MX2007004732A - Antibody formulation in histidine-acetate buffer - Google Patents

Abstract

The present application describes antibody formulations, including monoclonal antibodies formulated in histidine-acetate buffer, as well as a formulation comprising an antibody that binds to domain II of HER2 (for example, Pertuzumab), and a formulation comprising an antibody that binds to DR5 (for example, Apomab).

Description

ANTIBODY FORMULATIONS Field of the Invention The present invention relates to antibody formulations, including monoclonal antibodies formulated in histidine-acetate buffer, as well as a formulation comprising an antibody that binds to domain II of HER2 (e.g., Pertuzumab ), and a formulation comprising an antibody that binds to DR5 (e.g., Apomab).

Background of the Invention In the last ten years, advances in biotechnology have made it possible to produce a variety of proteins for pharmaceutical applications using recombinant DNA techniques. Because proteins are larger and more complex than traditional organic and inorganic drugs (ie, those that have multiple functional groups in addition to complex three-dimensional structures), the formulation of such proteins poses special problems. For a protein to remain biologically active, a formulation must preserve intact the conformational integrity of at least one core sequence of the protein's amino acids while protecting the multiple functional groups of the protein against degradation. The degradation pathways for the proteins may involve chemical instability (i.e., any procedure involving the modification of the protein by bond formation or dissociation resulting in a new chemical entity). or physical instability (ie, changes in the higher order structure of the protein). Chemical instability can result in deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Physical instability can be the result, for example, of denaturation, aggregation, precipitation or adsorption. The three most common pathways of protein degradation are aggregation, deamidation and protein oxidation. Cleland et al. Critical Reviews in Therapeutic Drug Carrier Systems 10 (4): 307-377 (1993).

Antibody Formulations Antibodies are included in the proteins used for pharmaceutical application. An example of an antibody useful for therapy is an antibody that binds to the HER2 antigen, such as Pertuzumab. U.S. Patent No. 6,339,142 discloses an HER2 antibody composition comprising a mixture of the anti-HER2 antibody and one or more acid variants thereof, wherein the amount of the acid variants is less than about 25%. Trastuzumab is an HER2 antibody and is used. The North American Patents Nros. 6,267,958 and 6,685,940 (Andya et al.) Describe lyophilized antibody formulations, including HER2 and IgE ormulations. WO97 / 04807 and US Patent No. 2004 / 0197326A1 (Fick et al.) Describe methods for treating allergic asthma with an IgE antibody. WO99 / 01556 (Lowman et al.) Refers to an IgE antibody with aspartyl residues prone to isomerization, and improved variants thereof. US Patent 2002/0045571 (Liu et al.) Provides concentrated protein formulations of reduced viscosity, exemplified by the formulations of humanized IgE antibodies, rhuMAb E25 and E26. WO 02/096457 and U.S. Patent 2004/0170623 (Arvinte et al.) Describe stable liquid formulations comprising the anti-IgE antibody E25. See also, US Patent No. 2004/0197324 Al (Liu and Shire) relating to anti-IgE formulations in high concentrations. U.S. Patent No. 6,171,586 (Lam et al.) Discloses stable aqueous formulations of antibodies. An antibody F (ab) 2 rhuMAb CD18 was formulated in buffers of sodium acetate and histidine-HCl. The preferred formulation for rhuMAb CD18 was 10mM sodium acetate, 8% trehalose, 0.01% TWEEN 20 ™, pH 5.0. Acetate formulations (pH 5.0) of rhuMAb CD20 stored at 40 ° C for one month showed greater stability than those samples formulated in histidine (pH 5, 0 or 6.0). U.S. Patent 2003/0190316 (Kakuta et al.) Refers to the antibody formulated hPM-1, a humanized IL-6 receptor antibody. The loss of monomer was higher in sodium citrate (pH 6.7), followed by sodium phosphate (pH 6.8), Tris-HCl (pH 7.2), histidine-HCl (pH 7.2) and glycine (pH 7.6) in descending order. The effect of phosphate-Na (pH 6.5), phosphate-His (pH 6.0 or 6.5), His-HCl (pH 6.5), and phosphate-Na (pH 6.0) on stability of hPM-1 was verified. WO2004 / 071439 (Burke et al.) States that the impurities arose in a formulation of natalizumab (humanized anti-alpha4 integrin monoclonal antibody), from the degradation of polysorbate 80, apparently through an oxidation reaction involving metal ions and histidine. Therefore, a phosphate buffer was selected.

WO 2000/066160 (English-language counterpart of EP 1 174 148A1) (Okada et al.) Refers to a formulation of a humanized C4G1 antibody that binds to a fibrinogen receptor of a human platelet membrane glycoprotein GPIIb / IIIa, in a sodium phosphate or sodium citrate buffer WO2004 / 019861 (Johnson et al.) Refers to CDP870, a pegylated anti-TNF Fab fragment, formulated at 200 mg / ml in 50mM sodium acetate ( pH 5.5) and 125 mM sodium chloride. WO2004 / 004639 (Nesta, P.) refers to a formulation for huC242-D1, a tumor-activated immunotoxin, in 50 mM succinic acid buffer (pH 6.0) and sucrose (5% w / v) . WO03 / 039485 (Kaisheva et al.) Found that Daclizumab (a humanized IL-2 receptor antibody) had the highest stability in sodium succinate buffer at pH 6.0, and that it rapidly lost potency in histidine upon buffer oxidation . WO 2004/001007 refers to the monoclonal antibody CD80 in a HC1 buffer of histidine, sodium acetate or sodium citrate. US Pat. No. 6,252,055 (Relton, J.) refers to anti-CD4 and anti-CD23 antibodies, formulated in buffers of maleate, succinate, phosphate or sodium acetate, identifying phosphate as the preferred buffer. U.S. Patent No. 5,608,038 (Eibl et al.) Refers to highly concentrated polyclonal immunoglobulin preparations, glucose or sucrose, and sodium chloride therein.

WO03 / 015894 (Oliver et al.) Refers to an aqueous formulation of 100 mg / mL SYNAGIS®, 25 mM histidine-HCl, 1.6 mM glycine, pH 6.0, and lyophilized SYNAGIS® that when is formulated (before lyophilization) contains 25 mM histidine, 1.6 mM glycine and 3% w / v mannitol at pH 6.0. U.S. Patent 2004/0191243 Al (Chen et al.) Reports on a formulation of ABX-IL8, a human IgG2 antibody. US Patent 2003/0113316 Al (aisheva et al.) Refers to a lyophilized anti-IL2 receptor antibody formulation.

HER2 Antibodies The HER family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The recipient family includes four distinct members that include the epidermal growth factor receptor (EGFR, ErbBl, or HERI), HER2 (ErbB2 or pl85neu), HER3 (ErbB3) and HER4 (ErbB4 or tiro2). The EGFR, encoded by the er > Bl gene, has been causally implicated in human malignancies. In particular, an increase in EGFR expression has been observed in breast, gallbladder, lung, head, neck and stomach cancers as well as glioblastomas. Increased expression of the EGFR receptor is often associated with an increase in the production of the EGFR ligand, which transforms the growth factor alpha (TGF-), by the same tumor cells, which results in the activation of the receptor via a pathway. Autocrine stimulant, Baselga and Mendelsohn Pharmac. Ther. 64: 127-154 (1994). Monoclonal antibodies directed against EGFR or its ligands, TGF-a and EGF, have been evaluated as therapeutic agents for the treatment of said malignant tumors. See, for example, Baselga and Mendelsohn., Supra; Masui et al. Cancer Research 44: 1002-1007 (1984); and Wu et al. J. Clin. Invest. 95: 1897-1905 (1995). The second member of the HER family, pl85neu, was originally identified as the product of the transforming gene of neuroblastomas from chemically treated rats. The activated form of the neu proto-oncogene is the result of a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu is observed in breast and ovarian cancers and is correlated with a poor prognosis (Slamon et al., Science, 235: 177-182 (1987); Slamon et al., Science, 244: 707 712 (1989) and U.S. Patent No. 4,968,603). Currently, no point mutation analogous to that of the neu proto-oncogene has been reported for human tumors. Overexpression of HER2 (frequently but not uniformly due to gene amplification), has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and the gallbladder See among others, King et al., Science, 229: 974 (1985); Yokota et al., Lancet: 1: 765-767 (1986); Fukushige et al., Mol Cell Biol., 6: 955-958 (1986); Guerin et al., Oncogene Res., 3: 21-31 (1988); Cohen et al., Oncogene, 4: 81-88 (1989); Yonemura et al., Cancer Res., 51: 1034 (1991); Borst et al., Gynecol. Oncol., 38: 364 (1990); einer et al., Cancer Res., 50: 421-425 (1990); Kern et al., Cancer Res., 50: 5184 (1990); Park et al., Cancer Res., 49: 6605 (1989); Zhau et al., Mol. Carcinog., 3: 254-257 (1990); Aasland et al. Br. J. Cancer 57: 358-363 (1988); Williams et al Pathobiology 59: 46-52 (1991); and McCann et al., Cancer, 65: 88-92 (1990). HER2 may have hyperexpression in prostate cancer (Gu et al., Lett. 99: 185-9 (1996); Ross et al., Hum. Pathol., 28: 827-33 (1997); Ross et al., Cancer 79). : 2162-70 (1997); and Sadasivan et al., J. Urol. 150: 126-31 (1993)). Antibodies directed against the human protein HER2 and rat pl85neu products have been described. Drebin and his colleagues have created antibodies against the rat neu gene product, pl85neu See, eg, Drebin et al., Cell 41: 695-706 (1985); Myers et al., Meth. Enzym. 198: 277-290 (1991); and W094 / 22478. Drebin et al. Oncogene 2: 273-277 (1988) report that mixtures of antibodies reactive with two distinct regions of pl85neu result in anti-tumor synergistic effects on neutro-transformed NIH-3T3 cells implanted in nude mice. See also U.S. Patent 5,824,311 issued October 20, 1998. Hudziak et al., Mol. Cell. Biol. 9 (3): 1165-1172 (1989) describe the generation of a panel of HER2 antibodies that were characterized using the SK-BR-3 line of human breast tumor cell. The relative proliferation of cells, in SK-BR-3 cells, after exposure to the antibodies, was determined by staining with methylrosaniline chloride of the monolayers after 72 hours. Using this assay, maximum inhibition was obtained with the antibody termed 4D5 which inhibited cell proliferation by 56%. Other panel antibodies reduced cell proliferation to a lesser degree in this assay. Additionally, it was found that the 4D5 antibody sensitized the overexpression of HER2 in the lines of breast tumor cells to the cytotoxic effects of TNF-α. See also U.S. Patent No. 5,677,171 issued October 14, 1997. The HER2 antibodies discussed in Hudziak et al. they are additionally characterized in Fendly et al. Cancer Research 50: 1550-1558 (1990); Kotts et al. In Vitro 26 (3): 59A (1990); Sarup et al.

