen binding site that is destroyed after removal of the corresponding non-human chain in the primary clone of the phage representation, that is, the epitope determines (fixes) the choice partner for the human chain. If this process is repeated to replace the remaining non-human chain, then a human antibody can be obtained (see PCT application WO 93/06213, published April 1, 1993). Unlike the traditional method of humanizing non-human antibodies by attaching a CDR, this method allows you to get fully human antibodies that do not have FR or CDR residues derived from non-human.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized antibodies that have binding specificity for at least two different antigens. In this case, one of the binding specificities is the specificity of binding to DLL4, the other specificity is specificity to any other antigen. Representative bispecific antibodies can bind to two different epitopes of the DLL4 protein. Bispecific antibodies can also be used to determine the localization of cytotoxic agents in cells that express DLL4. Such antibodies have a DLL4-binding branch and a branch that binds to a cytotoxic agent (e.g., saporin, anti-interferon-α antibody, vinca alkaloid, ricin A chain, methotrexate or hapten linked to a radioactive isotope). Bespecifically antibodies can be obtained in the form of full-sized antibodies or fragments of antibodies (for example, bespecifically F (ab ') ₂ antibodies).

Methods for producing bispecific antibodies are known in the art. The traditional recombinant production of bispecific antibodies is based on coexpression of two pairs of the heavy chain — the immunoglobulin light chain, where the two heavy chains have different specificities (Millstein & Cuello, Nature , 305: 537 (1983)). Due to the randomized set of immunoglobulin heavy and light chains, these hybridomas (quadroms) produce a potential mixture of 10 different antibody molecules, of which only one molecule has the “correct” bispecific structure. The purification of such a “correct” molecule, which is usually carried out by stepwise affinity chromatography, is rather difficult and gives a low yield of the product. Similar procedures are described in WO93 / 08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655 (1991).

In accordance with another and more preferred approach, the variable domains of an antibody with the desired binding specificities (combined antibody-antigen sites) are attached to immunoglobulin constant domain sequences. Such a fusion is preferably carried out with the constant domain of an immunoglobulin heavy chain containing at least a portion of the hinge region, CH2 and CH3. In this case, it is preferable that at least one of the hybrids contains the first constant region of the heavy chain (CH1) containing the site necessary for binding to the light chain. DNA encoding immunoglobulin heavy chain hybrids and, if necessary, immunoglobulin light chain hybrids are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides a high degree of flexibility in correcting the ratios of three polypeptide fragments in those embodiments of the invention in which unequal contents of the three polypeptide chains used in this design provide optimal yields. However, coding sequences for two or all three polypeptide chains can be inserted into one expression vector if expression of at least two polypeptide chains in equal proportions gives high yields, or if such ratios are not critical.

In a preferred embodiment of this approach, the bispecific antibodies consist of a hybrid immunoglobulin heavy chain having a first binding specificity in one branch, and an immunoglobulin “heavy chain - light chain” hybrid pair (providing a second binding specificity) in another branch. It was found that such an asymmetric structure facilitates the separation of the desired bispecific compound from undesirable combinations of immunoglobulin chains, since the presence of the immunoglobulin light chain in only one half of the bispecific molecule provides an easy way to isolate it. This method is described in WO 94/04690. A more detailed description of the preparation of bispecific antibodies can be found, for example, in Suresh et al., Methods in Enzymology , 121: 210 (1986).

According to another approach, the boundary between a pair of antibody molecules can be constructed in order to maximize the percentage of heterodimers isolated from recombinant cell culture. Such a boundary preferably contains at least a portion of the C _H 3 domain of the constant domain of an antibody. In this method, the small side chains of one or more amino acids in the boundary region of the first antibody molecule are replaced by larger side chains (e.g. tyrosine or tryptophan). In the boundary region of the second molecule of the antibody, compensating “cavities” are created having a size identical or similar to the size of the larger side chain (s) by replacing the large side chains of amino acids with smaller side chains (for example, alanine or threonine) . This allows you to increase the yield of heterodimers in relation to other undesirable end products, such as homodimers.

Bespecifically antibodies are cross-linked or "heteroconjugate" antibodies. So, for example, one of the antibodies in the heteroconjugate may be associated with avidin, the other with biotin. It has been suggested that such antibodies can, for example, target cells of the immune system to unwanted cells (US Pat. No. 4,676,980), so they can be used to treat HIV infections (WO 91/00360, WO 92/00373 and EP 03089) . Heteroconjugate antibodies can be prepared using any standard cross-linking methods. Suitable crosslinking agents are well known in the art and are described in US Pat. No. 4,676,980, along with various crosslinking methods.

Methods for producing bispecific antibodies from antibody fragments have also been described in the literature. So, for example, bespecifically antibodies can be obtained by chemical binding. Brennan et al., Science , 229: 81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to form F (ab ') ₂ fragments. These fragments are reduced in the presence of a dithiol complex forming agent, such as sodium arsenite, to stabilize adjacent dithiols and prevent the formation of an intermolecular disulfide bond. Then the obtained Fab'-fragments are converted into derivatives of thionitrobenzoate (TNB). Thereafter, one of the Fab'-TNB derivatives is again converted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative, whereby a bispecific antibody is obtained. Such bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent advances in this field allow direct isolation of Fab'-SH fragments from E. coli that can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the production of a fully humanized F (ab ') ₂ molecule of a bispecific antibody. Each Fab'fragment was separately secreted from E. coli and subjected to direct chemical binding in vitro with the formation of bespecifically antibodies. The bispecific antibody thus obtained has the ability to bind to cells overexpressing the HER2 receptor and normal human T cells, and also to trigger the lytic activity of human cytotoxic lymphocytes directed against human breast tumor target cells.

Other methods for producing and isolating fragments of a bispecific antibody directly from a recombinant cell culture have also been described. For example, bispecific antibodies were produced using “leucine zippers”. Kostelny et al., J. Immunol ., 148 (5): 1547-1553 (1992). Leucine zipper peptides derived from Fos and Jun proteins were coupled to the Fab ′ portions of two other antibodies by ligation of genes. Antibody homodimers were reduced in the hinge region to form monomers, then oxidized again to form antibody heterodimers. This method can also be used to produce antibody homodimers. Diantibody technology described by Hollinger et al., Proc. Natl. Acad. Sci., USA, 90: 6444-6448 (1993), provides an alternative mechanism for producing bispecific antibody fragments. These fragments contain the variable domain of the heavy chain (V _H ) connected to the variable domain of the light chain (V _L ) via a linker that is too short to create a pair between two domains of the same chain. Accordingly, 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 has also been described for producing bispecific antibody fragments using single chain Fv (sFv) dimers. See Gruber et al., J. Immunol., 152: 5368 (1994).

Antibodies with more than two valencies are also contemplated. So, for example, trispecific antibodies can also be obtained. Tutt et al., J. Immunol. 147: 60 (1991).

Polyvalent Antibodies

A polyvalent antibody can be internalized more quickly (and / or it can undergo catabolism faster) than a divalent antibody due to the expression of the antigen in the cell to which the antibodies bind. Antibodies according to the invention can be polyvalent antibodies (not belonging to the IgM class) with three or more antigen-binding sites (e.g., tetravalent antibodies), which can be easily produced by recombinant expression of a nucleic acid encoding the polypeptide chains of this antibody. A polyvalent antibody may contain a dimerization domain and three or more antigen binding sites. A preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this case, the antibody may contain an Fc region and three or more antigen binding sites located from the amino terminus with respect to the Fc region. The preferred multivalent antibody described herein contains (or consists of) from three to about eight antigen binding sites, preferably four antigen binding sites. A polyvalent antibody contains at least one polypeptide chain (and preferably two polypeptide chains), where the specified polypeptide (s) chain (s) contains (at) two or more variable domains. So, for example, the polypeptide (s) chain (s) may (may) contain VD1- (X1) n-VD2- (X2) n-Fc, where VD1 represents the first variable domain, VD2 represents the second variable domain, Fc represents represents one polypeptide chain of the Fc region, X1 and X2 are an amino acid or a polypeptide, and n is 0 or 1. So, for example, the polypeptide (s) chain (s) may (may) contain: a chain "VH-CH1-flexible linker "VH-CH1-Fc region"; or the chain "VH-CH1-VH-CH1-Fc region." The polyvalent antibody of the invention also preferably contains at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody described herein may, for example, contain from about two to eight polypeptides of the light chain variable domain. The light chain variable domain polypeptides described herein comprise a light chain variable domain, also, but not necessarily, a CL domain.

Antibody Options

In some embodiments of the invention, modification (s) of the amino acid sequence of the antibodies described herein is contemplated. So, for example, it may be desirable to increase the binding affinity and / or other biological properties of the antibody. Variants of the amino acid sequence of an antibody are obtained by introducing appropriate nucleotide substitutions into the nucleic acid encoding the antibody, or by peptide synthesis. Such modifications are, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequences of antibodies. In this case, any combination of deletions, insertions and substitutions can be introduced into the final structure, provided that such a final structure will have the desired properties. Amino acid modifications can be introduced into the considered amino acid sequence of an antibody in the process of obtaining such a sequence.

The method used to identify certain residues or regions of an antibody that have a preferred location for mutagenesis is called “alanine scanning mutagenesis” and is described by Cunningham & Wells (1989), Science, 244: 1081-1085. In this method, a residue or group of desired residues (e.g., charged residues such as Arg, Asp, His, Lys and Glu) is identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to influence the interaction of these amino acids with the antigen. Then, the localization of amino acids exhibiting functional sensitivity to such substitutions is specified by introducing additional or other modifications to the substitution position. So, for example, the position for introducing a substitution in the amino acid sequence can be determined in advance, while the nature of the mutation per se need not be determined in advance. So, for example, to analyze the effectiveness of a mutation at a given site, Ala-scanning or nonspecific mutagenesis is performed in the desired codon or in the desired region, and expressed immunoglobulins are screened for the desired activity.

Insertions into amino acid sequences are insertions at the amino and / or carboxy terminus, forming hybrids having a length from one residue to polypeptides containing one hundred or more residues, and also inserts within a sequence consisting of one or multiple amino acid residues. Examples of terminal insertions are an antibody with an N-terminal methionyl residue or an antibody attached to a cytotoxic polypeptide. Other insertion variants of the antibody molecule are hybrids, for example the N- or C-terminus of an antibody with an enzyme (for example, for ADEPT) or a polypeptide that increases the half-life of the antibody in serum.

Glycosylation of polypeptides is usually either N-linked or O-linked. N-linked glycosylation means the attachment of a carbohydrate group to the side chain of an aspartic moiety. The tripeptide sequences “asparagine-X-serine” and “asparagine-X-threonine”, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in the polypeptide contributes to the creation of a potential glycosylation site. O-linked glycosylation means the addition of one of the sugars, such as N-acetylgalactosamine, galactose or xylose, to a hydroxy amino acid, mainly to serine or threonine, although they may also be 5-hydroxyproline or 5-hydroxylysine.

The attachment of glycosylation sites to an antibody is usually carried out by modifying the amino acid sequence such that the amino acid sequence will contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Such a modification can also be carried out by adding to the sequence one or more serine or threonine residues or replacing them in the sequence of the original antibody (for O-linked glycosylation sites).

If the antibody contains an Fc region, a carbohydrate attached to this region can be modified. For example, antibodies having a mature carbohydrate structure in which there is no fucose and which is attached to the Fc region of an antibody are described in US patent application No. 2003/0157108, (Presta, L.). See also US 2004/0093621 A1 (Kyowa Hakko Kogyo Co., Ltd). Antibodies containing N-acetylglucosamine (GlcNAc) in their carbohydrate, bisecting the antibody molecule in half, where the carbohydrate is attached to the Fc region of the antibody, are described in WO 03/011878, Jean-Mairet et al., And in US patent No. 6602684, Umana et al. Antibodies having at least one galactose residue in an oligosaccharide attached to the Fc region of an antibody are described in WO 1997/30087, Patel et al. See also WO 98/58964 (Raju, S.) and WO 99/22764 (Raju, S.), which relate to modified carbohydrate antibodies attached to the Fc region of an antibody. See also U.S. Application 2005/0123546 (Umana et al.) For antigen binding molecules with modified glycosylation.

A preferred glycosylation variant from the point of view of the present invention includes the Fc region, where the carbohydrate structure attached to the Fc region does not contain fucose. Such options have an improved ADCC function. The Fc region also contains, but not necessarily, one or more amino acid substitutions that also improve ADCC function, for example, substitutions at positions 298, 333 and / or 334 of the Fc region (residue numbering is given according to the European numbering system). Publications that describe "defucosylated" or "deficient in fucose" antibodies are, for example, US patent application No. 2003/0157108; WO 2001/61739; WO 2001/29246; US 2003/0115614; US2002 / 0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; 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 defucosylated antibody producing cell lines are Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Patent Application No. 2003/0157108 A1, Presta, L ; and WO 2004/056312 A1, Adams et al ., in particular Example 11), and deficient cell lines, such as CHO cells, deficient in the alpha-1,6-fucosyltransferase gene, FUT8, (Yamane-Ohnuki et al. Biotech Bioeng. 87: 614 (2004)).

A variant of another type is the amino acid substitution variant. Such antibody variants have in their molecule at least one amino acid residue replaced by another residue. The sites that are of most interest for mutagenesis by substitutions are hypervariable regions, however, FR modifications can also be considered. Conservative substitutions are presented in table 1 under the heading "Preferred substitutions". If such substitutions lead to a change in biological activity, more significant substitutions can be introduced, referred to in Table 1 as “representative substitutions”, or also described below in the section relating to the class of amino acids, then the resulting products can be screened.

Table 1 Original Residues Representative Substitutions Preferred Substitutions Ala (A) Val; Leu Ile Val Arg (R) Lys; Gln; Asn Lys Asn (n) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (c) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu Val; Met; Ala; Phe; norleucine Leu Leu (L) Norleucine; Ile Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu Phe; Ile Leu Phe (f) Trp; Leu Val; Ile Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (t) Val; Ser Ser Trp (w) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile Leu Met; Phe; Ala; norleucine Leu

Significant changes in the biological properties of antibodies can be achieved by selecting substitutions that significantly differ in their effect on (a) the structure of the polypeptide backbone in the substitution region, for example, a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the desired site, or (c) side chain volume. Natural residues are divided into the following groups according to the general properties of the side chains:

(1) hydrophobic residues: norleucine, 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 lead to replacements of a member of one of these classes with a member of another class.

One representative replacement option is the replacement of one or more residues of the hypervariable region of the parent antibody (e.g., a humanized or human antibody). In general terms, the obtained option (s) selected for further development will improve the biological properties compared to the properties of the parent antibody from which they originate. The standard way of generating such variants with substitutions involves the maturation of affinity using the phage representation method. For this, several hypervariable region sites (for example, 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. Antibodies generated in this way can be represented on filamentous phage particles in the form of hybrids with the product of gene III, packaged in each particle of phage M13. Then the options presented on the phage, screened for their biological activity (for example, binding affinity), as described in this application. To identify sites of the hypervariable region that are candidates for modification, alanine scanning mutagenesis can be carried out, which allows the identification of residues of the hypervariable region, which play an important role in binding to the antigen. Alternatively or additionally, it may be useful to analyze the crystal structure of the antigen-antibody complex to identify contact sites between the antibody and antigen. Such contact residues and adjacent residues are candidates for replacement carried out in accordance with the methods developed here. After generating such variants, a panel of these variants is subjected to the screening described in this application, and antibodies exhibiting excellent properties in one or more relevant assays can be selected for further study.

Nucleic acid molecules encoding variants of the amino acid sequences of an antibody are prepared by various methods known to those skilled in the art. Such methods include, but are not limited to, isolation from a natural source (in the case of natural variants of amino acid sequences) or obtaining, using an oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cluster mutagenesis of a previously obtained variant or unmodified antibody variant.