Growth Regulation 1: 72-82 (1991); Shepard et al. J. Clin. Immunol. 11 (3): 117-127 (1991); Kumar et al. Mol. Cell. Biol. 11 (2): 979-986 (1991); Lewis et al. Cancer Immunol. Immunother. 37: 255-263 (1993); Pietras et al. Oncogene 9: 1829-1838 (1994); Vitetta et al. Cancer Research 54: 5301-5309 (1994); Sliwkowski et al. J. Biol. Chem. 269 (20): 14661-14665 (1994); Scott et al. J. Biol. Chem. 266: 14300-5 (1991); D'souza et al. Proc. Nati Acad. Sci. 91: 7202-7206 (1994); Lewis et al. Cancer Research 56: 1457-1465 (1996); and Schaefer et al. Oncogene 15: 1385-1394 (1997). A recombinant humanized version of the murine HER2 4D5 antibody (huMAb4D5-8, rhuMAb HER2, Trastuzumab or HERCEPTIN®, US Patent No. 5,821,337) is clinically active in patients with metastatic breast cancers that hyper-express HER2, who have received a prolonged anti-cancer therapy. (Baselga et al., J. Clin. Oncol. 14: 737-744 (1996)). Trastuzumab received marketing approval from the Food and Drug Administration on September 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors hyper-expressed the HER2 protein. Other HER2 antibodies with various properties have been described in Tagliabue et al. Int. J. Cancer 47: 933-937 (1991); McKenzie et al. Oncogene 4: 543-548 (1989); Maier et al. Cancer Res. 51: 5361-5369 (1991); Bacus et al. Molecular Carcinogenesis 3: 350-362 (1990); Stancovski et al. PNAS (USA) 88: 8691-8695 (1991); Bacus et al. Cancer Research 52: 2580-2589 (1992); Xu et al. Int. J. Cancer 53: 401-408 (1993); WO94 / 00136; Kasprzyk et al. Cancer Research 52: 2771-2776 (1992); Hancock et al. Cancer Res. 51: 4575-4580 (1991); Shawver et al. Cancer Res. 54: 1367-1373 (1994); Arteaga et al. Cancer Res. 54: 3758-3765 (1994); Harwerth et al. J. Biol. Chem. 267: 15160-15167 (1992); U.S. Patent No. 5,783,186; and Klapper et al. Oncogene 14: 2099-2109 (1997). The screening of homology detection has resulted in the identification of two other members of the HER receptor family; HER3 (U.S. Patent Nos. 5,183,884 and 5,480,968 as well as Kraus et al., PNAS (USA) 86: 9193-9197 (1989)) and HER4 (European Patent Application No. 599,274; Plowman et al., Proc. Nati, Acad. Sci. USA, 90: 1746-1750 (1993), and Plowman et al., Nature, 366: 473-475 (1993)). Both receptors exhibit an increase in expression in at least some lines of breast cancer cells. HER receptors are generally found in various combinations in cells, and it is believed that heterodimerization increases the diversity of cellular responses for a variety of HER ligands (Earp et al., Breast Cancer Research and Treatment 35: 115-132 (1995)). . The EGFR is linked by six different ligands; epidermal growth factor (EGF), transforming growth factor alpha (TGF-OI), anfiregulin, heparin-binding epidermal growth factor (HB-EGF), betacellulin and epiregulin (Groenen et al., Growth Factors 11: 235 -257 (1994)). A family of heregulin proteins resulting from the alternative splicing of a single gene are the ligands for HER3 and HER4. The family of heregulin includes alpha, beta and gamma heregulins (Holmes et al., Science, 256: 1205-1210 (1992); US Patent No. 5,641,869; and Schaefer et al., Oncogene 15: 1385-1394 (1997); ); neu differentiation factors (NDFs), glial growth factors (GGFs); acetylcholine receptor-inducing activity (ARIA); and factor derived from motor and sensory neurons (SMDF). For a review, see Groenen et al. Growth Factors 11: 235-257 (1994); Lemke, G. Molec. & Cell. Neurosci. 7: 247-262 (1996) and Lee et al. Pharm. Rev. 47: 51-85 (1995). Recently, three additional HER ligands have been identified; neuregulin-2 (NRG-2) that has been reported to bind to either HER3 or HER4 (Chang et al., Nature 387 509-512 (1997); and Carraway et al. Nature 387: 512-516 (1997)); neuregulin-3 which binds HER4 (Zhang et al., PNAS (USA) 94 (18): 9562-7 (1997)); and neuregulian-4 that binds to HER4 (Harari et al., Oncogene 18: 2681-89 (1999)) HB-EGF, betacellulin and epiregulin that also bind to HER4.

Although EGF and TGFα do not bind to HER2, EGF stimulates EGFR and HER2 to form a heterodimer, which activates EGFR and the result is the transphosphorylation of HER2 in the heterodimer. Dimerization and / or transphosphorylation appear to activate HER2 tyrosine kinases. See Earp et al., Supra. Likewise, when HER3 is co-expressed with HER2, an active signal complex is formed and antibodies directed against HER2 are able to disintegrate this complex (Sliwkowski et al., J. Biol. Chem., 269 (20): 14661 -14665 (1994)). Additionally, the affinity of HER3 for heregulin (HRG) is increased to a state of higher affinity when co-expressed with HER2. See also, Levi et al., Journal of Neuroscience 15: 1329-1340 (1995); Morrissey et al., Proc. Nati Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al., Cancer Res., 56: 1457-1465 (1996) with respect to the HER2-HER3 protein complex. HER4, such as HER3, forms an active signal complex with HER2 (Carraway and Cantley, Cell 78: 5-8 (1994)). To target the HER signal pathway, rhuMAb 2C4 (Pertuzumab, OMNITARG ™) was developed as a humanized antibody that inhibits the dimerization of HER2 with other HER receptors, thereby inhibiting ligand-driven phosphorylation and activation, and activation downstream of the RAS and AKT pathways. In a phase I trial of Pertuzumab as a single agent for the treatment of solid tumors, three subjects with advanced ovarian cancer were treated with Pertuzumab. One had a durable partial response, and one additional subject had a stable disease for 15 weeks Agus et al. Proc Am Soc Clin Oncol 22: 192, Abstract 771 (2003).

DR5 Antibodies Several ligands and receptors that belong to the tumor necrosis factor (TNF) superfamily have been identified in the art. Included among said ligands are tumor necrosis factor-alpha ("TNF-alpha"), tumor necrosis factor-beta ("TNF-beta" or "lymphotoxin-alpha"), lymphotoxin-beta ("LT-beta"), ligand CD30, ligand CD27, ligand CD40, ligand OX-40, ligand 4-1BB, LIGHT, ligand Apo-1 (also called Fas ligand or CD95 ligand), Apo-2 ligand (also called Apo2L or TRAIL), Apo ligand -3 (also called TWEAK), APRIL, ligand OPG (also called ligand RANK, ODF, or TRANCE), and TALL-1 (also called BlyS, BAFF or THANK) (See for example eg, Ashkenazi, Nature Review, 2: 420-430 (2002), Ashkenazi and Dixit, Science, 281: 1305-1308 (1998), Ashkenazi and Dixit, Curr Opin, Cell Biol., 11: 255-260 (2000), Golstein, Curr. Biol., 7: 750-753 (1997) Wallach, Cytokine Reference, Academic Press, 2000, pages 377-411; Locksley et al., Cell, 104: 487-501 (2001); Gruss and Dower, Blood, 85: 3378-3404 (1995), Schmid et al., Proc. Nati, Acad. Sci., 83: 1881 (1986), Dealtry et al., Eu J. Immunol., 17: 689 (1987); Pitti et al. , J. Biol. Chem., 271: 12687-12690 (1996); Wiley et al.r Immunity, 3: 673-682 (1995); Browning et al. , Cell, 72: 847-856 (1993); Armitage et al. Nature, 357: 80-82 (1992), WO 97/01633 published January 16, 1997, WO 97/25428 published July 17, 1997; Marsters et al., Curr. Biol., 8: 525-528 (1998); Chicheportiche et al., Biol. Chem., 272: 32401-32410 (1997); Hahne et al., J. Exp. Med., 188: 1185-1190 (1998); W098 / 28426 published July 2, 1998; W098 / 46751 published October 22, 1998; WO / 98/18921 published May 7, 1998; Moore et al., Science, 285: 260-263 (1999); Shu et al., J. Leukocyte Biol., 65: 680 (1999); Schneider et al., J. Exp. Med., 189: 1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem., 274: 15978-15981 (1999)). The induction of several cellular responses mediated by said ligands of the TNF family are typically initiated by their binding to specific cell receptors. Some, but not all, ligands of the TNF family bind to and induce various biological activities through "cell surface deadly receptors" to activate caspases, or enzymes that are carriers of the apoptosis or cell death pathway (Salvesen et al., Cell, 91: 443-446 (1997)). Included among the members of the TNF receptor superfamily, currently identified are TNFR1, TNFR2, TACI, GITR, CD27, OX-40, CD30, CD40, HVEM, Fas (also called Apo-1 or CD95), DR4 (also called TRAIL -R1), DR5 (also referred to as Apo-2 or TRAIL-R2), DcR1, DcR2, osteoprotegerin (OPG), RANK and Apo-3 (also referred to as DR3 or TRAMP). Most of these members of the receiving family TNFs share the typical structure of all cell surface receptors including the extracellular, transmembrane and intracellular regions, while others are found naturally as soluble proteins lacking intracellular and transmembrane domain. The extracellular portion of typical TNFRs contains a repeating amino acid sequence pattern of multiple cysteine rich domains (CRDs), starting from the term NH2-. The ligand designated Apo-2L or TRAIL was identified several years ago as a member of the TNF cytokine family (see, for example, iley et al., Immunity, 3: 673-682 (1995); Pitti et al., J Biol. Chem., 271: 12697-12690 (1996); O 97/01633; WO 97/25428; US Patent 5,763,223 issued June 9, 1998; US Patent 6,284,236 issued September 4, 2001; ). The full-length native sequence human Apo2L / TRAIL polypeptide is a type II transmembrane protein, 281 amino acids in length. Some cells can produce a natural soluble form of the polypeptide, through the enzymatic dissociation of the extracellular region of the polypeptide (Ariani et al., J. Cell, Biol., 137: 221-229 (1997)). Crystallographic studies of the soluble forms of Apo2L / TRAIL revealed a homotrimeric structure similar to the structures of TNF and other related proteins (Hymowitz et al., Molec. Cell, 4: 563-571 (1999); Cha et al., Immunity. , 11: 253-261 (1999), Mongkol sapaya et al., Nature Structural Biology, 6: 1048 (1999), Hymowitz et al., Biochemistry, 39: 633-644 (2000)). The Apo2L / TRAIL, unlike other members of the TNF family however, showed that it had a unique structural characteristic in the sense that the three cysteine residues (at the position position 230 of each subunit in the homotrimer) jointly coordinated an atom of zinc, and that zinc binding is important for trimer stability and biological activity (Hymowitz et al., supra; Bodmer et al., J. Biol. Chem., 275: 20632-20637 (2000)). It has been reported in the literature that Apo2L / TRAIL can play a role in the modulation of the immune system, including in immune diseases such as rheumatoid arthritis (see, for example., Thomas et al., J. Immunol., 161: 2195 -2200 (1998), Johnsen et al., Cytokine, 11: 664-672 (1999), Griffith et al., J. Exp. Med., 189: 1343-1353 (1999), Song et al., J. Exp. Med., 191: 1095-1103 (2000)).

It has also been reported that soluble forms of Apo2L / TRAIL induce apoptosis in a variety of cancer cells, including tumors of the colon, lung, breast, prostate, gallbladder, kidney, ovary and brain, as well as melanomas, leukemia, and myeloma. multiple (see, for example, Wiley et al., supra, Pitti et al., supra, US Patent 6,030,945 issued February 29, 2000, US Patent 6,746,668 issued June 8, 2004, Rieger et al. al., FEBS Letters, 427: 124-128 (1998), Ashkenazi et al., J. Clin. Invest., 104: 155-162 (1999), Walczak et al., Nature Med., 5: 157-163. (1999), Keane et al., Cancer Research, 59: 734-741 (1999), Izutani et al., Clin Cancer Res., 5: 2605-2612 (1999), Gazitt, Leukemia, 13: 1817-1824. (1999), Yu et al., Cancer Res., 60: 2384-2389 (2000), Chinnaiyan et al., Proc. Nati, Acad. Sci., 97: 1754-1759 (2000)). In vivo studies in murine tumor models further suggest that Apo2L / TRAIL, alone or in combination with radiation therapy or chemotherapy, can exert substantial anti-tumor effects (see, for example, Ashkenazi et al., Supra; Walzcak et al. ., supra, Gliniak et al., Cancer Res., 59: 6153-6158 (1999), Chinnaiyan et al., supra, Roth et al., Biochem. Biophys. Res. Comm., 265: 1999 (1999); PCT Application US / 00/15512; PCT Application US / 01/23691). In contrast to many types of cancer cells, most types of normal human cells appear to be resistant to the induction of apoptosis of several recombinant forms of Apo2L / TRAIL (Ashkenazi et al., Supra).; alzcak et al., supra). Jo et al. reported that a soluble form labeled with polyhistidine from Apo2L / TRAIL induced apoptosis in vitro in isolated normal humans, but not in non-human hepatocytes (Jo et al., Nature Med., 6: 564-567 (2000); see also, Nagata, Nature Med., 6: 502-503 (2000)). It is believed that certain recombinant Apo2L / TRAIL preparations may vary in terms of biochemical properties and biological activities in diseased versus normal cells, depending, for example, on the presence or absence of a papilloma molecule, the zinc content, and the percentage of trimer content (see, Lawrence et al., Nature Med., Letter to the Editor, 7: 383-385 (2001); Qin et al., Nature Med., Letter to the Editor, 7: 385-386 (2001 )). It has been found that Apo2L / TRAIL binds to at least five different receptors. At least two of the receptors that bind to Apo2L / TRAIL contain a functional, functional cytoplasmic domain. One of said receptors has been designated "DR4" (and alternatively as TR4 or TRAIL-R1) (Pan et al., Science, 276: 111-113 (1997); see also W098 / 32856 published July 30, 1998 099/37684 published July 29, 1999, WO 00/73349 published December 7, 2000, US Patent 6,433,147 issued August 13, 2002, and US Patent 6,461,823 issued on 8 October of 2002, and US Patent 6,342,383 granted on January 29, 2002). Another such receptor for Apo2L / TRAIL has been designated DR5 (it has also been alternatively referred to as Apo-2, TRAIL-R or TRAIL-R2, TR6, Tango-63, hAP08, TRICK2 or KILLER) (see, for example, Sheridan et al., Science, 277: 818-821 (1997), Pan et al., Science, 277: 815-818 (1997), W098 / 51793 published November 19, 1998; W098 / 41629 published on 24 of September 1998, Screaton et al., Curr. Biol., 7: 693-696 (1997), alczak et al., EMBO J., 16: 5386-5387 (1997), Wu et al., Nature Genetics, 17: 141-143 (1997); W098 / 35986 published August 20, 1998; EP870,827 published October 14, 1998; W098 / 46643 published October 22, 1998; WO99 / 02653 published January 21; 1999, O99 / 09165 published February 25, 1999; W099 / 11791 published March 11, 1999; North American Patent 2002/0072091 published August 13, 2002; US Patent 2002/0098550 published December 7, 2001; US Patent 6,313,269 issued on December 6, 2001; North American Patent 2001/0010924 published August 2, 2001; U.S. Patent 2003/01255540 published July 3, 2003; North American Patent 2002/0160446 published October 31, 2002, North American Patent 2002/0048785 published April 25, 2002; U.S. Patent No. 6,342,369 granted in February 2002; U.S. Patent 6,569,642 granted May 27, 2003, U.S. Patent 6,072,047 issued June 6, 2000; granted June 6, 2000, US Patent 6,642,358 granted November 4, 2003; IS 6,743,625 issued June 1, 2004). As DR4, it has been reported that DR5 contains a deadly cytoplasmic domain and that it is capable of apoptosis signal by ligand binding (or by binding to a molecule such as an agonist antibody, which mimics the activity of the ligand). The crystal structure of the complex formed between Apo-2L / TRAIL and DR5 has been described in Hymowitz et al., Molecular Cell, 4: 563-571 (1999). By binding to the ligand, both DR4 and DR5 can trigger apoptosis independently by recruiting and activating the apoptosis initiator, caspase-8, through the adapter molecule containing the mortal domain, called FADD / Mortl (Kischkel et al., Immunity , 12: 611-620 (2000), Sprick et al., Immunity, 12: 599-609 (2000), Bodmer et al., Nature Cell Biol., 2: 241-243 (2000)). It has been reported that Apo2L / TRAIL also binds to those receptors termed DcRl, DcR2 and OPG, which are believed to function as inhibitors, rather than signal transducers (see, eg, DcRl (also referred to as TRID, LIT or TRAIL-R3) (Pan et al., Science, 276: 111-113 (1997)); Sheridan et al., Science, 277: 818-821 (1997); McFarlane et al., J. Biol. Chem., 272: 25417-25420 (1997); Schneider et al., FEBS Letters, 416: 329-334 (1997); Degli-Esposti et al., J. Exp. Med., 186: 1165-1170 (1997); and Mongkolsapaya et al., J. Immunol., 160: 3-6 (1998)); DcR2 (termed TRUNDD or TRAIL-R4) (Marsters et al., Curr. Biol., 7: 1003-1006 (1997); Pan et al., FEBS Letters, 424: 41-45 (1998); Degli-Esposti et al., Immunity, 7: 813-820 (1997)), and OPG. In contrast to DR4 and DR5, the DcRl and DcR2 receptors do not signal apoptosis. Some antibodies that bind to DR4 receptors and / or DR5 have been mentioned in the literature. For example, anti-DR4 antibodies directed to the DR4 receptor and having agonist or apoptotic activity in certain mammalian cells have been described in for example WO 99/37684 published July 29, 1999; WO 00/73349 published July 12, 2000; WO 03/066661 published August 14, 2003. See also, for example Griffith et al., J. Immunol., 162: 2597-2605 (1999); Chuntharapai et al., J. Immunol., 166: 4891-4898 (2001); WO 02/097033 published December 2, 2002; WO 03/042367 published May 22, 2003; W0 03/038043 published May 8, 2003; W0 03/037913 published May 8, 2003. Some anti-DR5 antibodies have also been described see, for example, WO 98/51793 published November 8, 1998; Griffith et al., J. Immunol., 162: 2597-2605 (1999); Ichikawa et al., Nature Med., 7: 954-960 (2001); Hylander et al. , "An Ancuerpo for DR5 (TRAIL-Receptor 2) Suppresses the Growth of Gastrointestinal Tumors Derived from Patients, Cultured in SCID Mice", Abstract, 2nd International Congress on Monoclonal Antibodies in Cancers, August 29-September 1, 2002, Banff, Alberta, Canada; W0 03/038043 published May 8, 2003; W0 03/037913 published May 8, 2003. In addition, some antibodies that are cross-reactive for both the DR4 and DR5 receptors have been described (see, for example, US Patent 6,252,050 issued June 26, 2001) .

SUMMARY OF THE INVENTION The invention relates here, at least in part, to the identification of histidine-acetate, pH 5.5 to 6.5, as a particularly useful buffer for formulating monoclonal antibodies, especially full-length IgGl antibodies which are susceptible to deamidation and / or aggregation. The formulation retards the degradation of the antibody therein. Therefore, in a first aspect, the invention relates to a stable pharmaceutical formulation comprising a monoclonal antibody in histidine-acetate buffer, at pH 5.5 to 6.5, the monoclonal antibody is preferably bound to a selected antigen of the group consisting of HER2, CD20, DR5, BR3, IgE, and VEGF. Furthermore, the invention relates to a method for the treatment of a disease or disorder in a subject which comprises administering the formulation to a subject in an amount effective to treat the disease or disorder. In another aspect, the invention relates to a pharmaceutical formulation comprising: (a) a full-length IgGl antibody susceptible to deamidation or aggregation in an amount of from about 10mg / mL to about 250mg / mL; (b) histidine-acetate buffer, pH 5.5 to 6.5; (c) saccharide selected from the group consisting of trehalose and sucrose, in an amount of from about 60mM to about 250mM; and (d) polysorbate 20 in an amount of from about 0.01% to about 0.1%. The invention also provides a method for reducing the deamidation or aggregation of a therapeutic monoclonal antibody, which comprises formulating the antibody in a histidine-acetate buffer, pH 5.5 to 6.5. In a further aspect, the invention relates to a pharmaceutical formulation comprising an antibody that binds to domain II of HER2 in a histidine buffer at a pH of from about 5.5 to about 6.5, a saccharide and a surfactant .

The invention also relates to a pharmaceutical formulation comprising Pertuzumab in an amount of from about 20mg / mL to about 40mg / mL, histidine-acetate buffer, sucrose, and polysorbate 20, where the pH of the formulation is from about 5, 5 to about 6.5. The invention also relates to a pharmaceutical formulation comprising a DR5 antibody in a histidine buffer at a pH of from about 5.5 to about 6.5, a saccharide, and a surfactant. In another aspect, the invention relates to a pharmaceutical formulation comprising Apomab in an amount of from about 10mg / mL to about 30mg / mL, histidine-acetate buffer, trehalose, and polysorbate 20, where the pH of the formulation is from about 5.5 to about 6.5. In a further aspect, the invention provides a method for treating cancer in a subject, comprising administering the pharmaceutical formulation to the subject in an amount effective to treat cancer. The invention also relates to a bottle with a pierceable stopper with a syringe or a stainless steel tank comprising the formulation inside the bottle or tank, optionally in frozen form.

In addition, the invention provides a method for preparing a pharmaceutical formulation comprising: (a) preparing the monoclonal antibody formulation; and (b) evaluation of the physical stability, chemical stability, or biological activity of the monoclonal antibody in the formulation.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 describes Domains I-IV (SEQ ID Nos. 19-22, respectively) of the extracellular domain of HER2. Figures 2A and 2B describe alignments of the amino acid sequences of the variable light domains (VL) (Fig. 2A) and variable heavy domains (VH) (Fig. 2B) of the murine monoclonal antibody 2C4 (SEQ ID Nos. 1 and 2) , respectively); VL and VH domains of humanized 2C4 version 574 (SEQ ID Nos. 3 and 4, respectively), and the human consensus structures VL and VH (hum kl, subgroup I light kappa, humIII, heavy subgroup III) (SEQ ID. Nos. 5 and 6, respectively). The asterisks identify the differences between the humanized version 574 2C4 and the murine monoclonal antibody 2C4 or between the humanized version 574 2C4 and the human structure. The Complementarity Determination Regions (CDRs) are in brackets. Figures 3A and 3B show the amino acid sequences of the light chain of Pertuzumab and the heavy chain (SEQ ID Nos. 15 and 16, respectively). The CDRs are shown in bold. The calculated molecular weights of the light chain and the heavy chain are 3,526.22 Da and 49,216.56 Da (cysteines in reduced form). The carbohydrate moiety is attached to Asn 299 of the heavy chain. Figures 4A and 4B show the amino acid sequences of the heavy and light chain of Pertuzumab, where each includes an intact amino terminal signal peptide sequence (SEQ ID Nos. 17 and 18, respectively). Figure 5 depicts, schematically, the binding of 2C4 at the heterodimeric binding site of HER2, thereby avoiding heterodimerization with activated EGFR or HER3. Figure 6 describes the coupling of HER2 / HER3 to the APK and Akt pathways. Figure 7 compares the activities of Trastuzumab and Pertuzumab. Figure 8 describes the stability of the Pertuzumab formulation by ion exchange analysis (IEX). Figure 9 describes the stability of the Pertuzumab formulation by size exclusion chromatography (SEC) analysis. Figure 10 reflects the physical stability of Pertuzumab in different formulations. Figure 11 is from a shaking study of liquid formulations of Pertuzumab.

Figure 12 is from another study of agitation of liquid formulations of Pertuzumab. Figure 13 is from a freeze-thaw study of Pertuzumab formulation. Figures 14A and 14B show the amino acid sequences of the light chain of Trastuzumab (SEQ ID No. 13) and of the heavy chain (SEQ ID No. 14). Figures 15? and 15B describe a variant of the light chain sequence of Pertuzumab (SEQ ID No. 23) and a variant of the heavy chain sequence of Pertuzumab (SEQ ID No. 24). Figure 16 shows the oligosaccharide structures commonly observed in IgG antibodies. Figures 17A and 17B show the sequences of the light and heavy chains (SEQ ID Nos. 37-44) of the specific anti-IgE antibodies E25, E26, HAE1 and Hu-901. In fig. 17A, the variable light domain ends with residues VEIK, residue 111. In FIG. 17B, the variable heavy domain ends with the VTVSS residues, around residue 120. Figure 18A is a sequence alignment that compares the amino acid sequences of the variable light domain (VL) of each murine 2H7 (SEQ ID No. 25), humanized 2H7vl6 variant (SEQ ID No. 26), and subgroup I of human light chain kappa (SEQ ID No. 27). The VL CDRs of 2H7 and hu2H7vl6 are as follows: CDR1 (SEQ ID No. 57), CDR2 (SEQ ID No. 58), and CDR3 (SEQ ID No. 59). Figure 18B is a sequence alignment that compares the amino acid sequences of the variable heavy domain (VH) of each two murine 2H7 (SEQ ID No. 28), humanized 2H7vl6 variant (SEQ ID No. 29), and the sequence of human consensus of heavy chain subgroup III (SEQ ID No. 30). The VH CDRs of 2H7 and hu2H7vl6 are as follows: CDR1 (SEQ ID No. 60), CDR2 (SEQ ID No. 61), and CDR3 (SEQ ID No. 62). In fig. 18A and Fig. 18B, the CDR1, CDR2 and CDR3 in each chain are enclosed in brackets, flanked by the structural regions FR1-FR4, as indicated. 2H7 refers to the murine 2H7 antibody. The asterisks between two rows of sequences indicate the positions that are different between the two sequences. The numbering of waste is carried out according to Kabat et al. Sequences oí Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), with the insertions shown as a, b, c, d, and e. Figure 19 describes variable domain sequences of three different VEGF antibodies with SEQ ID Nos. 31-36. Figure 20 shows the elution profile of size exclusion chromatography (SEC) of the following Apomab samples: (a) control and formulations prepared in (b) pH 4.0, (c) pH 5.0, (d) pH 6.0 and (e) pH 7.0. The formulated samples were stored at 40 ° C for 2 months before analysis. Figure 21 describes the profile of the pH regime for loss of Apomab antibody monomer during storage. The monomeric kinetics by SEC was monitored during storage at 30 ° C and 40 ° C, and the constants of first order regime were calculated. Figure 22 provides the ion exchange chromatography (IEC) elution profile of Apomab samples as follows: (a) control and formulations prepared at (b) pH 4.0, (c) pH 5.0, (d) ) pH 6.0 and (e) pH 7.0. The formulated samples were stored at 40 ° C for 2 months before analysis. Figure 23 shows the profile of the pH regime for the loss of IEC main peak during storage. The main peak kinetics by IEC was monitored during storage at 30 ° C and 40 ° C and the constants of first order regime were calculated. Figure 24 shows the nucleotide sequence of the human Apo-2 ligand cDNA (SEQ ID NO: 45) and its derived amino acid sequence (SEQ ID NO: 46). The "N" at the position of nucleotide 447 (in SEQ ID NO: 45) was used to indicate that the nucleotide base may be a "T" or "G".