It may be desirable to introduce one or more modifications of the amino acids in the Fc region of the immunoglobulin polypeptides of the invention to produce a variant Fc region. An Fc region variant may comprise a sequence of a human Fc region (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions, including the position of the hinge cysteine. In accordance with the present description and prior art, it should be noted that in some embodiments of the invention, the antibody used in the methods of the invention may contain one or more modifications compared to a wild-type antibody analogue, for example, in the Fc region. However, such antibodies should essentially retain the properties of its wild-type analogue necessary for its therapeutic use. So, for example, it is believed that some modifications can be introduced into the Fc region, which will lead to a change (i.e., to improvement or weakening) of the binding to C1q and / or complement dependent cytotoxicity (CDC), for example, as described in WO 99/51642. See also Duncan & Winter Nature 322: 738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94/29351, which discusses other examples of variants of the Fc region. WO 00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with increased or decreased levels of binding to FcRs. The contents of these patent publications are specifically incorporated into this description by reference. See also Shields et al. J. Biol. Chem. 9 (2): 6591-6604 (2001). Antibodies with longer half-lives and with increased binding to the Fc receptor found in newborns (FcRn), which is responsible for the transmission of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) described in US 2005/0014934 A1 (Hinton et al.). Such antibodies include an Fc region with one or more of the substitutions described that increase the binding of the Fc region to FcRn. Polypeptide variants with modified amino acid sequences in the Fc region and with increased or decreased ability to bind to C1q are described in US patent No. 6194551B1 and in WO 99/51642. The contents of these patent publications are specifically incorporated into this description by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Derivatives of Antibodies

Antibodies according to the invention can be further modified so that they contain other non-protein molecules that are known in the art and are readily available. Preferred molecules suitable for derivatization of antibodies are water soluble polymers. Non-limiting examples of water-soluble polymers are polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene amide / ethylene amide copolymer homopolymers or random copolymers) and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g. glycerol), poly vinyl alcohol and mixtures thereof. In industrial production, preference should be given to propionaldehyde of polyethylene glycol due to its stability in water. This polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, then these polymers can have the same or different molecules. In general terms, the number and / or type of polymers used for derivatization can be selected based on several factors, including, but not limited to, specific properties or functions of the antibody that need to be improved, regardless of the particular treatment conditions under which the specified derivative of an antibody or the like is used.

Screening for antibodies with desired properties

Antibodies according to the invention can be characterized by their physical / chemical properties and biological functions using various assays known to those skilled in the art. In some embodiments of the invention, antibodies are characterized by any one or more of a property, such as binding to DLL4; and / or attenuation or blocking of Notch receptor activation; and / or attenuation or blocking of the molecular signal transmitted after transmission of the Notch receptor signal, and / or disruption or blocking of Notch receptor binding to DLL4; and / or stimulation of proliferation of endothelial cells; and / or inhibition of differentiation of endothelial cells; and / or inhibition of arterial differentiation; and / or inhibition of vascular tumor perfusion; and / or treating and / or preventing the development of a tumor, cell proliferative disorder, or cancer; and / or treating or preventing a disorder associated with the expression and / or activity of DLL4; and / or treating or preventing a disorder associated with Notch receptor expression and / or activity.

Purified antibodies can also be characterized by a variety of assays, including but not limited to N-terminal sequencing, amino acid analysis, size exclusion chromatography, high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and hydrolysis papain.

In some embodiments of the invention, the antibodies produced herein are assayed for their biological activity. In some embodiments of the invention, the antibody of the invention is tested for their antigen binding activity. Antigen binding assays that are known to those skilled in the art and which can be used in this application include, but are not limited to, any of the direct or competitive binding assays performed by methods such as Western blot assays, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay analysis), sandwich immunoassays, immunoprecipitation assays, fluorescence immunoassays and protein A immunoassays. Representative antigen binding assays are described below in the section “Pr measures. "

In yet another embodiment, the present invention relates to monoclonal anti-DLL4 antibodies that compete with antibodies 26.6, 26.14, 26.20, 26.34 and / or 26.82 for binding to DLL4. Such competing antibodies are antibodies that recognize the DLL4 epitope, which is similar to the epitope or overlaps with the DLL4 epitope recognized by antibodies 26.6, 26.14, 26.20, 26.34 and / or 26.82. Such competing antibodies can be obtained by screening supernatants for an anti-DLL4 hybridoma to competitively bind immobilized DLL4 with labeled antibodies 26.6, 26.14, 26.20, 26.34 and / or 26.82. A hybridoma supernatant containing a competitor antibody will help reduce the amount of bound labeled antibody detected in the competitive binding mixture in question compared to the amount of labeled labeled antibody detected in the binding control mixture containing an irrelevant antibody (or no antibody at all). Any of the competitive binding assays described herein is suitable for use in the above procedure.

In another aspect, the present invention relates to a monoclonal anti-DLL4 antibody comprising one or more (e.g. 2, 3, 4, 5 and / or 6) HVR antibodies 26.6, 26.14, 26.20, 26.34 and / or 26.82. A monoclonal anti-DLL4 antibody containing one or more HVR antibodies 26.6, 26.14, 26.20, 26.34 and / or 26.82 can be constructed by attaching one or more HVR antibodies 26.6, 26.14, 26.20, 26.34 and / or 26.82 to the antibody matrix sequence for example, the sequence of a human antibody that most closely matches the mouse sequence of the parent antibody or the consensus sequence of all human antibodies in a particular subgroup of the light chain or heavy chain of the parent antibody, and expression is obtained second (s) chimera (s) sequence (s) variable light and / or heavy chain in the presence or absence of concomitant (s) sequence (s) of the constant region in the recombinant host cells described herein.

Anti-DLL4 antibodies of the invention having the unique properties described herein can be obtained by screening anti-DLL4 hybrid clones for desired properties using any standard method. So, for example, if it is desirable to use a monoclonal anti-DLL4 antibody that blocks or nonblocks the binding of Notch receptors to DLL4, then such a candidate antibody can be tested in a competitive binding assay, such as competitive binding ELISA, where the wells of the plates are coated with DLL4, then an antibody solution in an excess of Notch receptor of interest is applied to these coated plates, and the bound antibody is subjected to enzymatic detection, for example, contacting the bound antibody with PX- conjugated anti-Ig antibody or biotinylated anti-Ig antibody with the development of the color PC reaction, for example, by developing tablets using streptavidin-PC and / or hydrogen peroxide, and detecting the color PC reaction using spectrophotometry at 490 nm in a tablet reader for ELISA.

In one embodiment, the present invention relates to a modified antibody that has some, but not all, effector functions, which makes it a desirable candidate antibody for various applications, where the half-life of the antibody in vivo is an important factor, some effector functions (such both complement dependent cytotoxicity and ADCC) are not necessary or even undesirable. In some embodiments, the Fc activity of the produced immunoglobulin is measured to ensure that only the desired properties are retained. In vitro and / or in vivo cytotoxicity assays can be performed to confirm attenuation / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody will not bind to FcγR (and therefore probably will not have ADCC activity), but will retain its ability to bind to FcRn . Primary cells mediating ADCC, i.e., NK cells, express FcγRIII only, while monocytes express FcγRI, FcγRII and FcγRIII. The data obtained for the expression of FcR on hematopoietic cells are summarized in table 3 on page 464 of the publication by Ravetch & Kinet Annu. Rev. Immunol. 9: 457-92 (1991). An example of an in vitro assay for evaluating the ADCC activity of a molecule of interest is described in US Pat. Nos. 5,500,362 or 5,821,337. Effector cells suitable for such assays are peripheral blood mononuclear cells (PBMCs) and natural killer cells (NKs). Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo , for example, in an animal model, such as the model described by Clynes et al. Proc. Natl. Acad. Sci. A 95: 652-656 (1998). To confirm that this antibody does not have the ability to bind to C1q, therefore, does not have CDC activity, tests for binding to C1q can also be performed. To assess complement activation, a CDC assay can be performed, for example, the assay described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). The determination of the level of binding to FcRn and clearance / half-life in vivo can also be carried out by methods known to those skilled in the art, for example, described in the Examples section.

Vectors, host cells and recombinant methods

For recombinant production of an antibody according to the invention, a nucleic acid encoding such an antibody is isolated and inserted into a replicated vector for subsequent cloning (DNA amplification) or expression. DNA encoding an antibody can be easily isolated and sequenced in accordance with standard procedures (for example, using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains). Many vectors are suitable for this purpose. The choice of vector depends in part on the host cell used. Typically, the preferred host cells are either prokaryotic cells or eukaryotic cells (usually mammalian cells). It should be noted that for this purpose, constant regions of any isotype can be used, including constant regions of IgG, IgM, IgA, IgD and IgE, and that such constant regions can be obtained from humans or animals of any species.

but. Generation of antibodies using prokaryotic host cells

i. Vector design

Polynucleotide sequences encoding the polypeptide components of an antibody of the invention can be obtained by standard recombinant methods. The desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternative polynucleotides can be synthesized using a nucleotide synthesizer or PCR methods. After obtaining sequences encoding the polypeptides, these sequences are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. For the purposes of the present invention, many available and known vectors can be used. The choice of the appropriate vector depends mainly on the size of the nucleic acids inserted into the specified vector, and on the specific host cell transformed by this vector. Each vector contains various components depending on its function (amplification or expression of a heterologous polynucleotide or both) and on its compatibility with the particular host cell in which it is located. The components of the vectors are typically, but are not limited to, the origin of replication, a selective marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insertion, and a transcription termination sequence.

Basically, plasmid vectors containing replicon and regulatory sequences derived from species compatible with the host cell are used together with the same hosts. Such a vector usually contains a replication site, as well as marker sequences that facilitate phenotypic selection in transformed cells. So, for example, E. coli is usually transformed with plasmid pBR322, derived from a certain type of E. coli . pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet), and makes it easy to identify transformed cells. pBR322 and its derivatives or other microbial plasmids or bacteriophage can also contain promoters that can be used by this microbial organism to express endogenous proteins, or they can be modified to contain such promoters. Examples of pBR322 derivatives used to express specific antibodies are described in detail in US Pat. No. 5,648,237 to Carter et al.

In addition, phage vectors containing replicon and regulatory sequences compatible with the host microorganism can be used as transforming vectors in combination with these hosts. For example, a bacteriophage, such as λGEM ^™ -11, can be used to produce a recombinant vector that can be used to transform susceptible host cells, such as E. coli LE392.

An expression vector according to the invention may contain two or more promoter-cistron pairs encoding each of the polypeptide components. The promoter is an untranslated regulatory sequence located above (from the 5'-end) from the cistron modulating its expression. Prokaryotic promoters are usually divided into two classes - inducible and constitutive. An inducible promoter is a promoter that initiates an increase in transcription levels of cistron under its control in response to changes in cultivation conditions, for example, in the presence or absence of nutrients or when the temperature changes.

Most promoters recognized by various potential host cells are well known in the art. The selected promoter may be operably linked to a cystron DNA encoding a light or heavy chain by isolating the promoter from the source DNA by hydrolyzing it with restriction enzymes and inserting the isolated promoter sequence into the vector of the invention. The native promoter sequence and many heterologous promoters can be used for direct amplification and / or expression of target genes. In some embodiments of the invention, heterologous promoters are used, since they can significantly increase the level of transcription and increase the yields of the expressed target gene compared to the native promoter for the desired polypeptides.

The promoters suitable for use in prokaryotic hosts are the PhoA promoter, β-galactamase and lactose promoter systems, tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter . However, other promoters that are functional in bacteria (for example, other known bacterial or phage promoters) can also be used. The nucleotide sequences of these promoters have been published, which makes it easier for those skilled in the art to carry out their functional ligation with cistrons encoding the desired light and heavy chains (Siebenlist et al. (1980) Cell 20: 269), using linkers or adapters to deliver all the required restriction sites.

In one aspect of the invention, each cistron in a recombinant vector contains a component such as a secretory signal sequence that regulates the translocation of expressed polypeptides across the membrane. In general terms, the indicated signal sequence may be a component of a vector, or such a sequence may be part of the desired DNA polypeptide inserted into this vector. The signal sequence selected for the implementation of the invention should be a sequence recognized and processed (i.e. cleaved by signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize and do not process signal sequences that are native to heterologous polypeptides, this signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group consisting of the leader sequence of alkaline phosphatase, penicillinase, lpp or thermostable enterotoxin II (STII), LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

In another aspect of the invention, the production of immunoglobulins according to the invention can occur in the cytoplasm of the host cell, therefore, in this case, the presence of secreting signal sequences in each cistron is not required. In accordance with this, light and heavy chains of immunoglobulin are expressed and subjected to folding and assembly with the formation of functional immunoglobulins in the cytoplasm. Some host strains (for example, trxB ^- strains of E. coli ) have appropriate cytoplasmic conditions that favor the formation of disulfide bonds, and thereby contribute to the proper folding and assembly of the expressed protein subunits. Proba & Pluckthun, Gene , 159: 203 (1995).

Prokaryotic host cells suitable for expressing antibodies of the invention are archaebacteria (Archaebacteria) and eubacteria (Eubacteria), such as gram-negative or gram-positive microorganisms. Examples of suitable bacteria are Escherichia (e.g., E.coli), Bacilli (e.g., B.subtilis), enterobacteria, bacteria of the species Pseudomonas (e.g., P.aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia , Vitreoscilla or Paracoccus . In one embodiment of the invention, gram-negative cells are used. In one embodiment of the invention, E. coli cells are used as hosts according to the invention. Examples of E. coli strains are strain W3110 (Bachmann, Cellular & Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC, deposit number No. 27325) and derivatives thereof, including strain 33D3 having the W3110 genotype Δ fhuA (ΔtonA) ptr3 laq Iq lacL8 ΔompTΔ (nmpc-fepE) degP41 kanR (US patent No. 5639635). Other strains and their derivatives are also suitable, such as E. coli 294 (ATCC 31446), E. coli B, E. coli λ 1776 (ATCC 31537) and E. coli RV308 (ATCC 31608). These examples are illustrative, but not limiting. Methods for constructing derivatives of any of the aforementioned bacteria having certain genotypes are known to those skilled in the art and are described, for example, in Bass et al., Proteins 8: 309-314 (1990). Usually, the appropriate bacteria must be selected taking into account the replicability of the replicon in the bacterial cells. Thus, for example, E. coli , Serratia or Salmonella bacteria may be suitable for use as a host if well-known plasmids are used to deliver the replicon, such as pBR322, pBR325, pACYC177 or pKN410. Typically, host cells must secrete minimal amounts of proteolytic enzymes, therefore, additional protease inhibitors are desirable in the cell culture.

ii. Antibody production

The host cells are transformed with the above expression vectors and cultured in a standard nutrient medium, modified to be suitable for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences.

The term “transformation” means the introduction of DNA into a prokaryotic host so that the DNA is either replicated as an extrachromosomal element or integrated into the chromosome. Depending on the host cell used, the transformation is carried out by standard methods suitable for such cells. Bacterial cells containing strong barriers, such cell walls, are usually treated with calcium, namely calcium chloride. Another transformation method uses polyethylene glycol / DMSO. Another method of transformation is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention are cultured in a medium known to those skilled in the art and suitable for culturing selected host cells. Examples of suitable media are Luria broth (LB) with essential nutritional supplements. In some embodiments of the invention, such a medium also comprises a selection agent selected based on the construction of the expression vector that promotes the selective growth of prokaryotic cells containing the expression vector. Thus, for example, ampicillin is added to a medium for culturing cells expressing an ampicillin resistance gene.

In addition to sources of carbon, nitrogen and inorganic phosphate, any necessary additives in appropriate concentrations can be included in the medium, introduced either separately or in a mixture with other additives or medium, such as a complex source of nitrogen. The culture medium may, but not necessarily, contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol.

Prokaryotic host cells are cultured at appropriate temperatures. For culturing E. coli, a preferred temperature is, for example, a temperature of from about 20 ° C to 39 ° C, more preferably from about 25 ° C to 37 ° C, even more preferably at about 30 ° C. The pH of the medium can vary from about 5 to 9, depending mainly on the host organism. For E. coli, the pH is preferably about 6.8 to 7.4, more preferably about 7.0.

If an inducible promoter is used in the expression vector according to the invention, protein expression is induced under conditions suitable for activating the promoter. In one aspect of the invention, PhoA promoters are used to regulate the transcription of polypeptides. Accordingly, transformed host cells are cultured in a phosphate-limited induction medium. A preferred phosphate-limited medium is C.R.A.P. (see, for example, Simmons et al., J. Immunol. Methods (2002) 263: 133-147). Various other inductors known to those skilled in the art can be used in combination with the vector construction used.

In one embodiment of the invention, the expressed polypeptides of the invention are secreted into the periplasm of the host cells and can be isolated from this periplasm. Protein isolation is mainly carried out by disrupting the microorganism, usually by methods such as osmotic shock, sonication or lysis. After cell disruption, cell debris or whole cells can be removed by centrifugation or filtration. These proteins can be further purified, for example, by chromatography on affinity resin. Alternatively, proteins can be transported to and separated from the cell medium. Cells can be removed from the culture, the culture supernatant can be filtered and concentrated to further purify the resulting proteins. Expressed polypeptides can then be isolated and identified by well-known methods, such as polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.

In one aspect of the invention, antibodies are produced in large quantities by fermentation. To obtain recombinant proteins can be carried out various large-scale fermentation procedures in culture with water. Large-scale fermentation is carried out in a fermenter with a capacity of at least 1000 liters, preferably from about 1000 to 100,000 liters. Such fermenters are equipped with a paddle mixer for uniform distribution of oxygen and trace elements, in particular glucose (a preferred carbon / energy source). The term "laboratory fermentation", in General terms, means fermentation in a fermenter with a volume of not more than about 100 l, and such a volume can vary from about 1 l to 100 l.

In the fermentation process, the induction of protein expression is usually initiated after cells cultured under suitable conditions have reached the desired density, for example, OD _{550 of} about 180-220, i.e. the early stationary phase. Various other inductors known to those skilled in the art and described above can be used in combination with the vector construction used. Before induction, cells can be cultured for a shorter period of time. Cells usually induce for about 12-50 hours, although the induction time can be increased or decreased.

To increase the yield of the product and to improve the quality of the polypeptides according to the invention, various fermentation conditions can be changed. For example, to ensure the “correct” assembly and folding of secreted antibody polypeptides, additional vectors can be used in which overexpression of chaperone proteins such as Dsb (DsbA, DsbB, DsbC, DsbD and / or DsbG) or FkpA (peptidylprolyl - cis, trans isomerase with chaperone activity), and which are intended for co-transformation of prokaryotic host cells. Chaperone proteins have been shown to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J. Bio. Chem . 274: 19601-19605; Georgiou et al., US patent No. 6083715; Georgiou et al., US patent No. 6027888; Bothmann & Pluckthun (2000) J. Biol. Chem. 275: 17100-17105; Ramm & Pluckthun (2000) J. Biol. Chem. 275: 17106-17113; Arie et al. (2001) Mol. Microbiol. 39: 199-210.

To minimize proteolysis of expressed heterologous proteins (and in particular, proteins that are proteolytically sensitive), some host strains deficient in proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified to introduce genetic mutation (s) into genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI and their combinations. Some protease-deficient E. coli strains are available and are described, for example, in Joly et al. (1998), see above; Georgiou et al., US patent No. 5264365; Georgiou et al., US patent No. 5508192; Hara et al., Microbial Drug Resistance, 2: 63-72 (1996).

In one embodiment of the invention, E. coli strains deficient in proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the invention.

iii. Antibody purification

Standard protein purification methods known to those skilled in the art can be applied. Examples of suitable purification methods are the following purification procedures: fractionation on immunoaffinity or ion exchange columns, precipitation with ethanol, reverse phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation -filtration using, for example, Sephadex G-75.

In one aspect of the invention, protein A immobilized on a solid phase is used for immunoaffinity purification of the full length antibody products of the invention. Protein A is a 41 kD cell wall protein derived from Staphylococcus aureus , which binds to the Fc region of antibodies with high affinity. Lindmark et al. (1983), J. Immunol. Meth. 62: 1-13. The solid phase on which Protein A is immobilized is preferably a column having a glass surface or a silicon-containing surface, more preferably a glass column with an adjustable pore size or a column coated with silicic acid. In some embodiments, said column is coated with a reagent, such as glycerin, to prevent nonspecific addition of impurities.

In the first purification step, a preparation obtained from the cell culture described above is applied to the solid phase with protein A immobilized on it to specifically bind the antibody of interest to protein A. Then the solid phase is washed to remove impurities that are not specifically bound to the solid phase. Finally, the antibody of interest is separated from the solid phase by elution.

b. Antibody Production Using Eukaryotic Host Cells

The components of the vectors are usually, but are not limited to, one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

(i) Component signal sequence

The vector used in eukaryotic host cells may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein, or a polypeptide of interest. The selected heterologous signal sequence is preferably a sequence that is recognized and processed (i.e. cleaved by signal peptidase) by the host cell. For expression in mammalian cells, mammalian signal sequences are used, as well as viral secretory leader sequences, for example, the herpes simplex virus gD signal sequence.

The DNA for such a precursor region is ligated to the antibody coding DNA while maintaining the reading frame.

(ii) Origin of replication

Typically, a component such as the origin of replication is not necessary for mammalian expression vectors. So, for example, the origin of SV40 replication is usually used only because it contains an early promoter.

(iii) Component Selective Gene

The expression and cloning vectors may contain a selective gene, also called a selective marker. Typically, selective genes encode proteins that (a) show resistance to antibiotics or to other toxins, such as ampicillin, neomycin, methotrexate or tetracycline, (b) compensate for the deficiency caused by auxotrophy, if necessary, or (c) provide important nutrient substances that do not come from complex environments.

One example of a screening scheme is the use of a drug that stops the growth of a host cell. Cells that have been successfully transformed with a heterologous gene produce a protein that reports drug resistance and thereby contributes to the survival of these cells in a selective medium. For such dominant selection, drugs such as neomycin, mycophenolic acid and hygromycin can be used.

Other examples of suitable selective markers for mammalian cells are markers that allow the identification of cells capable of incorporating an antibody encoding nucleic acid into their genome, for example, markers such as the DHFR gene, thymidine kinases, metallothionein-I and II, preferably genes encoding primate metallothionein, adenosine deaminase gene, ornithine decarboxylase, etc.

Thus, for example, cells transformed with the DHFR selective gene are first identified by culturing all transformants in culture medium containing methotrexate (Mtx), a competitive DHFR antagonist. If wild-type DHFR is used, then a suitable host cell is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CL-9069).

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

(iv) Component promoter

Expression and cloning vectors typically contain a promoter recognized by the host organism and operably linked to a nucleic acid encoding an antibody polypeptide. Promoter sequences suitable for eukaryotes are known to those skilled in the art. In fact, all eukaryotic genes have an AT-rich region, located approximately in the region located 25-30 nucleotides higher from the site of transcription initiation. Another such sequence, located approximately in the region located 70-80 nucleotides higher from the transcription initiation site of many genes, is the CNCAAT region, where N can be any nucleotide (SEQ ID NO: 60). At the 3 ′ end of most eukaryotic genes is the AATAAA sequence, which can serve as a signal to attach the poly A tail to the 3 ′ end of the coding sequence (SEQ ID NO: 61). All of these sequences can be appropriately inserted into eukaryotic expression vectors.

The transcription of antibody polypeptides from vectors present in mammalian host cells is regulated, for example, by promoters derived from the genomes of viruses such as poliomavirus, poultry smallpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus retrovirus, hepatitis B virus and monkey virus 40 (SV40); heterologous mammalian promoters, for example an actin promoter or an immunoglobulin promoter; and promoters of heat shock proteins, provided that such promoters are compatible with host cell systems.

The early and late promoters of the SV40 virus are usually obtained as an SV40 restriction fragment, which also contains the SV40 origin of replication. The early early promoter of human cytomegalovirus is usually obtained in the form of a restriction HindIII fragment E. The DNA expression system in mammalian host cells, derived from bovine papillomavirus used as a vector, is described in US Pat. No. 4,419,446. A modification of this system is described in US Pat. No. 4,601,978. Alternatively, a Routh sarcoma virus having a long terminal repeat may be used as a promoter.

(v) Component enhancer element

Transcription of DNA encoding an antibody polypeptide according to the invention in higher eukaryotes is often enhanced when an enhancer sequence is inserted into a vector. Currently, there are many enhancer sequences derived from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin genes). However, an enhancer from a eukaryotic cell virus is commonly used. Examples are the SV40 virus enhancer located in the late region of the origin of replication (100-270 bp), the cytomegalovirus early promoter enhancer, the polyomavirus enhancer located in the late region of the replication origin, and adnovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982), which describes enhancer elements for activating eukaryotic promoters. An enhancer can be inserted into the vector at the 5'- or 3'-position with respect to the sequence encoding the antibody polypeptide, but it is preferable that it be located at the 5'-position from the promoter.

(vi) Component transcription termination site

The expression vectors used in eukaryotic host cells usually also contain the sequences needed to terminate transcription and to stabilize mRNA. Such sequences are usually located on the 5'-end, sometimes on the 3'-end of the untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. As a component, such as a transcription termination site, the polyadenylation region of bovine growth hormone can be used. See WO 94/11026 and the description of expression vectors therein.

(vii) Selection and transformation of host cells

Host cells suitable for cloning or expression of DNA in the above vectors are higher eukaryotic cells described herein, including vertebrate host cells. Reproduction of vertebrate cells in culture (in tissue culture) has already become a routine procedure. Examples of suitable mammalian host cell lines are monkey transfected cell line CV1 transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); hamster cub kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); Sertoli mouse cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CVCC ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); Buffalo laboratory rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); murine mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annal N.Y. Acad. Sci. 383: 44-68 (1982)); MRC5 cells; FS4 cells and human hepatoma cell line (Hep G2).

Host cells are transformed with the above expression or cloning vectors to produce antibodies and cultured in suitable growth media, modified, if necessary, to induce promoters, select transformants or amplify genes encoding the desired sequences.

(viii) Cultivation of host cells

The host cells used to produce the antibodies of the invention can be cultured in various media. Media suitable for culturing host cells are commercially available media such as Hams F10 (Sigma), Minimum Support Medium ((MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle Medium (DMEM) , Sigma). In addition, any medium described in Ham et al., Meth. Can be used as a culture medium for culturing host cells . Enz . 58:44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), in US Pat. in WO 90103430; in WO 87/00195 or in US patent Re.No 30985. Hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride and calcium and magnesium phosphate), buffers (such as HEPES), nucleotides (such as adnosine and thymidine), antibiotics (such as the drug gentamicin ^TM ), trace elements (defined as inorganic compounds, usually present in final concentrations in micromolar doses ) and glucose or equivalent sources energy. Any other necessary additives may also be included at appropriate concentrations known to those skilled in the art. Culturing conditions, such as temperature, pH, and the like, are similar to those previously used to express selected host cells, and are known to one of ordinary skill in the art.

(ix) Purification of antibodies

Using recombinant DNA techniques, an antibody can be produced inside the cells or it can be directly secreted into the medium. If in the first stage the antibody is produced inside the cells, then the cell debris or host cells or their lysed fragments are removed, for example, by centrifugation or ultrafiltration. If the antibody is secreted into the medium, then supernatants obtained from such expression systems are usually first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon® ultrafiltration device. A protease inhibitor such as PMSF can be used to inhibit proteolysis in any of the preceding steps, antibiotics can be used to prevent the propagation of randomly introduced impurity microorganisms.

The antibody composition obtained from the cells can be purified, for example, by hydroxyappatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being the preferred method of purification. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies containing heavy chains of human immunoglobulin γ1, γ2 or γ4 (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). For all murine isotypes and for the human γ3 chain, G protein is recommended (Guss et al., EMBO J. 5: 15671575 (1986)). Agarose is most commonly used as the matrix to which the affinity ligand binds, but other matrices can be used. However, mechanically stable matrices, such as glass with an adjustable pore size or poly (styrene divinyl) benzene, provide a higher flow rate and can reduce processing time than can be achieved using agarose. If the antibody contains a C _H 3 domain, then Bakerbond ABX ^™ resin (JT Baker, Phillipsburg, NJ) can be used to purify it. Other methods of protein purification, such as fractionation on an ion exchange column, precipitation with ethanol, reverse phase HPLC, chromatography on silica, chromatography on heparin-Sepharose ^TM , chromatography on anion or cation exchange resin, can also be applied depending on the antibody to be isolated ( for example, on a column with polyaspartic acid), chromatofocusing, SDS-PAGE electrophoresis and precipitation with ammonium sulfate.

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

Immunoconjugates

The present invention also relates to immunoconjugates (which are synonymous with the terms “antibody-drug conjugates” or “ADC”), including any of the anti-DLL4 antibodies described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, drug, growth an inhibitory agent, a toxin (e.g., an enzymatically active toxin of a bacterial, fungal, plant or animal origin or fragments thereof), or a radioactive isotope (i.e., a radioactive conjugate).

Use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, that is, drugs for killing or suppressing tumor cells in the treatment of cancer (Syrigos & Epenetos (1999) Anticancer Research 19: 605-614; Niculescu-Duvaz & Springer (1997) Adv. Drg. Del. Rev. 26: 151-172; US Pat. No. 4,975,278), theoretically allows targeted delivery of the drug to the tumor and its accumulation within the cells, while systemic administration of these unconjugated drugs can lead to the production of toxicity levels that are unacceptable for normal cells, also insufficient for the tumor cells that need to be destroyed (Baldwin et al., 1986, Lancet pp. (Mar. 15, 1986): 603-05; Thorpe, 1985, “ Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review ”in Monoclonal Antobodies 84: Biological And Clinical Applications, A. Pinchera et al. (Eds.), Pp. 475-506). Thus, it is desirable that maximum effectiveness is combined with minimal toxicity. It has been reported that both polyclonal and monoclonal antibodies can be used in these strategies (Rowland et al., 1986, Cancer Immunol. Immunother. 21: 183-87). The drugs used in these methods are daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., (1986), see above). The toxins used in antibody-toxin conjugates are bacterial toxins such as diphtheria toxin, plant toxins such as ricin, and small toxin molecules 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) , maytansinoids (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). Toxins can exert their cytotoxic and cytostatic effects in accordance with mechanisms including binding to tubulin, binding to DNA, or inhibition of topoisomerase. Some cytotoxic drugs, when conjugated to large antibodies or protein receptor ligands, tend to lose activity or decrease activity.

Zevalin (Zevalin®) (ibritumab tiuksetan, Biogen / Idec) is an antibody-radioisotope conjugate consisting of a mouse monoclonal antibody IgG1-kappa directed against the CD20 antigen present on the surface of normal and malignant B-lymphocytes and ¹¹¹ radioactive isotopes In or ⁹⁰ Y associated with the thiourea-linker chelating complex (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 . 20 (15): 3262-69) . Although zevalin has activity against B-cell non-Hodgkin lymphoma (NHL), however, its administration leads to severe and chronic cytopenia in most patients. In 2000, permission was granted to use the drug Mylotarg (Mylotarg ^TM (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), that is, the antibody-drug conjugate consisting of an anti-human CD33 antibody bound to calicheamicin for the treatment of acute myeloid leukemia by injection of the specified drug ( Drugs of the Future (2000) 25 (7): 686; US Pat. - drug ”, consisting of antibodies against human CD42, linked posre Using a disulfide linker, CPP with a maytansinoid drug, DM1, was used in phase II trials for the treatment of cancers expressing CanAg, such as cancers of the colon, pancreas, stomach, etc. MLN-2704 (Millenium Pharm., BZL Biologics, Immunogen Inc.), an antibody-drug conjugate consisting of a monoclonal antibody against a membrane-bound prostate antigen (PSMA) attached to a maytansinoid drug, DM1, is under investigation for its possible Menenius for treating prostate tumors. Auristatin peptides, auristatin E (AE) and monomethylauristatin (MMAE), synthetic dolastatin analogues, were conjugated to chimeric monoclonal antibodies cBR96 (specific for Lewis Y antigen on carcinomas) and cAC10 (specific for CD30 on hematologic (malignant tumors 2003) Nature Biotechnology 21 (7): 778-784) and are at the developmental stage of therapeutic agents.

Chemotherapeutic agents used to prepare immunoconjugates are described herein (e.g., above). Enzymatically active toxins and their fragments that can be used for these purposes are diphtheria toxin A-chain, non-binding active fragments of diphtheria toxin, exotoxin A-chain (from Pseudomonas aeruginosa ), ricin A-chain, abrin A-chain, A- modecine chain, alpha-sarcinol, Aleurites fordii proteins, diantin protein, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcine, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocycin eritin, phenomycin . See, for example, WO 93/21232, published October 28, 1993. Various radionuclides can be used to produce radioactively conjugated antibodies. Examples of such radionuclides are ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re. Antibody and cytotoxic drug conjugates are prepared using various bifunctional protein binding agents, such as N-succinimidyl-3- (2-pyridylthiol) propionate (SPDP), iminothiolan (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate-HCl) active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) ethylenediamine), diisocyanates So e as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Thus, for example, ricin immunotoxin can be prepared as described in Vitetta et al. Science, 238: 1098 (1987). ¹⁴ C-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminopentaacetic acid (MX-DTPA) is a representative chelating agent for conjugating a radionuclide with an antibody. See WO 94/11026.

The present invention also contemplates conjugates of an antibody and one or more low molecular weight toxins, such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecene and CC1065, and derivatives of these toxins having toxic activity.

i. Maytansin and maytansinoids

In one embodiment of the invention, the immunoconjugate comprises an antibody (full length antibody or fragments thereof) according to the invention conjugated to one or more maytansinoid molecules.

Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrew Maytenus serrata (US patent No. 3896111). It was then discovered that some microbes also produce maytansinoids, such as maytansinol and C-3-maytansinol esters (US Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogues are described, for example, in US patent No. 4137230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4322348; 4,331,598; 4,316,650; 4,364,866; 4,424,219; 4,450,254; 4362663 and 4371533.