Figures 25A to 25C show the 411 amino acid sequence of the human DR5 receptor (SEQ ID NO: 47) published in WO 98/51793 on November 19, 1998, and the nucleotide coding sequence (SEQ ID NO: 48). Figures 26A to 26C show the 440 amino acid sequence of the human DR5 receptor (SEQ ID NO: 49) and the coding nucleotide sequence (SEQ ID NO: 50), also published in WO 98/35986 on August 20, 1998.

Figure 27 shows the amino acid sequence of heavy chain 7.3 Apomab (SEQ ID NO: 51). Figure 28 shows the light chain amino acid sequence 7.3 of Apomab (SEQ ID NO: 52). Figures 29A and 29B show the alignment of the heavy chain 16E2 (SEQ ID No. 53) and the heavy chain amino acid sequences 7.3 of Apomab (SEQ ID No. 51). Figure 30 shows the alignment of the light chain amino acid sequences 16E2 (SEQ ID No. 54) and light chain 7.3 of Apomab (SEQ ID No. 52). Figures 31A and 31B describe the variable heavy amino acid sequences (Fig. 31A; SEQ ID No. 55) and the variable light amino acid sequence (Fig. 31B; SEQ ID No. 56) of Apomab 7.3. The CDR residues were identified in bold. Figure 32 shows an alignment of mature light chains 2H7vl6 and 2H7v511 (SEQ ID Nos. 63 and 64, respectively). The sequences are shown with Kabat variable domain residue numbering and Eu constant domain residue numbering. Figure 33 shows an alignment of the heavy chains 2H7vl6 and 2H7v511 mature (SEQ ID Nos. 65 and 66, respectively). The sequences are shown with Kabat variable domain residue numbering and Eu constant domain residue numbering.

Detailed Description of Preferred Embodiments I. Of Initions The term "pharmaceutical formulation" refers to a preparation that is in such a form that allows the biological activity of the active ingredient to be effective, and that does not contain any additional component that is unacceptably toxic to a subject to which the formulation would be administered. . Said formulations are sterile. A "sterile" formulation is aseptic or free of any living microorganism and its spores. Here, a "frozen" formulation is one that is at a temperature below 0 ° C. In general, the frozen formulation is not freeze-dried, nor is it subjected previously or subsequently to lyophilization. Preferably, the frozen formulation comprises frozen pharmacological substances for storage (in stainless steel tanks) or frozen pharmacological products (in final package configuration). A "stable" formulation is one in which the protein thereof essentially retains its physical stability and / or its chemical stability and / or biological activity during storage. Preferably, the formulation retains essentially its physical and chemical stability, as well as its biological activity in storage. The storage period is generally selected based on the storage life for which the formulation is intended. Various analytical techniques for measuring protein stability are available in the art and have been made in, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed. , Marcel Dekker, Inc., New York, New York, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). The stability can be mediated at a selected temperature for a selected period of time. Preferably, the formulation is stable at about 40 ° C for at least about 2-4 weeks, and / or is stable at about 5 ° C and / or 15 ° C for at least 3 months, and / or is stable to approximately -20 ° C for at least 3 months or at least 1 year. In addition, the formulation is preferably stable after freezing (at, for example, -70 ° C), and thawing the formulation, for example after 1, 2 or 3 cycles of freezing and thawing. Stability can be assessed qualitatively and / or quantitatively in a variety of different ways, including the evaluation of aggregate formation (for example using size exclusion chromatography, turbidity measurement and / or visual inspection); verifying the heterogeneity of the load using cation exchange chromatography or capillary zone electrophoresis; analysis of the amino-terminal or carboxy-terminal sequence; mass spectrometric analysis; SDS-PAGE analysis to compare the reduced antibody in intact; peptide map (for example tryptic or LYS-C); evaluation of the biological activity or the antigen-binding function of the antibody; etc. The instability may involve one or more of: aggregation, deamidation (e.g., deamidation of Asn), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomerization), clipping / hydrolysis / fragmentation (e.g. the hinge region), succinimide formation, unpaired cysteine (s), N-terminal extension, C-terminal processing, glycocylation differences, etc. A "deamidated" monoclonal antibody is here one in which one or more asparagine residues thereof have been derivatized, for example to an aspartic acid or to an iso-aspartic acid. An antibody that is "susceptible to deamidation" is one that comprises one or more residues that have been shown to be susceptible to deamidation. An antibody that is "susceptible to aggregation" is one that has been shown to aggregate with other antibody molecules, especially by freezing and / or agitation. An antibody that is "susceptible to fragmentation" is one that has been shown to dissociate into two or more fragments, for example in a hinge region thereof. By "reducing the deamidation, aggregation, or fragmentation" is intended to avoid or decrease the amount of deamidation, aggregation, or fragmentation in relation to the monoclonal antibody formulated at a different pH or in a different buffer. Here, "biological activity" of a monoclonal antibody refers to the ability of the antibody to bind to the antigen and result in a measurable biological response that can be measured in vitro or in vivo. Said activity can be antagonist (for example when the antibody is an HER2 antibody) or agonist (for example when antibody binds to DR5). In the case of Pertuzumab, in one embodiment, biological activity refers to the ability of the antibody formulated to inhibit the pluriferation of the human breast cancer cell line DA-MB-175-VII. When the antibody is Apomab, the biological activity may relate, for example, to the ability of the antibody formulated to kill the colon carcinoma, Colo205 cells. By "isotonic" it is indicated that the formulation of interest essentially has the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure of from about 250 to 350mOsm. The isotonicity can be measured for example using a vapor pressure or an ice-type freezing type osmometer. As used herein, "buffer" refers to a pH-regulated solution that resists pH changes by the action of its conjugated acid-base components. The buffer of this invention preferably has a pH in the range of from about 5.0 to about 7.0, preferably from about 5.5 to about 6.5, for example from about 5.8 to about 6.2, and more preferably has a pH of about 6.0. Examples of buffers that will control pH in this range include acetate, succinate, gluconate, histidine, citrate, glycolglycine and other organic acid buffers. The preferred buffer is here a histidine buffer.

A "histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate. The preferred histidine buffer identified in the examples given here proved to be histidine acetate. In the preferred embodiment, the histidine acetate buffer is prepared by titration of L-histidine (free base, solid) with acetic acid (liquid). Preferably, the histidine buffer or the histidine acetate buffer is at a pH of 5.5, at 6.5, preferably at a pH of 5.8 to 6.2. A "saccharide" here comprises the general composition (CH20) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. Examples of saccharides include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, silitol, sorbitol, mannitol, melibiose, melezitose, raffinose, manotriose, stachyose, maltose, lactulose, maltulose , glucitol, maltilol, lactilol, iso-maltulose, etc. The preferred saccharide is here a non-reducing disaccharide such as trehalose or sucrose. Here, a "surfactant" refers to a surfactant, preferably a nonionic surfactant.

Examples of surfactants include polysorbates here (e.g., polysorbates 20 and, polysorbate 80); poloxamer (for example poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-zarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, 1-indoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (for example lauroamidopropyl); Myristamidopropyl-palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT ™ series (Mona Industries, Inc., Paterson, New Jersey); polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (for example Pluronics, PF68 etc), etc. The preferred surfactant is polysorbate 20. A "HER receptor" is a receptor tyrosine kinase protein that belongs to the HER receptor family and includes the EGFR, HER2, HER3 and HER4 receptors and other members of this family that will be identified in the future. The HER receptor will generally comprise an extracellular domain, which can bind an HER ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a terminal carboxyl signal domain containing various tyrosine residues that can be phosphorylated. Preferably the HER receptor is a human HER receptor of natural sequence. The extracellular domain of HER2 comprises four domains, Domain I (amino acid residues of about 1-195), Domino II (amino acid residues from about 196-320), Domino III (amino acid residues from about 321-488), and Domino IV ( amino acid residues from about 489-632) (residue numbering without signal peptide). See Garrett et al. Mol. Cell .. 11: 495-505 (2003), Cho et al. Nature 421: 756-760 (2003), Franklin et al. Cancer Cell 5: 317-328 (2004), or Plowman et al. Proc. Nati Acad. Sci. 90: 1746-1750 (1993). See also Fig. 1 given here. The terms "ErbBl," "HERI", "epidermal growth receptor factor" and "EGFR" are used interchangeably herein and refer to EGFR as described, for example, in Carpenter et al. Ann. Rev. Biochem. 56: 881-914 (1987), including the natural mutant forms thereof (for example a mutant EGFR with deletion as in Humphrey et al., PNAS (USA) 87: 4207-4211 (1990)). er £ > Bl refers to the gene that encodes the EGFR protein product. The expression "ErbB2" and "HER2" is used interchangeably herein and refers to the human HER2 protein described, for example in Semba et al., PNAS (USA) 82: 6497-6501 (1985) and Yamamoto et al. Nature 319: 230-234 (1986) (accession number of Genebank X03363). The term "er¿ >"B2" refers to the gene encoding human ErbB2 and "neu" refers to the gene encoding rat pl85neu.The preferred HER2 is the natural human sequence HER2. "ErbB3" and "HER3" refers to the receptor polypeptide such as which has been described, for example in US Patents Nos. 5,183,884 and 5,480,968 as well as in Kraus et al., PNAS (USA) 86: 9193-9197 (1989) .The terms "ErbB4" and "HER4" which are given herein refer to the receptor polypeptide described, for example in European Patent Application No. 599,274, Plowman et al., Proc. Nati, Acad. Sci. USA, 90: 1746-1750 (1993), and Plowman et al. al., Nature, 366: 473-475 (1993), including isoforms thereof, for example those described in W099 / 19488, published April 22, 1999. By "ligand HER" is meant a polypeptide that binds and / or activates a HER receptor The HER ligand of particular interest herein is a natural sequence human HER ligand such as an epidermal growth factor (EGF) (Sav. age et al., J. Biol. Chem. 247: 7612-7621 (1972)); an alpha transforming growth factor (TGF-α) (Marquardt et al., Science 223: 1079-1082 (1984)); anfiregulin also known as schwannoma or keratinocytic autocrine growth factor (Shoyab et al., Science 243: 1074-1076 (1989); Kimura et al., Nature 348: 257-260 (1990); and Cook et al., Mol.

Cell. Biol. 11: 2547-2557 (1991)); betacellulin (Shing et al., Science 259: 1604-1607 (1993); and Sasada et al., Biochem. Biophys., Res. Commun. 190: 1173 (1993)); heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al., Science 251: 936-939 (1991)); epiregulin (Toyoda et al., J. Biol. Chem. 270: 7495-7500 (1995); and Komurasaki et al., Oncogene 15: 2841-2848 (1997)); a heregulina (see below); neuregulin-2 (NRG-2) (Carraway et al., Nature 387: 512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc. Nati. Acad. Sci. 94: 9562-9567 (1997)); neuregu 1 ina-4 (NRG-4) (Harari et al., Oncogene 18: 2681-89 (1999)) or crypto (CR-1) (Kannan et al., J. Biol. Chem. 272 (6): 3330- 3335 (1997)). HER ligands that bind to EGFR include EGF, TGF-α, amphiphulline, betacellulin, HB-EGF and epiregulin. HER ligands that bind to HER3 include heregulins. HER ligands capable of binding to HER4 include betacellulin, epiregulin, HB-EGF, NRG-3, NRG-4 and heregulin. "Heregulin" (HRG) when used herein refers to a polypeptide encoded by the heregulin gene product described in the US Patent. No. 5,641,869 or Marchionni et al., Nature, 362: 312-318 (1993). Examples of heregulins include heregulin-a, heregulin-β? , heregulin ^ 2 and heregulin ^ 3 (Holmes et al., Science, 256: 1205-1210 (1992); and US Patent No. 5,641,869); Neu differentiation factor (NDF) (Peles et al.