Maytansinoid drug molecules are attractive drug molecules for use in antibody-drug conjugates since they (i) can be relatively easily prepared by fermentation or chemical modification and derivatization of fermentation products, (ii) functional groups suitable for derivatization, suitable for conjugation by attaching non-disulfide linkers to antibodies, (iii) are stable in plasma and (iv) are effective waged against various tumor cell lines.

Immunoconjugates containing maytansinoids, methods for their preparation and their therapeutic use are described, for example, in US patents Nos. 5208020, 5416064 and in European patent EP 0425235B1, which in their entirety are incorporated into this application by reference. In the publication of Liu et al. Proc. Natl. Acad. Sci., USA, 93: 8618-8623 (1996) describes immunoconjugates containing a maytansinoid designated DM1 that has been coupled to a C242 monoclonal antibody directed against human colon cancer. It was found that the conjugate is highly cytotoxic for cultured colon cancer cells and has antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52: 127-131 (1992) describe immunoconjugates in which a maytansinoid is conjugated by a disulfide linker to a murine antibody A7 that binds to an antigen present on human colon cancer cell lines or another murine TA.1 monoclonal antibody, which binds to the HER-2 / neu oncogen. The cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3 × 10 ⁵ HER-2 surface antigens per cell. This drug conjugate achieved a degree of cytotoxicity similar to that of the free maytansinoid drug, and such cytotoxicity can be enhanced by increasing the number of maytansinoid molecules per antibody molecule. The conjugate “A7-maytansinoid” showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically binding an antibody to a maytansinoid molecule, wherein said binding does not significantly reduce the biological activity of the antibody or maytansinoid molecule. See, for example, US patent No. 5208020 (which in its entirety is introduced into the present description by reference). The maytansinoid molecules present in this conjugate, on average, in the amount of 3-4 molecules per antibody molecule, show effectiveness in increasing cytotoxicity with respect to target cells, but do not adversely affect the function or solubility of the antibody, although it is assumed that even one toxin molecule per antibody, compared to an unconjugated antibody, will increase the cytotoxicity of the conjugate. Maytansinoids are well known in the art and can be synthesized by known methods, or they can be isolated from natural sources. Suitable maytansinoids are described, for example, in US Pat. No. 5,208,020 and in other patents and non-patent publications described above. Preferred maytansinoids are maytansinol and maytansinol analogues having modifications in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.

Many linker groups known to those skilled in the art can be used to create antibody-maytansinoid conjugates, including, for example, the groups described in US Pat. No. 5,208,020 or in European Patent EP 0425235B1, and in Chari et al., Cancer Research 52: 127 -131 (1992) and U.S. Patent Application No. 10/960602, filed Oct. 8, 2004, which are hereby incorporated by reference in their entirety. Antibody-maytansinoid conjugates containing the linker component of SMCC can be prepared as described in US Patent Application No. 10/960602, filed October 8, 2004. Linker groups are disulfide groups, thioether groups, acid sensitive groups, photochemically unstable groups, peptidase sensitive groups, or esterase sensitive groups described in the aforementioned patents, with disulfide and thioether groups being preferred. Additional linker groups are described and illustrated in this application.

Antibody-maytansinoid conjugates can be prepared using various bifunctional protein binding agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolan (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate-HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido-compounds (such as bis (p-azidene) bis-diazonium derivatives (such as bis- (p-d azoniybenzoil) -ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred binding agents for creating a disulfide bond are N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173: 723-737 [1978]) and N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP).

The linker can be attached to the maytansinoid molecule in various positions depending on the type of bond. For example, an ester bond can be formed by reaction with a hydroxyl group by standard coupling methods. Such a reaction can be carried out at position C-3 having a hydroxyl group, at position C-14 modified with hydroxymethyl, at position C-15 modified with a hydroxyl group, and at position C-20 having a hydroxyl group. In a preferred embodiment of the invention, said bond is formed at the C-3 position of maytansinol or its analogue.

ii. Auristatins and Dolastatins

In some embodiments of the invention, the immunoconjugate comprises an antibody of the invention conjugated to dolastatins or to peptide analogs of dolastatin and their derivatives, also to auristatins (US Pat. Nos. 5,635,483, 5,780,588). It was found that dolastatins and auristatins adversely affect the dynamics of microtubule formation, GTP hydrolysis, and nuclear and cell fission (Woyke et al (2001) Antimicrob. Agents and Chemother. 45 (12): 3580-3584) and have anti-cancer (US Pat. 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). Molecules of dolastatin or auristatin drugs can be attached to the antibody at the N (amino) end or C (carboxy) end of the peptide drug molecule (WO 02/088172).

Representative variants of auristatin are drug molecules containing monomethylauristatin, DE and DF attached to the N-terminus, and are described in US Pat. No. 10/983340, entitled "Monomethylvaline Compounds Capable of Conjugation to Ligands" and filed November 5, 2004, where the description of this application in its entirety is incorporated into this description by reference.

Typically, peptide molecules of a drug can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be formed, for example, by liquid-phase synthesis (see E. Schröder and K. Lübke, “The Peptides”, volume 1, pp. 76-136, 1965, Academic Press), well known to those skilled in the art peptide synthesis. Auristatin / dolastatin drug molecules can be prepared by methods described in US Pat. Nos. 5,635,483 and 5,780,588; and Pettit et al (1989) J. Am. Chem. Soc. 111: 5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13: 243-277; Pettit, GR, et al. Synthesis , 1996, 719-725; and Pettit et al (1996) J. Chem. Soc. Perkin Trans. 1 5: 859-863. See also Doronina (2003) Nat. Biotechnol. 21 (7): 778-784; and US Patent Application Reg. No. 10/983340, entitled "Monomethylvaline Compounds Capable of Conjugation to Ligands" and filed November 5, 2004, where this application is hereby incorporated by reference in its entirety (this application describes, for example, linkers and methods for producing monomethyl valine compounds such as MMAE and MMAF conjugated to linkers).

iii. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of the invention conjugated to one or more calicheamicin molecules. Antibiotics of the calicheamicin family are capable of producing double-stranded DNA breaks in subpicomolar concentrations. A description of the preparation of calicheamicin family conjugates can be found in US Pat. Nos. 5,712,354, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001, 5977296 (all patents are owned by American Cyanamid Company). Structural analogues of calicheamicin that can be used for this purpose include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ ₁ ^I , PSAG, and θ ₁ ^I (see Hinman et al ., Cancer Research 53: 3336-3342 (1993), Lode et al., Cancer Research 58: 2925-2928 (1998) and the aforementioned U.S. patents owned by American Cyanamid). Another antitumor drug with which the antibody can be conjugated is a QFA, which is an antifolate. Calicheamicin and QFA have intracellular active sites and hardly pass through the plasma membrane. Therefore, the absorption of these agents by cells through internalization mediated by the antibody leads to a significant increase in their cytotoxic effects.

iv. Other cytotoxic agents

Other antitumor agents that can be conjugated to antibodies of the invention are BCNU, streptozocin, vincristine and 5-fluorouracil, a family of agents known collectively as the LL-E33288 complex described in US Pat. Nos. 5,053,394, 5,770,710, and also esperamycins (patent US No. 5877296).

Enzymatically active toxins and their fragments that can be used for this purpose are diphtheria toxin A-chain, non-binding active diphtheria toxin fragments, exotoxin A-chain (from Pseudomonas aeruginosa ), ricin A-chain, abrin A-chain, A- modecine chain, alpha-sarcinol, Aleurites fordii proteins, Diantin protein, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcine, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocycin eritin, phenomycin . See, for example, WO 93/21232, published October 28, 1993.

The present invention also contemplates an immunoconjugate formed by an antibody and a compound having nucleolytic activity (e.g., ribonuclease or DNA endonuclease, such as deoxyribonuclease; DNase).

For selective destruction of the tumor, an antibody can be used that may contain a highly radioactive atom. Various radioactive isotopes can be used to produce radioactively conjugated antibodies. Examples of such radionuclides are ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb and radioactive isotopes Lu. If the indicated conjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example ^99m Tc or ¹²³ I, or a spin tag for imaging by nuclear magnetic resonance (NMR) (also known as MR imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radioactive or other labels may be incorporated into the conjugate by known methods. For example, a peptide can be synthesized by biological methods or it can be synthesized by chemical synthesis of amino acids using suitable amino acid precursors, including, for example, fluorine-19 instead of hydrogen. Labels such as ^99m Tc or ¹²³ I, ¹⁸⁶ Re, ¹⁸⁸ Re and ¹¹¹ In can be attached via the cysteine residue of the peptide. Yttrium-90 may be attached via a lysine residue. For the introduction of iodine-123, the IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used. Other methods are described in detail in Monoclonal Antibodies in Immunoscintigraphy (Chatal, CRC Press 1989).

Antibody and cytotoxic drug conjugates can be prepared using various bifunctional protein binding agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolan (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate-HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido-compounds (such as bis (p-azidebenzene) derivatives bis iazoniya (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Thus, for example, ricin immunotoxin can be prepared as described in Vitetta et al. Science , 238: 1098 (1987). ¹⁴ C-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminopentaacetic acid (MX-DTPA) is a representative chelating agent for conjugating a radionuclide with an antibody. See WO 94/11026. The linker may be a “cleavable linker” that facilitates the release of the cytotoxic drug in the cell. For example, acid sensitive linkers may be used; peptidase-sensitive linkers, photochemically unstable linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131 (1992), US patent No. 5208020).

Compounds specifically contemplated by the present invention include, but are not limited to, ADCs prepared using crosslinking reagents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB , SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB and SVSB (succinimidyl- (4-vinyl sulfone) benzoate), which are commercially available (e.g. available from Pierce Biotechnology, Inc., Rockford, IL., USA). See Application Handbook and Catalog, 2003-2004, pp. 467-498.

v. Obtaining conjugates “antibody - drug”

In the antibody-drug conjugates (ADC) of the invention, the antibody (Ab) is conjugated to one or more molecules of the drug (D) via a linker (L), where, for example, from about 1 to 20 molecules per antibody molecule medicinal product. The ADC conjugates of formula I can be obtained in several ways, involving organic chemical synthesis reactions using conditions and reagents known to those skilled in the art, wherein said methods include: (1) reacting an antibody nucleophilic group with a divalent linker reagent to form Ab-L via a covalent bond , then the reaction of interaction with the drug molecule D; and (2) a reaction of interaction of a nucleophilic group of a drug molecule with a divalent linker reagent to form D-L via a covalent bond, then a reaction of interaction with a nucleophilic group of an antibody. Additional methods for producing ADC are described below:

Ab- (LD) _p (I)

The linker may consist of one or more linker components. Representative examples of linker components are 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB" ), N-succinimidyl-4- (2-pyridylthio) pentanoate ("SPP"), N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate ("SMCC") and N-succinimidyl- (4-iodoacetyl ) aminobenzoate ("SIAB"). Other linker components are known to those skilled in the art, some of which are described herein. See also US patent application reg. No. 10/983340, entitled "Monomethylvaline Compounds Capable of Conjugation to Ligands" and filed November 5, 2004, where this application is in its entirety incorporated herein by reference.

In some embodiments of the invention, the linker may contain amino acid residues. Representative amino acid linker components are a dipeptide, tripeptide, tetrapeptide or pentapeptide. Representative dipeptides are valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Representative tripeptides are glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid residues that make up the amino acid linker component are natural amino acid residues, also small amino acids and non-natural amino acid analogues, such as citrulline. Amino acid linker components can be designed and optimized for their selectivity for enzymatic cleavage by specific enzymes, for example, tumor-associated protease, cathepsin B, C and D or plasmin protease.

The nucleophilic groups present on antibodies are, but are not limited to: (i) N-terminal amino groups, (ii) side chain amino groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) hydroxyl or amino groups sugars, where the specified antibody is glycosylated. Amino groups, thiol groups, and hydroxyl groups are nucleophilic and can undergo an interaction reaction to form covalent bonds with electrophilic groups on linker molecules or linker reagents, including: (i) active esters, such as NHS esters, HOBt esters, haloformates and galena anhydrides acids; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl and maleimide groups. Some antibodies have reducible interchain disulfides, i.e. cysteine bridges. For conjugation with linker reagents, antibodies can be made reactive by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge can theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of interaction of lysines with 2-iminothiolane (Trout reagent), which will lead to the conversion of the amine to thiol. Reactive thiol groups can be incorporated into an antibody (or fragment thereof) by introducing one, two, three, four or more cysteine residues (for example, obtaining mutant antibodies containing one or more non-natural cysteine amino acid residues).

Antibody-drug conjugates according to the invention can also be obtained by modifying the antibody with electrophilic groups that can react with nucleophilic substituents on a linker reagent or drug. Sugar glycosylated antibodies can be oxidized, for example, with periodic oxidizing agents to form aldehyde or ketone groups, which can react with the amino group of linker reagents or drug molecules. The resulting imine groups of the Schiff base can form a stable bond or they can be reduced, for example, with borohydride reagents to form stable amine bonds. In one embodiment of the invention, the reaction of the interaction of the carbohydrate moiety of a glycosylated antibody with galactose oxidase or with sodium meta-periodate can lead to the formation of carbonyl (aldehyde and ketone) groups in the protein that can react with the corresponding groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment of the invention, proteins containing N-terminal serine or threonine residues can react with sodium metaperiodate to form an aldehyde instead of the first amino acid (Geoghegan & Stroh (1992), Bioconjugate Chem. 3: 138-146; US Pat. No. 5,362,852). Such an aldehyde may interact with a drug molecule or with a linker nucleophile.

Similarly, nucleophilic groups on a drug molecule include, but are not limited to, amino groups, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazinecarboxylate and aryl hydrazide groups capable of reacting with the formation of covalent bonds with linker electrophilic groups reagents, including: (i) active esters, such as NHS esters, HOBt esters, acid halides and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl and maleimide groups.

Alternatively, a fusion protein containing an antibody and a cytotoxic agent can be obtained, for example, by recombinant methods or by peptide synthesis. The length of the DNA can be corresponding regions encoding two parts of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide, which does not adversely affect the desired properties of the conjugate.

In yet another embodiment of the invention, said antibody can be conjugated to a “receptor” (such as streptavidin) to pre-target a tumor, where said antibody-receptor conjugate is administered to a patient, followed by removal of the unbound conjugate from the bloodstream using a clearance agent, then administer a “ligand” (eg, avidin) conjugated to a cytotoxic agent (eg, a radionucleotide).

Pharmaceutical Compositions

Therapeutic compositions containing the antibody of the invention are prepared in convenient storage forms by mixing the antibody of the desired purity with optional physiologically acceptable carriers, excipients or stabilizers ( Remington; The Science and Practice of Pharmacy , 20th edition (2000)) to obtain aqueous solutions, lyophilized preparations or other dry preparations. Acceptable carriers, excipients or stabilizers at the dosages and concentrations used should be non-toxic to the recipients, and these are buffers such as phosphate, citrate, histidine and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol alcohol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; 3-pentanol) low molecular weight polypeptides (having about less than 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions, such as sodium; metal complexes (for example, Zn - protein complexes); and / or nonionic surfactants, such as Tween ^TM , Pluronic ^TM, or polyethylene glycol (PEG).

The composition described herein may also contain several active compounds necessary for a particular indication, preferably having additional activities that do not adversely affect each other. Such molecules may be present in combination in amounts effective to be used for the intended purpose.

The active ingredients can also be enclosed in a microcapsule obtained, for example, by coacervation or by interfacial polymerization, for example, hydroxymethyl cellulose or gelatin microcapsule and polymethyl methacrylate microcapsule, respectively, in systems for the delivery of colloidal drugs (for example, in liposomes, albumin microsulfides nanoparticles and nanocapsules) or in macroemulsions. This technique is described in Remington: The Science and Practice of Pharmacy 20th edition (2000).

Compositions used for in vivo administration must be sterile. This can be easily achieved by filtration through sterile filtering membranes.

Sustained release preparations may be prepared. Suitable examples of sustained release preparations are semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin of the invention, wherein said matrices are in the form of finished products, for example films or microcapsules. Examples of sustained release matrices are polyesters, hydrogels (e.g. poly (2-hydroxyethyl methacrylate) or polyvinyl alcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, an indecomposable ethylene-vinyl acetate copolymer decomposable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ^™ (injection microspheres consisting of a lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate copolymer and lactic acid-glycolic acid copolymer are capable of releasing molecules for more than 100 days, some hydrogels release proteins in shorter periods of time. When encapsulated immunoglobulins retained in the body for a long period of time, they may denature or aggregate upon exposure to moisture at ³⁷ C, resulting in a loss of biological activity and possible changes in immunogenicity. To stabilize, depending on the particular mechanism, rational strategies can be developed. For example, if it was discovered that the aggregation mechanism is the formation of an intermolecular SS bond through thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acid solutions, regulating the moisture content using appropriate additives and obtaining specific polymer-based compositions matrices.

Applications

The antibody according to the invention can be used, for example, in therapeutic methods in vitro , ex vivo and in vivo .

In one aspect, the present invention relates to methods for treating or preventing a tumor, cancer, or cell proliferative disorder associated with increased expression and / or activity of DLL4, wherein said methods comprise administering an effective amount of an anti-DLL4 antibody to an individual in need of such treatment .

In one aspect, the present invention provides methods for attenuating, inhibiting, blocking, or preventing the growth of a tumor or cancer, wherein said methods comprise administering an effective amount of an anti-DLL4 antibody to an individual in need of such treatment.

In one of its aspects, the present invention relates to methods for treating a tumor, cancer and / or cell proliferative disorder, wherein said methods comprise administering an effective amount of an anti-DLL4 antibody to an individual in need of such treatment.

In one aspect, the present invention relates to methods for inhibiting angiogenesis, wherein said methods comprise administering an effective amount of an anti-DLL4 antibody to an individual in need of such treatment.

In one of its aspects, the present invention relates to methods for treating a pathological condition associated with angiogenesis, wherein said methods comprise administering an effective amount of an anti-DLL4 antibody to an individual in need of such treatment. In some embodiments of the invention, said pathological condition associated with angiogenesis is a tumor, cancer, and / or cell proliferative disorder. In some embodiments of the invention, said pathological condition associated with angiogenesis is an intraocular neovascular disease.

In addition, at least some antibodies of the invention may bind to antigens derived from other species. Accordingly, the antibodies of the invention can be used to bind to an antigen having specific activity, for example, in a cell culture containing the indicated antigen, and in humans or other mammals having the antigen with which the antibody of the invention cross-reacts (e.g. chimpanzees, baboons, marmosets, dog-like monkeys and rhesus monkeys, also pigs or mice). In one embodiment of the invention, an antibody of the invention can be used to inhibit antigenic activity by contacting said antibody with an antigen so that antigenic activity is inhibited. A preferred antigen is a human protein molecule.

In one embodiment of the invention, the antibody of the invention can be used in a method of antigen binding in an individual suffering from a disorder associated with an increased level of expression and / or antigen activity, wherein said method comprises administering to said individual an antibody of the invention to bind to the antigen. A preferred antigen is a human protein molecule, a preferred individual is a human. Alternatively, the specified individual may be a mammal expressing the antigen to which the antibody of the invention binds. In addition, the indicated individual may be a mammal to which the indicated antigen has been introduced (for example, by direct administration of an antigen or expression of an antigenic transgene). An antibody according to the invention can be administered to humans for therapeutic purposes. In addition, the antibody of the invention can be administered to a non-human mammal (e.g., a primate, pig or mouse) and expresses an immunoglobulin cross-reacting antigen to treat this mammal, or it can be administered to an animal with a human disease model. As for the animal model, such animals can be used to evaluate the therapeutic efficacy of antibodies according to the invention (for example, to determine doses and time of administration).

Antibodies according to the invention can be used to treat, inhibit, slow progression, prevent / slow the onset of relapse, improve the dynamics or prevention of diseases, disorders or conditions associated with the expression and / or activity of one or more antigen molecules.

Representative disorders are carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies. More specific examples of such cancers are 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, abdominal cancer, hepatocellular cancer, gastric cancer, including cancer gastrointestinal tract; pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, colon cancer, colon cancer, endometrial or uterine carcinoma, salivary carcinoma kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anus carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain cancer, also head and neck cancer, and associated metastases . In some embodiments of the invention, said cancer is selected from the group consisting of small cell lung cancer, neuroblastoma, melanoma, breast carcinoma, stomach cancer, colon cancer (CRC), and hepatocellular carcinoma. In some embodiments of the invention, said cancer is selected from the group consisting of non-small cell lung cancer, colon cancer, and breast carcinoma, including metastases of said cancer.

In some embodiments, an immunoconjugate comprising an antibody conjugated to one or more cytotoxic agents is administered to a specified patient. In some embodiments of the invention, the immunoconjugate and / or antigen with which it binds is internalized in the cell, which leads to an increase in the therapeutic effectiveness of the immunoconjugate aimed at killing the target cell with which it binds. In one embodiment of the invention, the cytotoxic agent inhibits the nucleic acid or inhibits its activity in the target cell. In one embodiment of the invention, said cytotoxic agent inhibits or inhibits microtubule polymerization. Examples of these cytotoxic agents are any of the chemotherapeutic agents described herein (such as maytansinoid, auristatin, dolastatin or calicheamicin), a radioactive isotope, or ribonuclease or DNA endonuclease.

Antibodies according to the invention, taken separately or in combination with other compositions, can be used for therapeutic purposes. So, for example, the antibody according to the invention can be introduced together with another antibody, chemotherapeutic (s) agent (s) (including mixtures of chemotherapeutic agents), with other (s) cytotoxic (s) agent (s), antiangiogenic (s) agent ( s), cytokines and / or growth inhibitory (s) agent (s). If an antibody of the invention inhibits tumor growth, it may be especially desirable to combine it with one or more other therapeutic agents that also inhibit tumor growth, for example, with anti-VEGF agents, including anti-VEGF antibodies. Alternatively or additionally, the patient may be subjected to combination radiation therapy (for example, external beam irradiation or therapy using a radioactively labeled agent such as an antibody). Such combination therapy as indicated above is combination administration (where two or more agents are included in the same or separate compositions) and separate administration, in which the antibody of the invention can be administered before and / or after adjuvant therapy.

Combination therapy

As indicated above, the present invention relates to combination therapy in which an anti-DLL4 antibody is administered concurrently with another therapy. So, for example, anti-DLL4 antibodies are used in combination with anticancer therapy or with therapy against the formation of new vessels to treat various neoplastic or non-neoplastic conditions. In one embodiment of the invention, said neoplastic or non-neoplastic condition is characterized by a pathological disorder associated with aberrant or unwanted angiogenesis. Anti-DLL4 antibodies can be administered sequentially or in combination with another agent effective for such purposes, either as a single composition or as separate compositions. Alternatively or additionally, multiple DLL4 inhibitors may be administered.

The introduction of anti-DLL4 antibodies can be carried out simultaneously with other means, for example, in the form of a single composition or in two or more separate compositions using the same or different methods of administration. Alternatively or additionally, administration can be carried out sequentially in any order. In some embodiments of the invention, the intervals between administration of two or more compositions may be from several minutes to several days or from several weeks to several months. So, for example, an anticancer agent can be introduced first, then a DLL4 inhibitor. However, simultaneous administration of an anti-DLL4 antibody with other agents or administration of an anti-DLL4 antibody first and then administration of another agent is also contemplated.

An effective amount of therapeutic agent administered in combination with an anti-DLL4 antibody is prescribed by a doctor or veterinarian. To achieve the maximum treatment effect for these conditions, the administered dose is adjusted accordingly. The dose also depends on factors such as the type of therapeutic agent used and the particular patient being treated. Suitable doses of the anticancer agent are those used in modern therapy, and these doses can be reduced due to the combined action (synergistic effect) of the anticancer agent and anti-DLL4 antibody. In some embodiments, a combination of inhibitors enhances the effectiveness of a single inhibitor. The term "potentiate" means to increase the effectiveness of a therapeutic agent administered in a normal or acceptable dose. See also the section of this application entitled “Pharmaceutical Compositions”.

Typically, anti-DLL4 antibodies and anti-cancer agents are suitable for treating the same or similar diseases by preventing or mitigating a pathological disorder such as tumor growth or cancer cell growth. in one embodiment of the invention, said anti-cancer agent is an antiangiogenic agent.

Angiogenic therapy for cancer is a cancer treatment strategy whose goal is to inhibit the development of the blood vessels of the tumor, which are necessary for supplying the nutrients required for tumor growth. Since angiogenesis occurs both with the growth of the primary tumor and with metastases, treatment with anti-angiogenic agents carried out in accordance with the present invention can lead to inhibition of tumor growth at the site of formation of the primary tumor, and also to prevent the development of tumor metastases that form in other areas, which allows you to affect the tumor with other therapeutic agents.

Many anti-angiogenic agents have been identified and known to those skilled in the art, including the agents listed in this application, for example, in the Definitions section and the agents described, for example, in Carmeliet & Jain, Nature 407: 249-257 (2000); Ferrara et al., Nature Reviews: Drug Discovery , 3: 391-400 (2004); and Sato Int. J. Clin. Oncol. 8: 200-206 (2003). See also U.S. Patent Application US 20030055006. In one embodiment of the invention, an anti-DLL4 antibody is used in combination with a neutralizing anti-VEGF antibody (or fragment thereof) and / or another VEGF antagonist or VEGF receptor antagonist, including but not limited to, for example, a soluble VEGF receptor (e.g., VEGFR-1, VEGFR-2, VEGFR-3, neuroprilins (e.g., NRP1, NRP2)), fragments thereof, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR- antibodies, small molecule VEGFR tyrosine kinase inhibitors (RTK), antisense sequences for VEGF, ribosis Imes against VEGF or VEGF receptors; VEGF antagonist variants; and any combination thereof. Alternatively or additionally, in addition to the VEGF antagonist and another agent, two or more angiogenesis inhibitors may be administered simultaneously, in addition to the patient. In some embodiments of the invention, one or more additional therapeutic agents, for example anti-cancer agents, can be administered in combination with an anti-DLL4 antibody, a VEGF antagonist, and an anti-angiogenic agent.

In some aspects of the invention, other therapeutic methods suitable for combination therapy of a tumor using an anti-DLL4 antibody are other anti-cancer therapy (e.g., surgery, radiological treatment (e.g., radiation therapy or administration of radioactive substances), chemotherapy, anti-cancer treatment listed in this application and known to specialists, or combinations thereof). Alternatively or additionally, two or more antibodies that bind to the same or to two or more different antigens described herein can be simultaneously administered to a patient. Sometimes it may also be preferable to administer one or more cytokines to the patient.

Chemotherapeutic agents

In some of its aspects, the present invention relates to a method for blocking or reducing the growth of a tumor or cancer cells, wherein said method comprises administering effective amounts of a DLL4 antagonist and / or angiogenesis inhibitor (s) and one or more chemotherapeutic agents to a cancer susceptible patient or patient who has been diagnosed with cancer. Various chemotherapeutic agents can be used in the combined treatment methods of the invention. A representative and non-limiting list of chemotherapeutic agents considered is provided herein in the Definitions section.

As is obvious to every person skilled in the art, the corresponding doses of chemotherapeutic agents are approximately the same as those already used in clinical therapy, where the indicated chemotherapeutic agents are administered alone or in combination with other chemotherapeutic agents. Doses may vary depending on the condition being treated. The doctor prescribing this treatment can independently calculate the appropriate dose for each individual individual.

The present invention also relates to methods and compositions for inhibiting or preventing the recurrent growth of a tumor or cancer cells. The term “recurrent tumor or cancer cell growth” is used to define a condition that a given individual suffers from or a condition that is treated with one or more methods of therapy (eg, anti-cancer therapy such as chemotherapy, radiation therapy, surgery, hormone therapy and / or biological therapy / immunotherapy, anti-VEGF antibody therapy, in particular the standard therapeutic method for the treatment of a specific cancer), is not clinically adequate or only gives short-term beneficial effect, therefore, such patients need additional effective therapy. As used herein, the term may also mean the patient’s “immunity / resistance” state, for example, the condition of a patient who has a definite response to the therapy but has side effects; or a patient who develops resistance, there is no response to this therapy or a satisfactory response to this therapy, etc. In various embodiments of the invention, the cancer is a recurrent growth of the tumor or cancer cells, in which there is no noticeable decrease in the number of cancer cells or no increase, or no decrease in the size of the tumor or increase, or no further decrease in the size of the cancer tumors or cancer cell numbers. Establishment of the recurrent growth of a cancerous tumor or cancer cells can be carried out by any in vivo or in vitro method known to those skilled in the art, which include analysis of the effectiveness of cancer cell suppression, where the terms “relapse” or “resistance” or “immunity” are used in their generally accepted sense . An example of a recurring tumor growth is a tumor resistant to treatment with an anti-VEGF antibody.

The present invention relates to methods for blocking or attenuating the recurrent growth of a tumor or cancer cells in an individual, wherein said methods comprise administering to the individual one or more anti-DLL4 antibodies to block or attenuate the recurrent growth of a tumor or cancer cells. In some embodiments, the antagonist may be administered after anticancer therapy. In some embodiments, anti-DLL4 antibodies are administered concurrently with anti-cancer therapy. Alternatively or additionally, anti-DLL4 antibody therapy is administered alternately with other anti-cancer therapy, and such therapy may be carried out in any order. The present invention also encompasses methods for administering one or more inhibitory antibodies to prevent the development or relapse of cancer in patients predisposed to develop such cancer. Typically, such an individual has undergone (or is undergoing) combination anticancer therapy. In one embodiment of the invention, anti-cancer therapy is treatment with an antiangiogenic agent, for example, a VEGF antagonist. Antiangiogenic agents are those known to those skilled in the art and listed in the Definitions section of this application. In one embodiment, the antiangiogenic agent is a neutralizing anti-VEGF antibody or fragment thereof (e.g., AVASTIN® (Genentech, South San Francisco, CA) or LUCENTIS® (Genentech, South San Francisco, CA)), Y0317, M4, G6 , B20, 2C3, etc.). See, for example, US patents Nos. 6582959, 6884879, 6703020; WO 98/45332; WO 96/30046; WO 94/10202; EP 0666868B1; 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 may be administered in combination with a VEGF antagonist and anti-DLL4 antibody to block or attenuate the recurrent growth of a tumor or cancer cells, for example, see the section entitled “Combination Therapy”.

An antibody according to the invention (and an adjuvant therapeutic agent) is administered in various ways, including parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal administration, if local treatment is necessary, the antibody is administered to the affected area or to the vitreous body. Parenteral infusion is intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, a suitable route of administration of the antibody is a periodic infusion, in particular an infusion of the antibody in decreasing doses. Such doses may be administered by any suitable method, for example by injection, such as intravenous or subcutaneous injection, and these doses depend in part on whether such administration is periodic or continuous.

An antibody-containing composition according to the invention is prepared, divided into doses and administered in accordance with well-known medical practice. Factors considered for the preparation of such compositions are the particular disorder being treated, the particular mammal being treated, the clinical condition of the particular patient, the etiological factor causing the disorder, the site to which the agent is directed, the route of administration, the route of administration, and other factors known to practitioners. The specified antibody should be, but not necessarily, prepared in the form of a composition with one or more modern means used to prevent or treat the disorder in question. An effective amount of such other agents depends on the amount of the antibodies of the invention present in the composition, the type of disorder or method of treatment, and other factors discussed above. These other agents are usually administered in the same doses and in the same manner as previously used, or these doses comprise about 1-99% of the previously used doses.

To prevent or treat a disease, suitable doses of an antibody according to the invention (used alone or in combination with other agents, such as chemotherapeutic agents) depend on the type of disease being treated, the type of antibody, the severity and the course of treatment of the disease, regardless of whether the antibody is administered for preventive or therapeutic purposes, from previous treatment, from the patient’s medical history and susceptibility to the antibody, and from the appointment of the attending physician. Such an antibody may be administered to a patient once or several times during a course of treatment. Depending on the type and severity of the disease, the initial preset dose of the antibody administered to the patient is from about 1 μg / kg to 15 mg / kg (e.g. 0.1 mg / kg to 10 mg / kg), regardless of whether single or multiple administration or continuous infusion of a given antibody. A typical daily dose may be from about 1 μg / kg to 100 mg / kg or more, depending on the above factors. For repeated administration over a period of several days or more, depending on the condition, treatment may be carried out until the desired suppression of disease symptoms is achieved. A representative dose of the antibody may be from about 0.05 mg / kg to 10 mg / kg. For example, 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) may be administered to a patient. Such doses may be administered periodically, for example weekly or every three weeks (for example, about two to twenty, for example, about six doses of an antibody may be administered to a patient). In this case, a higher dose may be administered first, then one or more lower doses may be administered. A representative dose schedule includes administering an initial dose of about 4 mg / kg, followed by a weekly administration of a maintenance dose of about 2 mg / kg of antibody. However, other administration regimens may also be used. Monitoring the effect of such therapy can be easily carried out using standard methods and using standard assays.

The anti-DLL4 antibodies of the invention can be used in assays to detect the level of expression of DLL4 (such as diagnostic or prognostic assays) in specific cells or tissues where these antibodies are labeled as described below and / or immobilized on an insoluble matrix.