Cell 69: 205-216 (1992)); acetylcholine receptor-inducing activity (ARIA) (Falls et al., Cell 72: 801-815 (1993)); glial growth factors (GGFs) (Marchionni et al., Nature, 362: 312-318 (1993)); factor derived from motor and sensory neurons (SMDF) (Ho et al., J. Biol. Chem. 270: 14523-14532 (1995)); ? -heregulin (Schaefer et al., Oncogene 15: 1385-1394 (1997)). The term includes fragments and / or variants of biologically active amino acid sequences of a natural sequence HRG polypeptide, such as an EGF-like domain fragment thereof (for example? - ^? A "HER dimer" is here a dimer Non-covalently associated agents comprising at least two different HER receptors, said complexes can be formed when a cell expressing 2 or more HER receptors is exposed to an HER ligand and can be isolated by immunoprecipitation and analyzed by SDS-PAGE as described in for example Sliwkowski et al., J. Biol. Chem., 269 (20): 14661-14665 (1994) Examples of such HER dimers include the heterodimers EGFR-HER2, HER2-HER3 and HER3-HER. Dimer HER may comprise two or more HER2 receptors combined with a different HER receptor, such as HER3, HER4 or EGFR.Other is proteins, such as the cytokine receptor subunit (eg gpl30) may be associated with the dimer.

A "heterodimeric binding site" in HER2 refers to a region in the extracellular domain of HER2 that contacts, or forms an inferium with, a region in the extracellular domain of EGFR, HER3 or HER4 by formation of a dimer therewith. The region is located in the Domino II of HER2. Franklin et al. Cancer Cell 5: 317-328 (2004). "HER activation" or "HER2 activation" refers to the activation, or phosphorylation, of one or more of the HER receptors, or the HER2 receptors. In general, HER activation results in signal transduction (e.g., that caused by the intracellular kinase domain of tyrosine phosphorylating HER receptor residues in a HER receptor or a substrate polypeptide). The HER activation can be mediated by the binding of the HER ligand to a HER dimer comprising the HER receptor of interest. The binding of the HER ligand to a HER dimer can activate a kinase domain of one or more of the HER receptors in the dimer and therefore can result in the phosphorylation of the tyrosine residues in one or more of the HER receptors and / or phosphorylation of tyrosine residues on additional substrate polypeptides such as the intracellular Akt or MAPK kinases. The term "antibodies" is used here in the broadest sense and specifically covers full-length monoclonal antibodies, polyclonal antibodies, fierce multispecies antibodies (for example, bispecific antibodies) formed from at least two full-length antibodies, and fragments of antibodies, with the proviso that they exhibit the desired biological activity. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie the individual antibodies that comprise the population are identical and / or bind to the same epitope except for possible variants that can arise during the production of the monoclonal antibody, where said variants are generally present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are not contaminated by other immunoglobulins. The "monoclonal" modifier indicates that the character of the antibody is obtained from a substantially homogeneous population, and that it should not be considered that it requires production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present 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 ( see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using for example the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). Monoclonal antibodies specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody , while the rest of the strands is identical to or well homologous to the corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of said antibodies, with the condition that they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Nati Acad. Sel. USA, 81: 6851-6855 (1984)). The chimeric antibodies of interest herein include "primed" antibodies that comprise variable domain antigen binding sequences, derived from a non-human primate (eg, Old World Monkey, Ape etc) and human constant region sequences.

The "antibody fragments" comprise a portion of a full length antibody, preferably comprising the antigen binding region or variable thereof. Examples of antibody fragments include the Fab, Fab ', F (ab') 2 fragments, and Fv fragments; diabodies; linear antibodies; Single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. A "full length antibody" is one that comprises a variable antigen binding region, as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be constant domains of natural sequences (e.g., constant domains of human natural sequences) or variants of amino acid sequences thereof. Preferably, the full-length antibody has one or more effector functions. The term "major species antibody" refers herein to the antibody structure in a composition that is an antibody molecule quantitatively predominant in the composition. In one embodiment, the main species antibody is an HER2 antibody, such as an antibody that binds to Domain II of HER2, an antibody that inhibits HER dimerization more effectively than Trastuzumab, and / or an antibody that binds to HER2. a heterodimeric binding site of HER2. The preferred embodiment here is a main species HER2 antibody comprising variable heavy and light amino acid sequences variable in SEQ ID Nos. 3 and 4 and more preferably comprising the heavy chain and light chain amino acid sequences in SEQ IDs. Nos 15 and 16 Pertuzumab). An "amino acid sequence variant antibody" is herein an antibody with an amino acid sequence that differs from a major species antibody. Commonly, variants of the amino acid sequences will possess at least about 70% homology with the main species antibody and will preferably be at least about 80%, more preferably at least about 90% homologous with the main species antibody . The amino acid sequence variants possess substitutions, deletions, and / or additions at certain positions within or adjacent to the amino acid sequence of the major species antibody. Examples of the amino acid sequence variants provided herein include the acid variant (for example the deamidated antibody variant), the basic variant, the antibody with an amino-terminal leader extension (for example HSV-) in one or two chains light thereof, antibody with a C-terminal lysine residue in one or two heavy chains thereof, etc., and includes combinations of amino acid sequence variations of the heavy and / or light chains. The antibody variant of particular interest herein is the antibody comprising an amino-terminal leader extension in one or two light chains thereof, optionally further comprising another amino acid sequence and / or glycosylation differences related to the major species antibody. A "therapeutic monoclonal antibody" is an antibody that is used for therapy of a human subject. The therapeutic monoclonal antibodies described herein include: HER2 antibodies for cancer and various diseases or non-malignant disorders; CD20 or BR3 antibodies for therapy of malignant B-cell tumors, autoimmune diseases, rejection of the grafts or blocking of an immune response to a foreign antigen; IgE antibodies for therapy of an IgE-mediated disorder; DR5 or VEGF antibodies for cancer therapy. A "variant glycosylation antibody" is herein an antibody with one or more carbohydrate moieties attached thereto which differ from one or more carbohydrate moieties linked to a major species antibody. Examples of glycosylation variants include the antibody with the structure of the oligosaccharide Gl or G2 instead of the oligosaccharide structure GO bound to a Fe region thereof., antibody with one or two carbohydrate moieties attached to one or two light chains thereof, antibody without any carbohydrate bound to one or two heavy chains of the antibody, etc., and combinations of the glycosylation alterations. When the antibody has a Fe region, an oligosaccharide structure such as that shown in Fig. 16 here may be bound to one or two heavy chains of the antibody, for example to residue 299 (298, Eu numbering of residues). For Pertuzumab, the GO was the predominant oligosaccharide structure, with other oligosaccharide structures such as GO-F, Gl, Man5, an6, Gl-1, Gl (1-6), Gl (l-3) and G2 which they were found in minor amounts in the composition of Pertuzumab. Unless otherwise indicated a "Gl oligosaccharide structure" herein includes the structures G-1, Gl-1, Gl (1-6) and Gl (1-3). An "amino-terminal leader extension" refers herein to one or more amino acid residues of the amino-terminal leader sequence that are present at the amino terminus of one or more heavy or light chains of an antibody. An amino-terminal leader extension that constitutes an example, comprises or consists of amino acid residues, HSV, which are present in one or both light chains of an antibody variant.

"Homology" is defined as the percentage of residues in the variant of amino acid sequences that are identical after the sequence alignment and that introduce, if necessary, openings, to achieve the maximum homology percentage. Computer methods and programs for alignment are well known in the art. One such computer program is the "Align 2" of Genentech, Inc., which was presented with user documentation in the United States Copyright Office, Washington, DC 20559, on December 10, 1991. The "effector functions" of the antibody refer to those biological activities attributable to the Fe region (a Fe region of natural sequence or a Fe region variant of amino acid sequence) of an antibody. Examples of antibody effector functions include Clq binding; cytotoxicity; dependent on the complement; Fe receptor binding; cytotoxicity mediated by antibody-dependent cells (ADCC); phagocytosis; down-regulation of cell surface receptors (eg, B-cell receptor, BCR), etc. Depending on the amino acid sequence of the constant domains of their heavy chains, the full-length antibodies can be assigned to different "classes". There are five major classes of full length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are designated, d, e,?, And μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fe (FcRs) receptors (e.g., Natural Killer (K) cells, neutrophils, and macrophages) recognize the antibody attached to a target cell and subsequently cause the lysis of the target cell. Primary cells to intermediate ADCC, NK cells, express FcyRIII only, while monocytes express FcyRl, FcyRII and FcyRIII. The expression FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, ñnnu. Rev. Immunol 9: 457-92 (1991). To verify the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337, can be carried out. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.

Alternatively or additionally, the ADCC activity of the molecules of interest can be verified in vivo, for example, in an animal model as described in Clynes et al. PNAS (USA) 95: 652-656 (1998). "Human effector cells" are leukocytes that express one or more FcRs and that perform effector functions. Preferably, the cells express at least FCYRIII and perform the ADCC effector function. Examples of human leukocytes that are ADCC intermediates include peripheral blood mononuclear cells (PBMC), Natural Killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; PB cells Cs and NK are preferred. Effector cells can be isolated from a natural source thereof, for example from blood or PBMCs as described herein. The terms "Fe receptors" or "FcR" are used to describe a receptor that binds to the Fe region of an antibody. Preferred FcRs consist of a human FcR of natural sequence. In addition, a preferred FcR is one that binds to an IgG antibody (a gamma receptor) and includes the subclasses of the FcyRI, FcyRII, and FcyRIII subclasses, including variants allelic and alternatively the spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibitory receptor") that have similar amino acid sequences that differ mainly in the cytoplasmic domains thereof. The activating receptor FCYRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitor receptor FCYRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see review M. in Daéron, Annu. Rev. Immunol. 15: 203-234 (1997)). The FcRs have been reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those that will be identified in the future, are covered here by the term "FcR". The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 249 (1994)). "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (for example an antibody) in complex with a related antigen. To verify the activation of complement, a CDC assay can be carried out, for example such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). "Natural antibodies" are usually heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, while the amount of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each light and heavy chain also has intra-chain disulfide bridges regularly spaced. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. The particular amino acid residues are considered to form an inferium between the variable domains of light chain and heavy chain. The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequences between the antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed throughout all the variable domains of antibodies. It is concentrated in three segments that are called hypervariable regions in the variable domains of light chain and heavy chain. The portions of the most highly conserved variable domains are called structural regions (FRs). The heavy chain and light chain variable domains each comprise four FRs, which largely adopt a β-sheet configuration, connected by three hypervariable regions, which form loops that are connected, and which in some cases are part of the sheet structure H.H. The hypervariable regions in each chain are held together in close proximity by the FRs, and with the hypervariable regions of the other chain, they contribute to the formation of the antigen binding site of the antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit several effector functions such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (eg residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain). , and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues of a "hypervariable curl" (eg, residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain, Chothia and Lesk J. Mol. Biol. 196: 901-917 ( 1987)). The "Structural Region" or "FR" residuals are those variable domain residues other than the hypervariable region residues defined here. The digestion of antibodies in papain produces two identical antigen binding fragments that are called "Fab" fragments, with a single antigen binding site, and a residual "Fe" fragment, whose name reflects its ability to rapidly crystallize. The pepsin treatment provides an F (ab ') 2 fragment that has two antigen binding sites and is still capable of cross-linking antigen.

"Fv" is the minimum antibody fragment that contains an antigen binding site and complete antigen recognition. This region consists of a dimer of a variable domain of heavy chain and one of light chain, in non-covalent, hermetic association. It is in this configuration that the three hypervariable variable domain chain regions interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind the antigen, albeit at a site of lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab 'fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation given here for Fab 'in which the cysteine residues of the constant domains carry at least one free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of the Fab' fragments having between them the hinge cysteines. Other chemical couplings of the antibody fragments are also known. The "light chains" of antibodies of any vertebrate species can be assigned to one of two clearly distinct types, called kappa (?) And lambda (?), Based on the amino acid sequences of their constant domains. The "single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of the antibody, where these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For a review of scFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. , Springer-Verlag, New York, pp. 269-315 (1994). The scFv fragments of the HER2 antibody have been described in 093/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. The term "diabodies" refers to small fragments of antibodies with two antigen-binding sites, wherein said fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. The diabodies are described more fully in, for example, European Patent 404,097; WO 93/11161; and Hollinger et al., Proc. Nati Acad. Sci. USA, 90: 6444-6448 (1993). The "humanized" forms of non-human antibodies (eg, rodents) are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulin. For the most part humanized antibodies are human immunoglobulins (receptor antibody) in which the residues of a hypervariable region of the receptor are replaced with residues from a hypervariable region of a non-human species (donor antibody) such as mice, rats, rabbits or non-human primates that have the desired specificity, affinity and capacity. In some cases, the residues of the structural region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are prepared by further refining the behavior of the antibody. In general, the humanized antibody will substantially comprise all of at least one and typically two variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of FRs consist of those of a human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin. For additional details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). Humanized HER2 antibodies include huMAb D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or Trastuzumab (HERCEPTIN®) as described in the Table 3 of U.S. Patent 5,821,337 which is expressly incorporated herein by reference; the humanized antibodies 520C9 (093/21319) and the humanized 2C4 antibodies are as described herein. For the present purposes, "Trastuzumab", "HERCEPTIN ©" and "hu Ab4D5-8" refer to an antibody comprising the light and heavy chain amino acid sequences in SEQ ID NOS. 13 and 14, respectively. Here, "Pertuzumab", "rhuMAb 2C4" and "OMNITARG ™" refers to an antibody comprising the variable light and variable heavy amino acid sequences in SEQ ID NOS. 3 and 4, respectively. When Pertuzumab is a full-length antibody, it preferably comprises the light chain and heavy chain amino acid sequences in SEQ ID NOS. 15 and 16, respectively. A "naked antibody" is an antibody (such as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic or radiolabeled portion.