In another aspect, the present invention relates to methods for detecting DLL4, where these methods include detecting the complex "DLL4 - anti-DLL4 antibody" in the sample. As used herein, the term “detection” includes qualitative and / or quantitative detection (determination of levels) in the presence or absence of control.

In another aspect, the present invention relates to methods for diagnosing a disorder associated with the expression and / or activity of DLL4, where these methods include the detection of the complex "DLL4 - anti-DLL4 antibody" in a biological sample taken from a patient suffering from the specified disorder, or a patient suspected of having this disorder. In some embodiments of the invention, the expression of DLL4 is an increased level of expression or abnormal (unwanted) expression. In some embodiments of the invention, said disorder is a tumor, cancer, and / or cell proliferative disorder.

In another aspect, the present invention relates to any of the anti-DLL4 antibodies described herein, wherein said anti-DLL4 antibody contains a detectable label.

In another aspect, the present invention relates to a complex consisting of any of the anti-DLL4 antibodies and DLL4 described herein. In some embodiments of the invention, said complex is present in vivo or in vitro . In some embodiments of the invention, said complex comprises a cancer cell. In some embodiments, the anti-DLL4 antibody is detectably labeled.

Anti-DLL4 antibodies can be used to detect DLL4 in any one of a number of well-known analytical detection methods. For example, a biological sample can be analyzed for DLL4 by taking a sample from the desired source, mixing the sample with an anti-DLL4 antibody to form the antibody / DLL4 complex with any DLL4 present in the mixture, and detecting any antibody / complex DLL4 ”present in this mixture. A biological sample can be obtained for analysis by methods known to those skilled in the art and suitable for analysis of a particular sample. The methods of mixing the sample with antibodies and the detection methods of the antibody / DLL4 complex are selected depending on the type of analysis used. Such analyzes are immunohistochemical analysis, competitive analysis and sandwich analysis, as well as steric inhibition assays.

DLL4 assay methods use one or more of the following reagents, such as: labeled DLL4 analog, immobilized DLL4 analog, labeled anti-DLL4 antibody, immobilized anti-DLL4 antibody, and steric conjugates. Labeled reagents are also known as “tags.”

The label used is any detectable functional group that does not affect the binding of DLL4 and anti-DLL4 antibodies. Various labels are known to be used in immunoassays, and examples of such labels are molecules that can be detected directly, such as fluorochrome, a chemiluminescent substance, and radioactive labels, as well as molecules such as enzymes that must undergo an interaction or derivatization reaction to detect them. . Examples of such labels are ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I radioisotopes, fluorophores such as rare earth metal chelate complexes and fluorescein and its derivatives, rhodamine and its derivatives, dansil, umbelliferone, luciferase, for example firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase, for example glucose oxidase, galactose oxidase 6-glucose, heterocyclic oxidases Such as uricase and xanthine oxidase, attached to an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP; lactoperoxidase or microperoxidase; biotin / avidin, spin labels, bacteriophage labels, stable free radicals, etc.

Standard methods are used to covalently bind these labels to proteins or polypeptides. So, for example, binding agents such as dialdehydes, carbodiimides, dimaleimides, bisimidates, bis-diazotized benzidine and the like can be used to label antibodies with the above-described fluorescent, chemiluminescent and enzyme labels. See, for example, US Pat. 3940475 (fluorimetry) and 3645090 (enzymes; Hunter et al. , Nature , 144: 945 (1962); David et al. , Biochemistry, 13: 1014-1021 (1974); Pain et al. , J. Immunol. Methods , 40: 219-230 (1981); and Nygren, J. Histochem. And Cytochem., 30: 407-412 (1982). Preferred labels used in this application are enzymes such as horseradish peroxidase and alkaline phosphatase. , including enzymes, with this antibody is a standard process used in immunoassay methods and is well known to one of ordinary skill in the art. See, for example, O'Sullivan et al. , "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay, "in Methods in Enzymology , ed. JJ Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166.

For some analytical methods, the immobilization of reagents is necessary. Immobilization results in the separation of the anti-DLL4 antibody from any DLL4 remaining in solution in the free state. This procedure is usually carried out by insolubilization of the anti-DLL4 antibody or analogue of DLL4 before the analysis procedure, as occurs when adsorption on a water-insoluble matrix or surface (Bennich et al ., US patent No. 3720760), by covalent binding (for example cross-linking with glutaraldehyde) or by subsequent insolubilization of an anti-DLL4 antibody or analogue of DLL4, for example by immunoprecipitation.

The expression of proteins in the sample can be evaluated in accordance with immunohistochemical analysis and staining protocols. It has been shown that immunohistochemical staining of tissue sections is a reliable method for assessing or detecting the presence of proteins in a sample. Immunohistochemical (IHC) methods involve the use of antibodies to probe and visualize cell antigens in situ , usually using chromogenic or fluorescent reagents. To prepare a sample, a tissue or cell sample taken from a mammal (usually a human) can be used. Examples of such samples include, but are not limited to, cancer cells, such as cancer cells of the colon, breast, prostate, ovary, lung, stomach, pancreas, lymphoma, and leukemia. The sample can be obtained using various procedures known to those skilled in the art, including but not limited to surgery, aspiration, or biopsy. The fabric may be fresh or frozen. In one embodiment of the invention, the sample is fixed and embedded in paraffin or the like. A tissue sample can be fixed (i.e., reserved) by standard methods. The average person skilled in the art knows that the choice of fixing substance depends on the goals of histological staining of the sample or some other analysis. The average person skilled in the art also knows that the fixation time depends on the size of the tissue sample and on the fixing substance used.

Immunohistochemical (IHC) analysis can be carried out in combination with other methods, such as morphological staining and / or in situ fluorescence hybridization. Two general methods of IHC analysis are known to specialists: direct and indirect analyzes. According to a first assay, the binding of an antibody to a target antigen (e.g., DLL4) is determined by a direct method. In this direct assay, a labeled reagent is used, such as a fluorescent label or an “first” antibody labeled with the enzyme, which can be visualized without interaction with another antibody. In a typical indirect assay, the unconjugated “first” antibody binds to the antigen, then the labeled “second” antibody binds to the first antibody. If the “second” antibody is conjugated to an enzyme tag, then a chromogenic or fluorogenic substrate is added to visualize the antigen. Amplification of the signal can occur as a result of the reaction of several "second" antibodies with different epitopes on the "first" antibody.

The “first” and / or “second” antibody used for immunohistochemical analysis is usually labeled with a detectable molecule. There are many labels that can be basically divided into groups according to the categories described below.

In addition to the sample preparation procedures discussed above, additional processing of tissue sections may be desirable prior to, during, or after the IHC analysis. Thus, for example, epitope recovery techniques such as heating a tissue sample in citrate buffer can be applied (see, for example, Leong et al. Appl. Immunohistochem . 4 (3): 201 (1996)).

After the optional blocking step, the tissue section is treated with the “first” antibody for a certain period of time and under suitable conditions sufficient to bind the first antibody to the target protein antigen in the tissue sample. Appropriate conditions suitable for achieving this goal can be determined through routine experimentation. The level of binding of the antibody to the sample is determined using any one of the detectable labels discussed above. A preferred label is an enzyme label (e.g., HRPO) that catalyzes the chemical conversion of a chromogenic substrate, such as a 3,3'-diaminobenzidine chromogen. Preferably, said enzymatic label is conjugated to an antibody that specifically binds to the “first” antibody (for example, the “first” antibody is a rabbit polyclonal antibody, the “second” antibody is a goat anti-rabbit antibody).

Thus prepared samples can be obtained in the form of histological sections and applied to slides. These slides are then analyzed under a microscope and evaluated according to staining intensity criteria commonly used by those skilled in routine analysis procedures. The staining intensity can be estimated by the following criteria in table 2:

table 2 Painting stain Rating Cell staining was not observed. 0 Very faint / barely noticeable staining was observed in more than 10% of the cells. 1+ Slight to moderate staining was observed in more than 10% of the cells. 2+ Moderate-intense staining was observed in more than 10% of the cells. 3+

Typically, in an immunohistochemical analysis, an assessment of a staining pattern of about 2+ or higher is diagnostic and / or prognostic. In some embodiments of the invention, in an immunohistochemical analysis, an assessment of a staining pattern of about 1+ or higher is diagnostic and / or prognostic. In other embodiments of the invention, an assessment of a staining pattern of about 3 or higher is diagnostic and / or prognostic. It should be noted that if cells and / or tissues of a tumor or colon adenomas are evaluated by immunohistochemical analysis, staining is usually determined or evaluated in tumor cells and / or tissues (and not in stromal or surrounding tissues that may be present in the sample )

There are also other analytical methods known as competitive assays or sandwich analyzes, and such methods are widely used in the commercially available diagnostic industry.

Competitive assays are based on the ability of a labeled DLL4 analog to compete with a DLL4 test sample for binding to a limited number of antigen-binding sites of an anti-DLL4 antibody. The anti-DLL4 antibody is usually solubilized before or after competitive binding, then the label and DLL4 associated with the anti-DLL4 antibody are separated from the unbound label and DLL4. This separation is carried out by decantation (where the specified binding partner was previously subjected to solubilization) or by centrifugation (where the binding partner was precipitated after a competitive reaction). The amount of DLL4 test sample is inversely proportional to the amount of bound label, as determined by the amount of marker substance. Dose-response curves were constructed from known amounts of DLL4, and the results were compared with test results to determine the amount of DLL4 present in the test sample. If enzymes are used as detectable markers, then such analyzes are called ELISA systems.

Another type of competitive analysis, called "homogeneous" analysis, does not involve phase separation. In this case, the enzyme conjugate with DLL4 is prepared and used so that the anti-DLL4 antibody, when linked to DLL4, can modify the enzymatic activity. In this case, DLL4 or immunologically active fragments thereof are conjugated to an enzyme, such as peroxidase, via a bifunctional organic bridge. Conjugates intended for use with an anti-DLL4 antibody are selected such that binding of the anti-DLL4 antibody results in inhibition or potentiation of the enzymatic activity of the label. This method per se is widely used in practice and is called EMIT.

In methods of homogeneous analysis using steric hindrance, steric conjugates are used. These conjugates are synthesized by covalent binding of a low molecular weight hapten to a small fragment of DLL4 so that the anti-hapten antibody is substantially unable to bind to the conjugate simultaneously with the anti-DLL4 antibody. In this analysis procedure, the DLL4 present in the test sample will bind to the anti-DLL4 antibody, which allows the anti-hapten antibody to bind to this conjugate and change the properties of the hapten conjugate, for example, change the fluorescence intensity if the hapten is a fluorophore .

Sandwich analyzes, in particular, can be used to detect DLL4 or anti-DLL4 antibodies. In sequential sandwich analyzes, an immobilized anti-DLL4 antibody is used to adsorb the test sample DLL4, then the test sample is removed by washing and the bound DLL4 is used to adsorb the “second” labeled anti-DLL4 antibody, after which the bound material is separated from the rest of the label. The number of linked tags is directly proportional to the number of sample DLL4 samples. In “simultaneously” conducted “sandwich” assays, the test sample is not separated from the label before the addition of labeled anti-DLL4 antibody. Samples tested for the presence of DLL4 use a sequential sandwich assay using a monoclonal anti-DLL4 antibody as one antibody and a polyclonal anti-DLL4 antibody as another antibody.

Only representative analyzes on DLL4 have been described above. Other methods currently in use, or methods that will be developed in the future and in which an anti-DLL4 antibody will be used to determine DLL4, are included in the scope of the methods considered, including the bioassays described here.

Industrial products

In another aspect, the present invention relates to an industrial product containing materials used for the treatment, prevention and / or diagnosis of the above disorders. The industrial product comprises a container and a label or liner enclosed in the package or glued to the container. Suitable containers are, for example, bottles, vials, syringes, and the like. Containers can be made of various materials, such as glass or plastic. This container contains a composition that, alone or in combination with other composition (s), is effective for treating, preventing and / or diagnosing this condition, and may have a sterile inlet (for example, such a container may be an intravenous bag the introduction of a solution or a vessel having a plug pierced by a needle for injection). In this composition, the at least one active agent is an antibody of the invention. On the label or in the insert enclosed in the package, it should be indicated that such a composition is used to treat this particular disease, such as cancer. Furthermore, said industrial product may include (a) a first container containing a composition comprising an antibody of the invention, and (b) a second container containing a composition comprising an additional therapeutic agent, including, for example, a chemotherapeutic agent or an antiangiogenic agent including, for example, an anti-VEGF antibody (e.g., bevacizumab). In this embodiment of the invention, the industrial product according to the invention may further comprise a package insert, which indicates that compositions containing the “first” and “second” antibody can be used to treat a particular condition, such as cancer. Alternatively or additionally, said industrial product may also comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. In addition, it may include other materials necessary for commercial production and for the consumer, including other buffers, diluents, filters, needles and syringes.

The following are examples of methods and compositions according to the invention. It should be noted that on the basis of the above general description of the invention, various other variants of the invention can be implemented.

EXAMPLES

Materials and methods

Commercially available reagents described in the examples were used in accordance with the manufacturer's instructions, unless otherwise indicated. The source of cells used in the following examples and throughout the description of the invention is the American Type Culture Collection, American Type Culture Collection Manassas, VA 20108, and these cells are identified by their ATCC® serial numbers. The works cited in the examples are listed after the examples. All of these works are introduced into the present description by reference.

Example 1: Materials and Methods

In the examples, the following materials and methods were used.

HUVEC analysis on fibrin gel spheres. HUVEC analysis on fibrin gel spheres is described in detail in the literature (Nakatsu, MN et al. Microvasc Res 66, 102-12 (2003)). Briefly, 3 Cytodex ™ spheres (Amersham Pharmacia Biotech) were coated with 350-400 HUVEC cells per sphere. Approximately 200 spheres coated with HUVEC were poured into a fibrin clot in one of the wells of a 12-well tissue culture plate. 8 × 10 ⁴ SF cells were seeded onto the top of the clot. Assays were completed on day 7-9 for immunostaining and imaging. In some experiments, the growth of HUVEC was visualized by staining with biotin-conjugated anti-CD31 antibody (clone WM59, eBioscience) and streptavidin-Cy3. To stain HUVEC nuclei, fibrin gels were fixed overnight in 2% paraformaldehyde (PFA) and stained with 4 ', 6-diamidino-2-phenylindole (DAPI, Sigma). To stain Ki67, the fibrin gels were treated with 10 × trypsin-EDTA for 5 minutes to remove the top layer of SF, then neutralized with 10% FBS in PBS and fixed overnight in 4% PFA. Fibrin gels were then blocked with 10% goat serum in PBST for 4 hours, then incubated overnight with rabbit anti-mouse Ki67 (ready-to-use Sp6 clone, LabVision), then detected using a “second” Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch). All overnight incubation procedures were performed at 4 ° C.

The study of the retina of newborn mice. Newborn CD1 mice from the same litter ip were injected with PBS or YW26.82 (10 mg / kg) on P1 and P3. Eyes were removed at P5 and fixed at 4% PFA in PBS overnight. Excised retinas were blocked with 10% goat serum in PBST for 3 hours, then incubated overnight with the “first” antibodies. The first mixture included biotinylated isolectin B4 (25 μg / ml, Bandeiraea simplicifolia ; Sigma), and one of the following components, such as rabbit anti-mouse Ki67 (1: 1, ready-to-eat clone Sp6, Lab Vision) or mouse Cy3-conjugated anti-mouse antibody alpha-SMA (1: 2000, Sigma-Aldrich) and 10% serum in PBLEC (1% Triton X-100, 0.1 mM CaCl ₂ , 0.1 mM MgCl ₂ , 0.1 mM MnCl ₂ in PBS, pH 6.8). The retinas were then washed in PBST and incubated overnight with a combination of the “second” antibody and streptavidin Alexa Fluor® 488 (1: 200; Molecular Probes) and with a Cy3-conjugated anti-rabbit IgG antibody (1: 200; Jackson ImmunoResearch). After retinal staining was completed, 4% PFA in PBS was fixed. All overnight incubation procedures were performed at 4 ° C. Images of histological sections of the retina were obtained on a slide using confocal fluorescence microscopy.