An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions thereof that results in an improvement in the affinity of an antibody to the antigen, as compared to a major antibody that does not possess these alterations. Preferred affinity-matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by methods known in the art. Marks et al. Bio / Technology 10: 779-783 (1992) describe maturation by affinity through the transfer of VH and VL domain. The random mutagenesis of CDR and / or structural residues has been described by: Barbas et al. Proc Nat. Acad. Sci, USA 91: 3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al., J. Immunol. 154 (7): 3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226: 889-896 (1992). An "agonist antibody" is an antibody that binds to and activates a receptor. In general, the activation capacity of the agonist antibody receptor will be at least qualitatively similar (and can be essentially quantitatively similar) to a natural agonist ligand of the receptor. An example of an agonist antibody is one that binds to a receptor in the TNF receptor superfamily, such as DR5, and induces apoptosis of cells expressing the TNF receptor (e.g., DR5). Assays for determining the induction of apoptosis have been described in W098 / 51793 and W099 / 37684, both of which are hereby expressly incorporated by reference. An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of their natural environment are materials that would interfere with the diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody determined by the Lowry method and more preferably more than 99% by weight, (2) to a sufficient degree to obtain at least 15 residues. of an N-terminal or internal amino acid sequence by use of a rotating cup sequencer, or (3) to homogeneity with SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibody includes the antibody in situ within recombinant cells because at least one component of the antibody's natural environment will not be present. Commonly, however, the isolated antibody will be prepared with at least one purification step. An HER2 antibody that "inhibits HER dimerization more effectively than Trastuzumab" is one that reduces or eliminates HER dimers more efficiently (eg at least about 2 times more efficiently) than Trastuzumab. Preferably, said antibody inhibits HER2 dimerization at least approximately as efficiently as an antibody selected from the group consisting of murine monoclonal antibody 2C4, a Fab fragment of murine monoclonal antibody 2C4, Pertuzumab and a Fab fragment of Pertuzumab. The inhibition of HER dimerization can be evaluated by direct study of HER dimers or by evaluation of HER activation, or by downstream signal, which will result in HER dimerization and / or evaluation of the binding site of HER. HER2-antibody, etc. Assays for the detection of antibodies capable of inhibiting HER dimerization more efficiently than Trastuzumab have been described in Agus et al. Cancer Cell 2: 127-137 (2002) and WO01 / 00245 (Adams et al.). By way of example only, it can be assayed for inhibition of HER dimerization by checking for example the inhibition of HER dimer formation (see, for example, Fig. 1A-B of Agus et al., Cancer Cell 2: 127-137 (2002 ); and WOOl / 00245); reduction in HER ligand activation of cells expressing HER dimers (eg, WOOl / 00245 and Fig. 2A-B of Agus et al.Cell Cell 2: 127-137 (2002)); blocking the binding of the HER ligand to the cells expressing the HER dimers (eg, (WOOl / 00245 and Fig. 2E of Agus et al.Cancer Cell 2: 127-137 (2002)): inhibition of cell growth in cells cancerous (e.g., MCF7, MDA-MD-134, ZR-75-1, MD-MB-175, T-47D) cells that express HER dimers in the presence (or absence) of the HER ligand (e.g., WOOl / 00245 and Figs 3A-D of Agus et al., Cancer Cell 2: 127-137 (2002)), inhibition of the downstream signal (eg, inhibition of HRG-dependent AKT phosphorylation or inhibition of MAPK-dependent phosphorylation). of HRG or TGFa) (see, for example, WOOl / 00245 and Fig. 2C-D of Agus et al., Cancer Cell 2: 127-137 (2002).) It can also be verified whether the antibody inhibits HER dimerization by studying from the HER2 binding site to the antibody, for example by evaluating a structure or model, such as a crystal structure, of the antibody bound to HER2 (see, for example, F Ranklin et al. Cancer Cell 5: 317-328 (2004)). The HER2 antibody can "inhibit HRG-dependent AKT phosphorylation" and / or inhibit MAPK-dependent phosphorylation of HRG or TGFa "more effectively (eg, at least twice as efficiently) than Trastuzumab (see, example mode Agus et al. Cancer Cell 2: 127-137 (2002) and WO01 / 00245) The HER2 antibody can be an antibody that "does not inhibit the dissociation of the HER2 ectodomain" (Molina et al., Cancer Res. 61: 4744-4749 (2001) An HER2 antibody that "binds to a heterodimeric binding site" of HER2, binds to residues in domain II (and optionally binds also to residues in other domains of the extracellular domain HER2, such as domains I and III), and can spherically prevent, at least to some degree, the formation of a heterodimer HER2-EGFR, HER2-HER3 or HER2-HER4, Franklin et al., Cancer Cell 5: 317-328 ( 2004) characterizes the crystal structure HER2-Pertuzumab, deposited in the protein data bank RCSB (Cod ID and IS78), illustrating an example of an antibody that binds to the heterodimeric binding site of HER2. An antibody that "binds to domain II" of HERS binds to residues in domain II and optionally to residues in other HER2 domains such as domains I and III. Preferably the antibody that binds to domain II binds to the junction between domains I, II and III of HER2. A "growth inhibitory agent", when used herein, refers to a compound or composition that inhibits the growth of a cell, especially a cancer cell that expresses HER either in vitro or in vivo. Therefore, the growth inhibitory agent can be one that significantly reduces the percentage of cells expressing HER in the S phase. Examples of growth inhibitory agents include agents that block the progress of the cell cycle (at a location other than the cell cycle). S phase), such as agents that induce the capture of Gl and the capture of the M phase. Classical M-phase blockers include vinca (vincristine and vinblastine), taxanes and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. Those agents that capture Gl are also spread over the capture of the S phase, for example, DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. Additional information can be found in The Molecular Basis of Cancer, endelsohn and Israel, eds. , Chapter 1, entitled "Regulation of the Cellar cycle, oncogenes and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. Examples of "growth inhibitory" antibodies are those that bind to HER2 and that inhibit the growth of cancer cells that hyperepress HER2. Preferred growth inhibitory HER2 antibodies inhibit the growth of breast tumor cells SK-BR-3 in cell cultures by more than 20%, preferably more than 50% (for example from about 50% to about 100%) in an antibody concentration of about 0.5 to 30 μg ml, where growth inhibition is determined six days after exposure of SK cells -BR-3 to the antibody (see U.S. Patent No. 5,677,171 issued October 14, 1997). The growth inhibition assay of SK-BR-3 cells is described in more detail in that patent and below. The preferred growth inhibitory antibody is a humanized variant of the murine monoclonal antibody 4D5, for example, Trastuzumab. An antibody that "induces apoptosis" is one that induces programmed cell death determined by the binding of annexin V, DNA fragmentation, cellular shrinkage, endoplasmic reticulum dilatation, cell growth and / or formation of membranous vesicles "called apoptotic bodies. The cell is usually a cell that expresses the antigen to which the antibody binds.Preferably, the cell is a tumor cell.For example, the translocation of serine phosphatidyl (PS) can be measured by annexin binding; DNA fragmentation can be assessed through DNA shift; and nuclear / chromatin condensation together with DNA fragmentation can be assessed by any increase in hypodiploid cells. Preferably, the antibody that induces apoptosis is an antibody that produces an induction in about 2 to 50 times, preferably about 5 to 50 times and more preferably about 10 to 50 times of binding of annexin relative to untreated cells in an assay of annexin binding using cells that express an antigen to which the antibody binds. Examples of antibodies that induce apoptosis are the HER2 7C2 and 7F3 antibodies and some DR5 antibodies. The "2C4 epitope" is the region in the extracellular domain of HER2 to which the 2C4 antibody binds. To detect antibodies that bind to the 2C4 epitope, a routine cross-block assay such as that described in ñntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David La (1988) can be performed. Alternatively, epitope mapping can be performed to verify if the antibody binds to the 2C4 epitope of HER2. The 2C4 epitope comprises residues of domain II in the extracellular domain of HER2. 2C4 and Pertuzumab bind to the extracellular domain of HER2 at the junction of domains I, II, and III. Franklin et al. Cancer Cell 5: 317-328 (2004). The "4D5 epitope" is the region in the extracellular domain of HER2 to which the antibody 4D5 (ATCC CRL 10463) and Trastuzumab binds. This epitope is close to the transmembrane domain of HER2 and within Domain IV of HER2. To screen for antibodies that bind to a 4D5 epitope, a routine cross-block assay can be performed as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, an epitope mapping can be performed to verify whether the antibody binds to the HER2 epitope 4D5 (eg, one or more residues in the region from about residue 529 to about residue 625, inclusive, of HER2). The "7C2 / 7F3 epitope" is the region in the amino terminus within Domain I of the extracellular domain of HER2 to which the 7C2 and / or 7F3 antibodies bind (each deposited in ATCC, see below). To detect antibodies that bind to the 7C2 / 7F3 epitope, a routine cross-block assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow, and David Lane (1988) can be performed. Alternatively, epitope mapping can be performed to establish whether the antibody binds to the epitope 7C2 / 7F3 in HER2 (for example one or more of the residues in the region from about residue 22 to about residue 53 of HER2). "Treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who already suffer from the disease as well as those in whom it is desired to avoid the disease. Therefore, the patient who must be treated here may have been diagnosed with a disease or may be predisposed or susceptible to the disease. The terms "cancer" and "cancerous" refer to or describe the physiological state of mammals that is typically characterized by irregular cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma and leukemia or lymphoid tumors. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer that includes small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer that includes gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, cancer liver, gallbladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, prostate cancer, vulval cancer, thyroid cancer, carcinoma hepatic, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, tumors of the biliary tract as well as cancer of the head and neck. The term "effective amount" refers to an amount of drug that is effective for a disease of the patient. When the disease is cancer, the effective amount of drug can reduce the amount of cancer cells; it can reduce the size of the tumor; inhibit (ie, slow down to a certain point and preferably stop) the infiltration of cancer cells into the peripheral organs; inhibit (ie, slow down to a certain point and preferably stop) tumor metastasis; inhibit, to some extent, the growth of the tumor; and / or alleviating to some extent one or more of the symptoms associated with cancer. To the extent that the drug can prevent the growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. The effective amount can extend the progression of free survival, result in an objective response (including a partial response, PR, or a complete response, CR), increase the overall survival time, and / or improve one or more symptoms of the Cancer.