Tumor models. Were used 8-10-week-old female "naked" mice with a congenital absence of natural killer cells. To generate subcutaneous tumors, mice were injected with 0.1 ml of a cell suspension containing 50% matrigel in the right hind flank (BD Bioscience). Each mouse was injected with 5 × 10 ⁶ cancer cells of the human colon HM7, 10 × 10 ⁶ cells of carcinoma of the human colon Colo205, 10 × 10 ⁶ cells of human lung carcinoma Calu6, 10 × 10 ⁶ cells of human lung carcinoma MV-522, 10 × 10 ⁶ WEHI-3 mouse leukemia cells, 10 × 10 ⁶ EL4 murine lymphoma cells, 10 × 10 ⁶ human ovarian cancer cells SK-OV-3 X1, 10 × 10 ⁶ LL2 murine lung cancer cells, 10 × 10 ⁶ leukemia / lymphoma cells EL4 or 10 × 10 ⁶ non-small cell lung cancer cells H1299. To obtain a model of human melanoma MDA-MB-435, mice were injected with 0.1 ml of cell (5 × 10 ⁶ ) suspension containing 50% matrigel in the fatty body of the mammary gland. Anti-DLL4 antibody YW26.82 was administered ip (10 mg / kg body weight, twice a week). To obtain the following tumor models of each test mouse, a tumor fragment (1 mm ³ ) was implanted subcutaneously in the right flank: SKMES-1 non-small cell lung cancer, MX-1 human breast cancer, SW620 human colon and colon cancer, and LS174T human adenocarcinoma. Tumor size was evaluated using a caliper. Tumor volume ^(mm3) was determined by measuring the length (l) and width (w) and calculated by the formula (V = lw ^2/2). Each group included 10-15 animals. Statistical comparison of treatment groups was performed using two-tailed student t-test.

Labeling and immunohistochemical analysis of tumor vessels.

Mice were anesthetized with isoflurane. Iv was then injected with an FITC-labeled Lycopersicon esculentum lectin (150 μg in 150 μl 0.9% NaCl; Vector Laboratories) and left for 5 minutes to enter the bloodstream, followed by systemic perfusion. The vasculature was transcardially perfused with 1% PFA in PBS for 3 minutes. Tumors were removed and fixed by immersion in the same fixing substance for 2 hours, after which they were incubated overnight in 30% sucrose for cryoprotection, then poured into OCT. Sections (4 μm thick) were stained with anti-mouse CD31 antibody (1:50, BD Pharmingen), then goat anti-rat IgG antibody Alexa 594 (1: 800, Molecular Probes).

Histological and immunohistochemical analysis of the mouse small intestine.

Formal fixed and paraffin embedded tissue sections of mouse small intestine with a thickness of 3 μm were obtained. Histochemical identification of small intestine cells was performed using alcian blue in accordance with the manufacturer's recommendations (PolyScientific). For staining with anti-Ki67 antibody, the sections were pretreated with the target reconstitution solution (S1700, DAKO) and incubated with rabbit anti-Ki67 antibody (1: 200, clone SP6, Neomarkers). A “second” goat anti-rabbit antibody at a concentration of 7.5 g / ml (Vector labs) was detected using the Vectastain ABC Elite Kit (Vector labs). All Ki67-stained sections were contrast stained with Meyer hematoxylin. An anti-rat HES-1 antibody (clone NM1, MBL, International), then TSA-HRP, was used to stain HES-1.

RNA interference. A set of short interfering RNA (siRNA) duplexes (SMART) targeting human DLL4 and a control non-targeted siRNA (Si Control Non-Target siRNA # 2) were purchased from Dharmacon. SiRNA duplexes (50 nM) were transfected into HUVEC cells at 40% confluency using OptiMEM-1® reagent and Lipofectamine ^TM 2000 (Invitrogen). FACS analysis was performed 48 hours after siRNA transfection. Below are the sequences of the SMART set of 4 siRNAs targeting DLL4:

CAACTGCCCTTATGGCTTTTT (SEQ ID NO: 62) (oligonucleotide 1, sense sequence),

AAAGCCATAAGGGCAGTTGTT (SEQ ID NO: 63) (oligonucleotide 1, antisense sequence),

CAACTGCCCTTCAATTTCATT (SEQ ID NO: 64) (oligonucleotide 2, sense sequence),

TGAAATTGAAGGGCAGTTGTT (SEQ ID NO: 65) (oligonucleotide 2, antisense sequence),

TGACCAAGATCTCAACTACTT (SEQ ID NO: 66) (oligonucleotide 3, sense sequence),

GTAGTTGAGATCTTGGTCATT (SEQ ID NO: 67) (oligonucleotide 3, antisense sequence),

GGCCAACTATGCTTGTGAATT (SEQ ID NO: 68) (oligonucleotide 4, sense sequence),

TTCACAAGCATAGTTGGCCTT (SEQ ID NO: 69) (oligonucleotide 4, antisense sequence).

Notch ligand: Notch blocking ELISA. 96-well microtiter plates were coated with recombinant rat Notch1-Fc (rrNotch1-Fc, R&D Systems) at a concentration of 0.5 μg / ml. This assay used a conditioned medium containing DLL4-AP (amino acids 1-404 DLL4 attached to human placenta alkaline phosphatase (AP)). To obtain a conditioned medium, 293 cells were temporarily transfected with a DLL4-AP-expressing plasmid using Fugen6 reagent (Roche Molecular Biochemicals). Five days after transfection, the conditioned medium was collected, filtered and stored at 4 ° C. The purified antibodies, which were titrated from 0.15 to 25 μg / ml, were pre-incubated for 1 hour at room temperature with a DLL4-AP-conditioned medium at dilution, which provided 50% maximum achievable binding to coated rrNotch1-Fc. The antibody / DLL4-AP mixture was then added to the rrNotch1-Fc-coated plate for 1 hour at room temperature, after which the plates were washed several times with PBS. Bound DLL4-AP was detected using 1-Step PNPP reagent (Pierce), used as a substrate, and absorbance (OD) was measured at 405 nm. Identical analysis was performed using DLL1-AP (human DLL1, amino acids 1-445). Similar analyzes were performed using purified DLL4-His (C-terminal His-labeled human DLL4, amino acids 1-404) and Jag1-His (R & D system). Bound His-labeled ligands were detected using mouse anti-His mAb (1 μg / ml, Roche Molecular Biochemicals), biotinylated goat anti-mouse antibody (Jackson ImmunoResearch) and streptavidin-AP (Jackson ImmunoResearch).

RNA extraction and quantitative RT-PCR in real time. Extraction of full-length RNA from HUVEC in a 2-dimensional culture was carried out using the RNeasy® Mini Kit (Qiagen) in accordance with the manufacturers instructions. To extract full-length RNA from HUVEC cells growing in fibrin gels, these fibrin gels were treated with 10 × trypsin-EDTA (Gibco) for 5 minutes to remove the top layer of fibroblasts, then neutralized with 10% FBS in PBS. Thereafter, fibrin clots were removed from tissue culture wells and centrifuged (10K for 5 minutes) in microtubes to remove excess fluid. The obtained gel "granules" were lysed using a lysis buffer (using the RNeasy® Mini Kit), then processed as in the case of HUVEC in a 2-dimensional culture. RNA quality was evaluated using 6,000 RNA nanochips and an Agilent 2100 bioanalyzer (Agilent Technologies). Real-time quantitative RT-PCR was performed in triplicate using a real-time PCR system of 7500 (Applied Biosystems). The human GAPDH gene was used as a reference gene for normalization. Expression levels were expressed as the average (± standard error) of the multiplicity of mRNA changes relative to the control for 3 separate determinations. The forward and reverse primer and probe sequences for VEGFR2, TGFβ2 and GAPDH are shown below.

TGFb2

Direct primer: GTA AAG TCT TGC AAA TGC AGC TA (SEQ ID NO: 70)

Reverse Primer: CAT CAT CAT TAT CAT CAT CAT TGT C (SEQ ID NO: 71)

Probe: AAT TCT TGG AAA AGT GGC AAG ACC AAA AT (SEQ ID NO: 72)

VEGFR2

Direct primer: CTT TCC ACC AGC AGG AAG TAG (SEQ ID NO: 73)

Reverse Primer: TGC AGT CCG AGG TCC TTT (SEQ ID NO: 74)

Probe: CGC ATT TGA TTT TCA TTT CGA CAA CAG A (SEQ ID NO: 75)

GAPDH

Direct primer: GAA GAT GGT GAT GGG ATT TC (SEQ ID NO: 76)

Reverse Primer: GAA GGT GAA GGT CGG AGT C (SEQ ID NO: 77)

Probe: CAA GCT TCC CGT TCT CAG CC (SEQ ID NO: 78)

Example 2: Generation of phage anti-DLL4 antibodies

Libraries of synthesized phage antibodies were constructed on a single framework region (humanized anti-ErbB2 antibody, 4D5) by introducing variability in the heavy and light chain hypervariable regions (CDRs) (Lee, CV et al. J Mol Biol 340, 1073-93 (2004) ; Liang, WC et al. J Biol Chem 281, 951-61 (2006)). Panning on tablets with source libraries was performed to analyze His-labeled human DLL4 (amino acids 1-404) immobilized on MaxiSorp ™ immuno-plates. After four rounds of enrichment, clones were randomly collected and specific binding clones were identified using phage ELISA. The resulting hDLL4-binding clones were then screened using His-labeled mouse DLL4 mouse protein to identify cross-linking clones. For each positive phage clone, the variable regions of the heavy and light chains were subcloned into pRK expression vectors that were designed to express full-length IgG chains. The heavy and light chain constructs were cotransfected into 293 or CHO cells, and the expressed antibodies were isolated from serum-free medium on an affinity column with protein A. Purified antibodies were tested by ELISA to block the interaction of DLL4 with rat Notch1-Fc and FACS to bind to stable cell lines expressing either full-sized human DLL4 or mouse DLL4. For affinity maturation, phage libraries with three different combinations of CDR loops (CDR-L3, -H1, and -H2) originating from the original clone of interest were constructed using a soft randomization strategy so that each selected position was modified to produce a non-residual wild-type or to maintain a ratio of non-wild-type residues to wild-type residues of about 50:50. Clones with high affinity were then identified in four rounds of liquid phase panning performed for analysis on human and mouse His-tagged DLL4 proteins under conditions of ever increasing stiffness.

Example 3: Characterization of anti-DLL4 antibodies

To determine the binding affinity of anti-DLL4 Mab, measurements were made using surface plasmon resonance (SRP) on a BIAcore ™ -3000 device (BIAcore, Inc., Piscataway, NJ). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the instructions of the suppliers. The anti-DLL4 antibody was diluted with 10 mM sodium acetate, pH 4.8, to 5 μg / ml, then injected at a flow rate of 5 μl / min until the response unit (RU) of the bound antibody was approximately 500. Then, to block unreacted groups were injected with 1M ethanolamine. To measure the reaction kinetics, two serial dilutions of human or murine DLL4-His molecules (0.7 nM-500 nM) were injected in PBS containing 0.05% ^TM 20 tween at 25 ° C and at a flow rate of 25 μl / min. Association rate (k _on ) and dissociation rate (k _off ) were calculated using a simple 1: 1 Langmuir binding model (BIAcore Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio k _off / k _on . The results of this experiment are presented in table.3. Antibody YW26.81 yielded similar Kd values for human and mouse DLL4 (Kd values were 0.1 nM and 0.09 nM, respectively), which allows an assessment of the effective effect of antibodies in mouse models.

Table 3
The affinity and kinetics of binding of anti-DLL4 antibodies to human and murine DLL4. “NA” means no measurements Clone mouse delta 4 (ECD5.5) human delta4 (ECD5.5) k _on / 10 ⁵ k _off / 10 ^-4 Kd (nM) k _on / 10 ⁵ k _off / 10 ^-4 Kd (nM) Yw26 0.57 16 28 0.3 7.4 25 YW26.6 4.1 0.61 0.15 2 0.49 0.25 YW26.14 3.3 0.73 0.22 1.8 0.66 0.37 YW26.20 3.3 one 0.3 1.7 0.79 0.46 YW26.34 3.6 0.65 0.18 1.9 0.67 0.35 YW26.82 5,4 0.46 0.09 2,4 0.37 0.15

Example 4: Additional characterization of anti-DLL4 antibodies

Mapping epitopes of anti-DLL4 Mab YW26.82: Anti-DLL4 Mab 26.82 recognized the binding determinant present in EGF-like repeat # 2 (EGL2) of the human extracellular domain DLL4 (ECD). EGL2 contains amino acids 252-282 of the human ECD DLL4. Briefly, ECD DLL4 mutants were expressed as alkaline phosphatase fusion proteins and their binding to the antibody was analyzed. 11 a is a schematic representation of a set of DLL4 mutants expressed as C-terminal fusion proteins with alkaline phosphatase (AP) in a human placenta. The parentheses indicate the DLL4 sequences included in the fusion proteins. 293T cell conditioned media containing fusion proteins was tested on 96-well microtiter plates coated with purified anti-DLL4 Mab (YW26.82, 0.5 μg / ml). The associated DLL4.AP was detected using 1-Step PNPP reagent (Pierce) as a substrate and OD absorbance was measured at 405 nm. Mab YW26.82 associated with constructs containing the EGL2 DLL4 domain, and did not bind to constructs containing the EGL2 DLL4 domain. These results showed that anti-DLL4 Mab YW 26.82 recognized the epitope in the EGL2 domain of human ECD DLL4.

Mab YW26.82 selectively binds to murine and human DLL4. Nunc Maxisorp 96-well plates were coated with purified recombinant proteins as described above (1 μg / ml). The binding of YW26.82 at the indicated concentrations was measured by ELISA. Bound antibodies were detected using a PX-conjugated anti-human antibody using TMB as a substrate and OD absorbance was measured at 450 nm. As an analytical control, an anti-HER2 antibody and recombinant ErbB2-ECD were used (Fig. 11b). The results of this experiment are presented in figure 11b. Mab YW26.82 binds to human and mouse DLL4 and does not bind to human DLL1 and human JAG1 at a detectable level. These results showed that Mab YW26.82 selectively binds to DLL4.

FACS analysis of 293 cells temporarily transfected with a vector full-sized DLL4, Jag1 or DLL1 also demonstrated that Mab YW26.82 selectively binds to DLL4. As shown in FIG. 11c, a significant level of binding of YW26.82 was detected only on DLL4-transfected cells (top panel). No significant binding was observed on DLL1 or Jag1 transfected cells. The expression of Jag1 and DLL1 was confirmed by binding of recombinant rat Notch1-Fc (rrNotch1-Fc, middle panel) and recombinant rat Notch2-Fc (rrNotch2-Fc, bottom panel), respectively. YW26.82, rrNotch1-Fc or rrNotch2-Fc (R & D system) was used at 2 μg / ml, then a PV-conjugated goat anti-human IgG antibody was used (1: 500, Jackson ImmunoResearch).

Competitive binding experiments have shown that Mab YW26.82 effectively and selectively blocks Notch interaction with DLL4, but not with other Notch ligands. As shown in FIG. 11d, the anti-DLL4 Mab blocks the binding of the DLL4-AP, but not the DLL1-AP, with rNotch1 covered, with the calculated IC50 ~ 12 nM (left panel). Anti-DLL4 Mab blocks the binding of DLL4-His, but not Jag1-His, to rNotch1 coated, with an IC50 of ~ 8 nM calculated (right panel).

Anti-DLL4 Mab YW26.82 specifically binds to endogenously expressed DLL4 in endothelial human umbilical vein cells (HUVEC). FACS analysis of HUVECs transfected with control or DLL4-specific siRNA was performed. YW26.82 was used at 2 μg / ml, then a PV-conjugated goat anti-human IgG antibody was used (1: 500, Jackson ImmunoResearch). The results of this experiment are presented in FIG. 11e. In this case, binding was observed with untransfected HUVEC (control) and with HUVEC transfected with control siRNA. In contrast, the level of binding to HUVEC transfected with DLL4 siRNA was significantly reduced. These experiments demonstrated that anti-DLL4 Mab YW26.82 specifically binds to endogenously expressed DLL4 in HUVEC.

Example 5: Treatment with anti-DLL4 antibody leads to increased in vitro endothelial cell proliferation

Human umbilical vein endothelial cells (HUVEC), growing in fibrin gels in the presence of co-cultured human skin fibroblast (SF) cells, produce processes that have a distinct cavity-like structure (Nakatsu, MN et al. Microvasc Res 66, 102-12 ( 2003)). The addition of the anti-DLL4 antibody YW26.82 leads to a marked increase in the length and number of processes (Fig. 7a). Proteolytic processing of Notch, catalyzed by the γ-secretase activity of the protein complex, is the main stage in the activation of Notch (Baron, M. Semin Cell Dev Biol 14, 113-9 (2003)). It is interesting to note that the dibenzazepine (DBZ) γ-secretase inhibitor (van Es, JH et al. Nature 435, 959-63 (2005); Milano, J. et al. Toxicol Sci 82, 341-58 (2004)) has this same effect on the overgrowth of HUVEC. Taking into account that these two treatments act by different mechanisms, it should be noted that a significant level of germination is clearly due to the weakening of Notch signal transmission. Ki67 staining showed that enhanced EC germination is caused by an increase in cell proliferation (Fig. 7b). In the initial fibrin gel assay, HUVEC growth and subsequent cavity formation were confirmed by co-culture with SF cells. When replacing SF cells with conditioned medium, anti-DLL4 Mab and DBZ still had the ability to enhance HUVEC proliferation (Fig. 1c), which confirms the autonomous role of EC in the transmission of DLL4 / Notch signal. In a reverse experiment, Notch activation by the immobilized DLL4 protein resulted in significant inhibition of cell growth (Fig. 7e). These results suggest that the activation state of DLL4 / Notch signaling is closely related to EC proliferation.