A "cancer expressing HER2" is one that comprises cells that have a HER2 protein present on its cell surface. A cancer that "hyperexpresses" an HER receptor is one that has significantly higher levels of HER receptor, such as HER2, on the cell surface thereof, compared to a non-cancerous cell of the same type of tissue. Such hyperexpression can be caused by gene amplification or by an increase in transcription or translation. Overexpression of the HER receptor can be determined in a diagnostic or prognostic assay by evaluating the increased levels of HER protein present on the surface of a cell (e.g., through an immunohistochemistry assay; IHC). Alternatively, or additionally, the levels of nucleic acid encoding HER can be measured in the cell, for example through fluorescence in situ hybridization (FISH; see W098 / 45479 published October 1998), southern blot or by reaction techniques in polymerase chain (PCR), such as quantitative real-time PCR (RT-PCR). It is also possible to study hyperexpression of the HER receptor by measuring the spread antigen (e.g., HER extracellular domain) in a biological fluid such as serum (see, for example, U.S. Patent No. 4,933,294 issued June 12, 1990; / 05264 published April 18, 1991, U.S. Patent 5,401,638 issued March 28, 1995, and Sias et al., J. Immunol, Methods 132: 73-80 (1990)). Apart from the above assays, several in vivo assays are available for the person skilled in the art. For example, one can expose cells within the patient's body to an antibody that is optionally labeled with a detectable label, eg, a radioactive isotope, and the binding of the antibody to cells in the patient can be evaluated for example by external scanning to determine radioactivity or by analyzing a biopsy taken from the patient before exposing it to the antibody. Conversely, a cancer that "does not overexpress the HER2 receptor" is one that does not express higher levels than the normal HER2 receptor, compared to a non-cancerous cell of the same type of tissue. A cancer that "hyperexpresses" an HER ligand is one that produces significantly higher levels of that ligand, compared to a non-cancerous cell of the same type of tissue. Such hyperexpression can be caused by gene amplification or by an increase in transcription or translation. Overexpression of the HER ligand can be determined diagnostically by evaluating the levels of ligand (or nucleic acid encoding it) in the patient, for example, in a tumor biopsy or by various diagnostic assays such as IHC, FISH, southern blot. , PCR or in vivo assays such as those described above. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes the destruction of cells. The term includes radioactive isotopes (eg, At211, T I131, TI125, vY90,, DRe188, Sc ™ m153, oBi-; 212, dP32"and isotopes" radioactive), chemotherapeutic agents and toxins such as small molecule toxins or enzycally active toxins of bacterial, fungal, plant or animal origin including fragments and / or variants thereof. A "chemotherapeutic agent" is a chemical compound that is useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylene imines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; acetogenins (especially bulatacin and bulatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacona lapacol; Colchicines; betulinic acid; a camptothecin (including the synthetic analog topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9-aminocamptothecin); Bryostatin; Callistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutorrobina; pancratistatin; a sarcodictine; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novembichin, phenesterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, photomustine, lomustine, nimustine and ranimnustine; antibiotics such as enedin antibiotics (eg, calicheamicin, especially gammall calicheamicin and omegall calicheamicin (see, for example, Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)), dinemicin, including dynemycin A; a esperamycin, as well as a neocarzino-statin chromophore and chromoprotein antibiotic antibiotics enedin), aclacinomisins, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorrubicin, 6-diazo-5-oxo -L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, injection of doxorubicin HC1 liposome (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), doxorubin liposomal pegylated (CAELYX®) and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, iv ivomycins, peplomycin, potfiromycin, puromycin, chelamicin, rodrububicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, tiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocythabin, floxuridine; anti-adrenergic agents such as aminoglutethimide, mitotane, trilostane; folic acid fillers such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabuchil; bisantrene; edatraxate; defofamin; demecolcine; diaziquone; elfornitin; eliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainin; inoitic maitans such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofirano; spirogermanium; tenuazonic acid; triaziquone; 2, 2 ', 2"-trichlorotriethylamine, trichothecenes (especially T-2 toxin, verracurin ?, roridin A and anguidine), urethane, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacitosin, arabinoside (" Ara-C "); thiotepa, taxoid, for example, paclitaxel (TAXOL ©), paclitaxel formulation in nanoparticles manipulated with albumin (ABRAXANE ™) and docetaxel (TAXOTERE®), chloranbucil, 6-thioguanine, mercaptopurine, methotrexate, platinum agents such as cisplatin, oxaliplatin and carboplatin; vincas, which prevent the polymerization of microtubule-forming tubulin, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®) and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovovina; novantrone; edatrexate; Daunomycin; aminopterin; ibandronate; RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®) ) or risedronate (ACTONEL®); troxacitabine (a cytosine analog 1,3-dioxolane nucleoside); antisense oligonucleotides, particularly those that inhibit the expression of genes in the signaling pathways involved in the aberrant proliferation of cells such as, for example, PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; Topoisomerase 1 inhibitor (for example, LURTOTECAN®); rmRH (for example, ABARELIX®); BAY439006 (Sorafenib, Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib), proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; inhibitor of Bcl-2 such as oblimersen sodium (GENASENSE®); pixantrono; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; as well as combinations of two or more of the foregoing such as CHOP, an abbreviation for a combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN) in combination with 5-FU and leucovovina. Also included in this definition are anti-hormonal agents that act to regulate or inhibit the action of hormones on tumors such as anti-estrogens with mixed agonist / antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxy tamoxifen, toremifene (FARESTON®), idoxifen, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SER 3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents can block the dimerization of the estrogen receptor (ER), inhibit DNA binding, increase ER turnover, and / or suppress ER levels); aromatase inhibitors, including spheroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and non-spheroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors including vorozole ( RIVISOR®), megestrol acetate (MEGASE®), fadrozole, imidazole; Luteinizing hormone-releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sexual spheroids, which include progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens / retinoids such as f-luoximesterone, the total trans-retic acid and fenretinide; onapristone; anti-progesterone; descending estrogen receptor regulators (ERDs); anti-androgen such as flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; as well as combinations of two or more of the above. As used herein, the term "drug directed to EGFR "refers to a therapeutic agent that binds EGFR and, optionally, inhibits EGFR activation, Examples of such agents include antibodies and small molecules that bind EGFR Examples of antibodies to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Patent No. 4,943,533, Mendelsohn et al.) And variants thereof, such as 225 chimerized (C225 or Cetuximab; ERBUTIX®) and reconfigured human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); antibodies that bind to mutant type II GFR (US Patent No. 5.212) .290), humanized and chimeric antibodies that bind to EGFR as described in US Patent No. 5,891,996, and human antibodies that bind to EGFR, such as ABX-EGF (see WO98 / 50433, Abgenix). The anti-EGFR antibody can be conjugated with a cytotoxic agent, thus generating an immunoconjugant (see, for example, EP659,439A2, Merck Patent GmbH). Examples of small molecules that bind to EGFR include ZD1839 or Gefitinib (IRESSA ™, Astra Zeneca), CP-358774 or Erlotinib HCL (TARCEVA ™, Genentech / OSI) and AG1478, AG1571 (SU 5271; Sugen).

A "tyrosine kinase inhibitor" is a molecule that inhibits to some extent the activity of tyrosine kinases in a tyrosine kinase such as an HER receptor. Examples of such inhibitors include the EGFR-targeted drugs indicated in the preceding paragraph as well as the small molecule HER2 tyrosine kinase inhibitor such as TAK165 obtainable in Takeda, dual HER inhibitors such as EKB-569 (obtainable in Wyeth) which bind preferably to EGFR but which inhibit both cells that overexpress HER2 & EGFR, GW572016 (obtained in Glaxo) an inhibitor of oral tyrosine kinase HER2 and EGFR, and PKI-166 (obtainable in Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as the antisense agent ISIS-5132 obtainable at ISIS Pharmaceuticals which inhibits the Raf-1 signal; TK inhibitors that do not target HER such as Imatinib mesylate (Gleevac ™) obtainable in Glaxo; CIK 1040 inhibitor of extracellular regulation MAPK kinase (obtainable from Pharmacia); quinazolines, such as PD 153035, 4- (3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2,3-d] pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4-fluoranilino) phthalimide); Tyrphostins containing nitrothiophene portions; PD-0183805 (Warner-Lamber); antisense molecules (for example, those that bind to a nucleic acid encoding HER); Quinoxalines (U.S. Patent No. 5,804,396); trifostins (U.S. Patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly); Imatinib mesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone); or as described in any of the following patent publications: U.S. Patent No. 5,804,396; WO99 / 09016 (American Cyanimid); WO98 / 43960 (American Cyanamid); W097 / 38983 (Warner Lambert); WO99 / 06378 (Warner Lambert); WO99 / 06396 (Warner Lambert); WO96 / 30347 (Pfizer, Inc); W096 / 33978 (Zeneca); W096 / 3397 (Zeneca); and WO96 / 33980 (Zeneca). An "anti-angiogenic agent" refers to a compound that blocks or interferes to a certain extent with the development of blood vessels. The anti-angiogenic factor can be, for example, a small molecule or an antibody that binds to a growth factor or a growth factor receptor involved in the promotion of angiogenesis. The preferred anti-angiogenic factor here is an antibody that binds to Vascular Endothelial Growth Factor (VEGF), such as Bevacizumab (AVASTIN®). The term "cytokine" is a generic term for proteins released by a population of cells that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monoquinas, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor, fibroblast growth factor, prolactin; placental lactogen; tumor necrosis factor-a and -ß; Muleriana inhibitory substance; peptide associated with mouse gonadotropin; inhibin; activin; Vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -β, and - ?; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 , IL-11, IL-12; a tumor necrosis factor such as TNF-a or TNF-β; and other polypeptide factors including LIF and an equipment ligand (KL). As used herein, the term "cytokine" includes proteins from natural sources or from recombinant cell cultures and biologically active equivalents of the naturally occurring cytokines. The antibody that is formulated is preferably essentially pure and convenient essentially homogeneous (ie, it is free of contaminating proteins etc.). "Essentially pure" antibody means a composition comprising at least about 90% by weight of the antibody based on the total weight of the composition, preferably at least about 95% by weight. "Essentially homogeneous" antibody means a composition comprising at least about 99% by weight of antibodies based on the total weight of the composition. A "B cell surface marker" or a "B cell surface antigen" is here an antigen expressed on the surface of a B cell that can be the target of an antibody that binds to it. Examples of markers of B cell surface include the surface markers of leukocyte CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a , CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 (for descriptions, see the Leukocyte Antigen Facts Book, 2nd Edition 1997, ed Barclay et al Academic Press, Harcourt Brace &... Co., New York). Other markers of B cell surface include RP105, FcRH2, B-cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, BTIG, NAG14, SLGC16270, FcRHl, IRTA2, AT D578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. The B cell surface marker of particular interest herein is preferably expressed in B cells compared to other tissues of non-B cells of a mammal and can be expressed in both B precursor cells and cells. mature B. The preferred B cell surface marker here is CD20 or BR3. The "CD20" antigen, or "CD20," is a non-glycosylated phosphoprotein of approximately 35-kDa, which is found on the surface of more than 90% of B cells from limfoid organs or peripheral blood. CD20 is present in both normal B cells and malignant B cells, but is not expressed in the stem cells. Other names for CD20 in the literature include "antigen restricted to B lymphocytes" and "Bp35". The CD20 antigen has been described for example in, Clark et al. Proc. Nati Acad. Sci. (USA) 82: 1766 (1985). Simply for purposes herein, "humanized 2H7" refers to a humanized variant of the 2H7 antibody, which CDR sequences have been described in U.S. Patent No. 5,500,362 the (Figs. 5 and 6), which is expressly incorporated herein by reference . Examples of these antibodies 2H7 humanized include the variants described in WO2004 / 056312, which are also incorporated herein expressly by reference, as well as other variants, which include, but are not limited to: 2H7vl6, 2H7v31, 2H7v73, 2H7v75, 2H7v96 , 2H7vll4, 2H7vll5, 2H7vll6, 2H7vl38, 2H7v477, 2H7v375, etc. In one embodiment, the humanized 2H7 antibody comprises one, two, three, four, or six belt of the following CDR sequences: CDR Ll sequence RASSSVSYXH wherein X is of M or L (SEQ ID No. 67), for example SEQ ID No 57 (Fig. 18A), CDR sequence L2 of SEQ ID No. 58 (Fig. 18A), CDR sequence L3 of QQ XFNPPT where X is S or A (SEQ ID No. 68), for example SEQ ID No. 59 (Fig. 18A), CDR sequence Hl of SEQ ID No. 60 (Fig. 18B), sequence CDR H2 of AI YPGNGXTSYNQKFKG where X is D or A (SEQ ID No. 69), for example SEQ ID No. 61 (Fig. 18B), and CDR sequence H3 of VVYYSXXYWYFDV where X at position 6 is N, A, Y , W or D, and the X in position 7 is S or R (SEQ ID No. 70), for example SEQ ID No. 62 (Fig. 18B). The above CDR sequences are generally present within the human light variable and heavy variable sequences, such as substantially the FR residues of human consensus of the light chain kappa I subgroup (VL6l), and substantially the human consensus FR residues of the subgroup III of human heavy chain (VHIII). See also WO 2004/056312 (Lowman et al.). The variable heavy region can be linked to a human IgG chain constant region, where the region can be, for example, IgG1 or IgG3, including the variant constant and natural sequence regions. In a preferred embodiment, said antibody comprises the variable heavy domain sequence of SEQ ID No. 29 (vl6, is as shown in FIG. 18B), optionally the variable light domain sequence of SEQ ID No. 26 (vl6, as shown in Fig. 18A), optionally comprising one or more amino acid substitutions at positions 56, 100, and / or 100a, for example D56A, N100A or N100Y, and / or SlOOaR in the domain variable chain and one or more amino acid substitutions at positions 32 and / or 92, for example M32L and / or S92A, in the light variable domain. Preferably, the antibody is an intact antibody comprising the light chain amino acid sequences of SEQ ID Nos. 63 or 64, and the heavy chain amino acid sequences of SEQ ID No. 65, 66, 71 or 72. A Humanized 2H7 antibody preferred is ocrelizumab (Genentech). The present antibody may further comprise at least one amino acid substitution in the Fe region that enhances ADCC activity, such as one in which the amino acid substitutions are at positions 298, 333, and 334, preferably S298A, E333A, and K334A, using Eu numbering of heavy chain residues. See also U.S. Patent No. 6,737,056B1, Presta. Any of the antibodies can comprise at least one substitution in the Fe region that enhances the binding of FcRn or the half-life in the serum, for example a substitution at position 434 of heavy chain, such as N434W. See also U.S. Patent No. 6,737,056B1, Presta.