Example 6: Treatment with anti-DLL4 antibody leads to increased in vivo endothelial cell proliferation

In the retina of mice, immediately after birth, a stereotypical vascular pattern develops in a well-defined sequence of events (Stone, J. & Dreher, Z. J Comp Neurol 255, 35-49 (1987); Gerhardt, H. et al. J. Cell Biol. 161 1163-77 (2003); Fruttiger, M. Invest Ophthalmol Vis Sci 43, 522-7 (2002)). The pronounced and dynamic expression of DLL4 in growing EC in the retina of newborn mice suggests a possible role for DLL4 in the regulation of retinal vascular development (Claxton, S. & Fruttiger, M. Gene Expr Patterns 5, 123-7 (2004)). Systemic delivery of YW26.82 led to a significant change in the retinal vasculature. Massive accumulation of EC occurred in the retina, resulting in the formation of a folded structure with primitive vascular morphology (Fig.7d). In EC, a significant increase in the intensity of Ki67-labeling was observed, which indicates an increase in EC proliferation (Fig.7h). Therefore, such a hyperproliferative phenotype of EC retina after blockade of DLL4 in newborn mice confirmed in vitro data.

Example 7: The main role of DLL4 / Notch in the regulation of epithelial cell proliferation

VEGF regulates several fundamental processes in EC (Ferrara, N. Exs , 209-31 (2005); Coultas, L. et al. Nature 438, 937-45 (2005)). However, in this regard, it is not very clear how VEGF signals are integrated in complex processes of vascular formation, such as arteriophenous (AV) differentiation and hierarchical vascular organization, that is, events that, obviously, require additional highly coordinated signal transmission paths. Genetic studies on zebra fish suggest that VEGF acts before a cascade of Notch reactions during arterial endothelial differentiation (Lawson, ND et al. Development 128, 3675-83 (2001)). The inventors of the present invention have found that VEGF stimulation of HUVEC leads to an increase in the surface expression level of DLL4 (data not shown), which is consistent with a recent report on activation of DLL4 mRNA by VEGF stimulation (Patel, NS et al. Cancer Res 65, 8690- 7 (2005)). It is interesting to note that DLL4 itself is activated after Notch activation (Fig. 12), which suggests the existence of a positive feedback mechanism, thanks to which DLL4 efficiently transfers the VEGF signal to the Notch path. Briefly, HUVEC was stimulated with immobilized C-terminal His-labeled human DLL4 (amino acids 1-404) in the absence or presence of DBZ (0.08 μM). 36 hours after stimulation, endogenous expression of DLL4 was evaluated by FACS analysis using anti-DLL4 antibody.

It should be noted that EC hyperproliferation resulting from blocking Notch signal transmission still depends on VEGF. In a 3-dimensional fibrin gel culture, treatment with anti-VEGF Mab terminates a significant level of EC growth in the presence or absence of DBZ (Fig. 7f), which increases the likelihood that hyperproliferative behavior may be due in part to amplification of the VEGF signal. Indeed, blocking Notch under the action of YW26.82 or DBZ leads to the activation of VEGFR2 (Fig.7g). Conversely, Notch activation by immobilized DLL4 inhibits the expression of VEGFR2 (Fig. 7g). Therefore, although VEGF can act before undergoing the cascade of DLL4 / Notch reactions, DLL4 / Notch is capable of fine-tuning the response by negatively regulating VEGFR2 expression.

Example 8: Treatment with anti-DLL4 antibody blocks endothelial cell differentiation and arterial development

In addition to increasing the level of EC proliferation, the antagonistic effect of DLL4 / Notch leads to a sharp morphological change in the processes of EC in the fibrin gel. Multicellular cavity-like structures were mostly absent (Fig. 8a), which suggests a violation of EC differentiation. In the retinas treated with Mab YW26.82 monoclonal antibody, the characteristic pattern of radially alternating arteries and veins was significantly impaired. Staining with anti-α-actin smooth muscle antibody (ASMA), which is associated with retinal arteries, was completely absent (Fig. 8c). This observation was very similar to impaired arterial development in DLL4 ^+/- embryos. These data, from all points of view, confirm that DLL4 / Notch plays a major role in the regulation of EC differentiation.

Example 9: Expression of TFGβ Associated with an Activated Notch State

Similar to the cascade of Notch reactions, TGFβ signal transmission depends on its environment and has various and often opposite effects on the inhibition of differentiation, proliferation and cell growth. In addition, the cascade of TGFβ reactions is involved in vascular development processes (Urness, LD et al, Nat Genet 26, 328-31 (2000); Oshima, M. et al, Dev Biol 179, 297-302 (1996); Larsson, J . et al. Embo J 20, 1663-73 (2001)). For example, a deficiency of kinase 1, similar to the activin receptor (ALK1), that is, a specific TGFβ receptor of type I EC, leads to the formation of a primitive network structure of EC in the yolk sacs and to insufficient arteriovenous function (AVM), that is, a common phenotype inherent in mice with impaired signal transmission Notch (Urness, LD et al, Nat Genet 26, 328-31 (2000); Iso, T. et al, Arterioscler Thromb Vasc Biol 23, 543-53 (2003)). This prompted the authors of the present invention to investigate the possible relationship between these two cascades of reactions. The inventors have found that the expression of TGFβ2 (Fig. 8b) is directly related to the activated state of Notch, suggesting that a cascade of TGFβ reactions may occur after a cascade of Notch reactions. In General, the data obtained by the authors of the present invention confirm the model in which the DLL4 / Notch system, serving as a "signal line", integrates VEGF signals by regulating the expression of DLL4 and directs the TGFβ pathway to stimulate differentiation of EC.

Example 10: Treatment with anti-DLL4 antibody inhibits tumor growth in vivo

To fully identify the possible role of DLL4 / Notch signal transmission in tumor angiogenesis, the authors of the present invention investigated the effect of DLL4 blocking on tumor growth in preclinical tumor models (FIGS. 9a-d). In models with xenografts of HM7, Colo205 and Calu6 tumors (Fig. 9a-c), treatment with antibody YW26.82 was initiated after stable tumor development (after reaching a tumor size of ≥250 mm ³ ). In all three models, the difference in tumor growth rates between the control and treatment groups became noticeable three days after the dose. The tumor volume in the treatment group remained unchanged during the two-week treatment. In addition to subcutaneous tumors, anti-DLL4 Mab also inhibited tumor growth in the fatty body of the murine mammary gland. In the MDA-MB-435 tumor model, treatment was started 14 days after tumor cell injection. The difference in the tumor growth curves between the control group and the treatment groups was observed six days after the dose and became more noticeable as treatment continued (Fig. 9d).

The authors of the present invention also investigated the effect of blocking DLL4 and / or VEGF on the growth of a large number of tumors in preclinical tumor models (Fig. 9e-f; ip). In tumor xenograft models MV-522 and WEHI3, treatment with YW26.82 and / or anti-VEGF antibody was initiated after stable tumor development (after reaching a tumor size of ≥250 mm ³ ). In the MV-522 model, treatment with YW26.82 antibody and anti-VEGF antibody led to inhibition of the growth of individual tumors, a combination of two treatments gave an even greater effect. In the WEHI3 model, treatment with anti-VEGF antibody did not have any effect on tumor growth; treatment with antibody YW26.82 led to significant inhibition of tumor growth. In the SK-OV-3X1, LL2, EL4, H1299, SKMES-1, MX-1, SW620 and LS174T models, treatment with YW26.82 antibody (5 mg / kg, i.r., twice a week) and / or treatment anti-VEGF antibody (5 mg / kg, i.p., twice a week) was performed after stable tumor development. In each of these models, treatment with YW26.82 single antibody resulted in inhibition of tumor growth. In addition, in all models in which this combination was tested, the YW26.82 antibody showed increased efficacy in combination with the anti-VEGF antibody.

Example 11: Treatment with anti-DLL4 antibody leads to increased proliferation of tumor endothelial cells

In studies on the inhibition of tumor growth, the authors of the present invention used a tumor model of murine lymphoma EL4 for histological analysis of the vascular system. The authors of the present invention, it was found that the treatment of anti-DLL4 Mab leads to a sharp increase in the density of endothelial cells (Fig.9g). In contrast, the anti-VEGF antibody gives the exact opposite effect (Fig. 9g), although both of these treatments show similar efficacy in this model.

Example 12: Treatment with anti-DLL4 antibody leads to inhibition of tumor vascular perfusion

Since in vitro blocking of the cascade of DLL4 / Notch reactions worsens the formation of a cavity-like structure by endothelial cells (Fig. 8a), a study was conducted to determine if treatment with anti-DLL4 Mab causes a similar violation of the tumor vascular system and whether it affects effective blood flow. Systemic perfusion with FITC-lectin showed that treatment with anti-DLL4 Mab leads to a significant decrease in the level of labeling of tumor vessels with lectin (Fig. 9h). It should be noted that, as shown, the violation of arteriovenous (AV) function in ALK1-deficient mice leads to circulatory disorders (Urness, LD et al, Nat Genet 26, 328-31 (2000)). Taking into account the important role of DLL4 / Notch signal transmission in AV differentiation, it should be noted that in the retina of embryos and newborn mice, anti-DLL4 Mab can affect specific events in the life cycle of tumor EC and cause impaired directed blood flow. Indeed, in Colo205 tumors treated with anti-DLL4 Mab, there are areas where high EC density is associated with a low content of viable tumor cells, due to weak vascular function. To better understand the nature of specific vascular disorders, additional studies have been carried out using methods of visualization of the vascular system.

Example 13: DLL4 / Notch does not play an important role in mouse small intestine homeostasis

Given the pleiotropic role of Notch signaling in the regulation of homeostasis of self-renewing systems in newborns, the main problem with general Notch inhibition is that such inhibition can have a negative effect. For example, Notch signaling is necessary to preserve undifferentiated crypt precursor cells in the small intestine (van Es, JH et al. Nature 435, 959-63 (2005); Fre, S. et al. Nature 435, 964-8 ( 2005)). Indeed, γ-secretase inhibitors, which should block all Notch activities without a distinct difference, cause unwanted side effects in rodents, due to the increase in the mass of goblet cells in the crypt compartment (Milano, J. et al. Toxicol Sci 82, 341-58 (2004 ); Wong, GT et al. J Biol Chem 279, 12876-82 (2004)). The authors of the present invention conducted immunohistochemical analyzes of the small intestine of mice treated with anti-DLL4 Mab. In contrast to the DBZ treatment, no differences in the differentiation or proliferation profiles of crypt epithelial cells were observed in the groups treated with anti-DLL4 Mab and in the control group after 6 weeks of treatment (Fig. 10). In addition, anti-DLL4 Mab did not affect the expression of the Notch target gene for HES-1 in the rapidly dividing and temporarily amplifying population (TA) (FIG. 10). These results confirm the view that DLL4 / Notch signaling is highly dependent on the vascular system.

Example 14: Treatment with anti-DLL4 antibody does not affect the retinal vasculature of adult mice

Although blocking of DLL4 has a strong influence on the development of the vascular system of the retina in newborn mice, however, the administration of anti-DLL4 antibody did not have a noticeable effect on the vascular system of the retina of an adult animal (Fig. 8d). Thus, DLL4 / Notch signaling plays a crucial role in the process of active angiogenesis, but it plays a less important role in maintaining a normal vascular system. In support of this opinion, it can be noted that during the treatment with anti-DLL4 Mab, no noticeable weight loss or death of tumor mice, which were injected with a dose of 10 mg / kg twice a week for 8 weeks, was observed. In tumor models, anti-DLL4 Mab and anti-VEGF antibody had the opposite effect on the vascular system of the tumor, which suggests the existence of non-overlapping mechanisms of their action.

Although the invention presented above is described in detail with examples that are provided to illustrate and better understand the present invention, the above description of the invention and examples of its implementation should not be construed as limiting the scope of the invention.

Claims (27)

1. The selected anti-DLL4 antibody containing six HVRs, where each HVR includes, consists or essentially consists of a sequence selected from the group consisting of SEQ ID NO: 1-18, wherein SEQ ID NO: 10 corresponds to HVR -L1, SEQ ID NO: 11 corresponds to HVR-L2, SEQ ID NO: 12, 13, 14, 15, 16, 17 or 18 corresponds to HVR-L3, SEQ ID NO: 1 or 2 corresponds to HVR-H1, SEQ ID NO : 3, 4, 5, 6, 7 or 8 corresponds to HVR-H2, and SEQ ID NO: 9 corresponds to HVR-H3. 2. The antibody according to claim 1, containing HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, where each of these areas respectively contains either SEQ ID NO: 10, 11, 14 , 2, 3, and 9; or SEQ ID NO: 10, 11, 15, 1.5, and 9; or SEQ ID NO: 10, 11, 16, 1, 6, and 9; or SEQ ID NO: 10, 11, 17, 1, 7, and 9; or SEQ ID NO: 10, 11, 18, 1.8, and 9. 3. The antibody according to claim 1 or 2, where at least part of the frame sequence is a human consensus frame sequence. 4. The antibody according to claim 1 or 2, containing the human consensus framework sequence of the Kappa subgroup. 5. The antibody according to claim 1 or 2, containing the human consensus framework sequence of the heavy chain of subgroup III. 6. The antibody according to claim 5, where the specified antibody has a substitution in one or more of the provisions of 71, 73 and 78, and these substitutions are, respectively, substitutions R71A, N73T and N78A. 7. Polynucleotide encoding the antibody according to any one of claims 1 to 6. 8. The expression vector containing the polynucleotide according to claim 7. 9. A host cell capable of expressing exogenous genetic material containing the vector of claim 8. 10. The host cell according to claim 9, which is a prokaryotic cell, a eukaryotic cell or a mammalian cell. 11. A method for producing an anti-DLL4 antibody, comprising (a) expressing the vector of claim 8 in a suitable host cell and (b) isolating said antibody. 12. The method according to claim 11, where the specified host cell is a prokaryotic cell or a eukaryotic cell. 13. A method for detecting DLL4, comprising detecting a complex of DLL4 and the anti-DLL4 antibody of claim 1 in a biological sample. 14. A method for diagnosing a disorder associated with the expression of DLL4, comprising detecting the complex of DLL4 and the anti-DLL4 antibody of claim 1 in a biological sample obtained from a patient suffering from said disorder, or from a patient suspected of having such a disorder. 15. The method of claim 13 or 14, wherein said anti-DLL4 antibody is labeled in a manner suitable for detection. 16. A composition for modulating pathological conditions associated with the expression and / or activity of DLL4, containing an effective amount of an anti-DLL4 antibody according to any one of claims 1 to 6. 17. A composition for modulating pathological conditions associated with the expression and / or activity of DLL4, containing an effective amount of polynucleotide according to claim 7. 18. The composition according to clause 16 or 17, which further comprises a carrier. 19. A method of treating a tumor, cancer or a disorder of cell proliferation, comprising administering to an individual in need of such treatment an effective amount of an antibody according to any one of claims 1 to 6. 20. The method according to claim 19, where the specified tumor, cancer or cell proliferative disease is colon cancer, lung cancer or breast cancer. 21. The method according to claim 19 or 20, further comprising administering an effective amount of an antiangiogenic agent. 22. The method according to item 21, where the specified antiangiogenic agent is an antagonist of vascular endothelial growth factor (VEGF). 23. The method of claim 22, wherein said VEGF antagonist is an anti-VEGF antibody. 24. The method according to item 23, where the specified anti-VEGF4 antibody is bevacizumab. 25. The method according to any one of claims 19-20 or 22-24, further comprising administering an effective amount of a chemotherapeutic agent. 26. A method of increasing the effectiveness of an antiangiogenic agent in an individual suffering from a pathological condition associated with angiogenesis, comprising administering to the individual, in addition to the antiangiogenic agent, an effective amount of an antibody according to any one of claims 1 to 6. 27. The method of claim 26, wherein said pathological condition associated with angiogenesis is a tumor, cancer, and / or a disorder of cell proliferation, or an intraocular neovascular disease.

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