Any of these antibodies may additionally comprise at least one amino acid substitution in the Fe region that increases CDC activity, for example, comprising at least one substitution at position 326, preferably K326A or K326W. See also U.S. Patent No. 6,528,624B1 (Idusogie et al.). Some preferred humanized 2H7 variants are those comprising the variable light domain of SEQ ID No. 26 and the variable heavy domain of SEQ ID No. 29, including those with or without substitutions in an Fe region (if present), and those that comprise a variable heavy domain with alteration N100A; or D56A and N100A; or D56A, N100Y, and SlOOaR; in SEQ ID No. 29 and a variable light domain with 32L alteration; or S92A; or M32L and S92A; in SEQ ID No. 26. The 34 in the variable heavy chain of 2H7vl6 has been identified as a potential source of antibody stability and is another potential candidate for substitution. Summarizing in a summary of several preferred embodiments of the invention, the variable region of the variants based on 2H7vl6 comprises the amino acid sequence of vl6 except at the positions of amino acid substitutions that are indicated in the following Table. Unless otherwise indicated, the 2H7 variants will have the same light chain as that of vl6.

Examples of Humanized 2H7 Variants and Antibodies A preferred humanized 2H7 comprises variable variable light domain sequence 2H7vl6: DIQMTQSPSSLSASVGDRVTITCRASSSVSY HWYQQKPGKAPKPLI YAPSNLASGVPSRF SGSGSGTDFTLTI SSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID No. 26); and the variable heavy domain sequence 2H7vl6: EVQLVESGGGLVQPGGSLRLSCAASGYT TSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYN QKFKGRFTI SVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS (SEQ ID No. 29). When the humanized 2H7vl6 antibody is an intact antibody, it may comprise the amino acid sequence of light chain: DIQMTQSPSSLSASVGDRVTITCRASSSVSY HWYQQKPGKAPKPLIYAPSNLASGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQ SFNPPTFGQGTKVEIKRTVAAPSVFI FPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID No. 63); And the heavy chain amino acid sequence of SEQ ID No. 65 or: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYN QKFKGRFTI SVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGT LVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY STY RVVSVLTVLHQD LNGKEYKCKVSNKALPAPIEK I SKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID No. 71). Another preferred humanized 2H7 antibody comprises the variable light domain sequence 2H7v511: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKR (SEQ ID No. 73) and variable heavy domain sequence 2H7v511: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYN QKFKGRFTI SVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSS (SEQ ID No. 74). When the humanized 2H7v511 antibody is an intact antibody, it may comprise the amino acid sequence of light chain: DIQMTQS PSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFI FPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID No. 64) and amino acid sequence of heavy chain of SEQ ID No. 66 or : EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYN QKFKGRFTI SVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRY YFDVWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATY RVVSVLTVLHQDWLNGKEYKCKVS AALPAPIAATI SKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID No. 72). A "malignant B cell tumor" includes here a non-Hodkin's lymphoma (NHL), including follicular / low quality NHL, small lymphocytic NHL (SL) NHL, follicular / intermediate quality NHL, intermediate quality diffuse NHL, high quality immunoblastic NHL, high quality lymphoblastic NHL, low quality non-dissociated NHL cells , NHL of bulky disease, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia; leukemia, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia; and other malignant hematological tumors. Such malignancies can be treated with antibodies directed against B-cell surface markers, such as CD20. The term "non-Hodquinian lymphoma" or "NHL", as used herein, refers to a cancer of the lymphatic system other than Hodgkin lymphomas. Hodgkin lymphomas can generally be distinguished from non-Hodgkin lymphomas by the presence of Reed-Sternberg cells in Hodgkin's lymphomas and the absence of such cells in non-Hodkin's lymphomas. Examples of non-Hodkin's lymphomas encompassed by the term used herein include any that could be identified as such by a person skilled in the art (eg, an oncologist or a pathologist) according to the classification schemes known in the art, such as as the Revised European-American Limfoma (REAL) scheme in Color Atlas of Clinical Hematology, Third Edition; A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Limited 2000) (see, in particular, Fig. 11.57, 11.58 and / or 11.59). More specific examples include, but are not limited to, refractory or relapsing NHL, low frontal line quality NHL, stage III / IV NHL, chemotherapy resistant NHL, precursor B lymphoblastic leukemia and / or lymphoma, lymphoma of small lymphocytes, chronic B-cell lymphatic leukemia and / or prolymphocytic leukemia and / or small lymphocyte lymphoma, proliffocytic B cell lymphoma, immunocytoma and / or lymphocytic lymphoma, marginal zone B-cell lymphoma, marginal zone lymphoma splenic, MALT lymphoma of marginal extranodal zone, nodal marginal zone lymphoma, hairy cell leukemia, plasma cell plasmacytoma and / or myeloma, follicular / low quality lymphoma, follicular NHL / intermediate quality, mantle cell lymphoma, lymphoma of follicular center (follicular), diffuse NHL of intermediate quality, diffuse large B-cell lymphoma, aggressive NHL (including aggressive frontal NHL and relapsed NHL agr esivo), NHL relapsing after or refractory to transplantation of autologous strain cells, mediastinal primary large B-cell lymphoma, primary fusion lymphoma, high-quality immunoblastic NHL, high-quality lymphoblastic NHL, high-quality small non-dissociated NHL , NHL of bilirubin disease, Burkitt's lymphoma, lymphoblastic leukemia / lymphoblastic lymphoblastic T-cell precursor (peripheral), lymphoma and / or T-cell leukemia, chronic T-cell lymphocytic leukemia and / or prolymphatic leukemia, large granular lymphocytic leukemia, fungoides of mycosis and / or Sezary syndrome, T-cell lymphoma / extranodal natural killers (nasal type), enteropathic T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis of T-cell lymphoma, lymphoma of the skin (cutaneous), anaplastic large cell lymphoma, angiocentric lymphoma, intestinal T cell lymphoma, peripheral T cell lymphoma rich (not otherwise specified) and antimoimmunoblastic T-cell lymphoma. An "autoimmune disease" as referred to herein is a disease or disorder that arises from and is directed against an individual's own tissues or a cosegregation or manifestation thereof or the resulting disorders thereof. Examples of autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis, juvenile onset rheumatoid arthritis, osteoarthritis, soreatic arthritis and ankylosing spondylitis), psoriasis, dermatitis, including atopic dermatitis, chronic idiopathic urticaria, including chronic autoimmune urticaria , polymyositis / dermatomyositis, toxic epidermal necrolysis, scleroderma (which includes systemic scleroderma), sclerosis such as progressive systemic sclerosisinflammatory bowel disease (IBD), (for example, Crohn's disease, ulcerative colitis, inflammatory bowel autoimmune disease), pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis), dyslexic syndrome, including acute dyslexic syndrome in adults (ARDS) ), meningitis, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen encephalitis, uveitis or autoimmune uveitis, colitis such as microscopic colitis and collagenous colitis, glomerulonephritis (GN) such as membranous GN (membranous nephropathy) , Idiopathic membranous GN, proliferative membranous GN (MPGN), which includes Type I and Type II, and rapidly progressive GN, allergic disorders, allergic reactions, eczema, asthma, disorders that involve T cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, adhesion deficiency of leukosites Systemic lupus erythematosus (SLE) such as cutaneous SLE, subacute cutaneous lupus erythematosus, lupus (including nephritis, cerebritis, pediatric, non-renal, discoid, alopecia), juvenile initiation diabetes mellitus (Type I) that includes pediatric dependent diabetes mellitus of insulin (IDDM), adult-onset diabetes mellitus (Type II diabetes), multiple sclerosis (MS) such as spino-optic MS, immune responses associated with delayed and acute hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis (including vasculitis of large blood vessels (including polymyalgia rheumatica and giant cell arteritis (Takayasu)), middle vessel vasculitis (including Kawasaki disease and polyarthritis nodosa), CNS vasculitis, systemic necrotizing vasculitis and vasculitis associated with ANCA such as Churg-Strauss vasculitis or syndrome (CSS)), temporal arteritis, aplastic anemia, positive Coombs anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), deficiency of Factor VIII, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocytic diapedesis, inflammatory CNS disorders, multiple organ lesion syndrome, diseases mediated by the antigen-antibody complex, glomerular-basement membrane disease, anti-phospholipid antibody, allergic neuritis, Bechet or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigus such as vesicular pemphigoid, pemphigus (which includes vulgaris, foliaceus and mucous membrane pemphigoid), autoimmune polyendocrinopathies, disease of Reiter, immune complex nephritis, chronic neuropathy such as polyneuropathies of IgM or neuropathy mediated by IgM, thrombocytopenia (developed for example by patients with myocardial infarction), including thrombotic thrombocytopenia purpura (TTP) and autoimmune thrombocytopenia or mediated by immunity such such as idiopathic thrombocytopenic purpura (ITP) that includes chronic or acute ITP, autoimmune disease of the testes and ovaries that includes autoimmune orchitis and oophortism, primary hypothyroidism, hypoparathyroidism, endocrine autoimmune diseases that include thyroiditis such as autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis) ), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Addison's disease, Grave, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinophatic syndromes), paraneo syndromes plastics, which include syndromes for neurological paraneoplastic such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, rigid man or rigid person, encephalomyelitis such as allergic encephalomyelitis, severe myiastemia, cerebellar degeneration, limbic encephalitis and / or brain stem, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (WHO), and sensory neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronqueolitis obliterans (non-transplant) vs NSIP, Guillain-Barré syndrome, Berger's disease (IgA efropathy), primary biliary cirrhosis, celiac stomatitis (gluten enteropathy), refractory stomatitis, dermatitis herpetiformis, cryoglobulinemia, amilotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune inner ear disease (AIED) T- or autoimmune hearing loss, opsoclonus myoclonus syndrome (WHO), polychondritis such as refractory polychondritis, pulmonary alveolar proteinosis, amyloidosis, hepatitis giant cells, scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis that includes monoclonal B-cell lymphocytosis (for example benign monoclonal gram-monophagus and monoclonal garnmopathy of undetermined significance MGUS), peripheral neuropathy, paraneoplastic syndrome, canelopathies such as epilepsy, migranea, arrhythmia , muscle disorders, deafness, blindness, periodic paralysis, and CNS carcinoid diseases, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine insufficiency, Schmidt adrenalitis syndrome, gastric atrophy, pre-senile dementia, demyelinating diseases, Dressler syndrome, arcata alopecia, CREST syndrome (calcinosis), Raynaud's phenomenon, esophageal dysmotility, sclerodactilism and telangiectasia ), male and female autoimmune infertility, ankylosing spondylitis, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, poultry disease, pneumonia syndrome of Alport, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter syndrome, Caplan syndrome, dengue, endocarditis, endomyocardial fibrosis , endof talmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman syndrome, Felty syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronous cyclitis or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection , ecovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan syndrome, autoimmune gonadal deficiency, Sydenham chorea, post-streptoal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, and giant cell polymyalgia. "Tumor necrosis factor receptor superfamily" or "TNF receptor superfamily" refers here to receptor polypeptides joined by cytokines in the TNF family. In general, these receptors are Type I transmembrane receptors with one or more repeat sequences rich in cysteine in their extracellular domain. The TNF receptor superfamily can be further divided into (1) deadly receptors; (2) decoy receivers; and (3) signaling receptors that lack deadly domains. "Deadly receptors" contain a cytoplasmic or intercellular region A "deadly domain" refers to a region or sequence that acts to transduce signals in the cell that can result in apoptosis or induction of certain genes. "Decoy receptors" lack a functional death domain and are unable to transduce signals that result in apoptosis. Examples of cytokines in the TNF gene family include Tumor Necrosis Alpha Factor (TNF-alpha), Necrosis Beta Factor (TNF-beta or lymphoxin), CD30 ligands, CD27 ligands, CD40 ligands, OX-40 ligand, 4-1BB ligand. , Apo-1 ligand (also referred to as Fas ligand or CD95 ligand), Apo-2 ligand (also referred to as TRAIL), Apo-3 ligand (also referred to as TWEAK), osteoprotegerin (OPG), APRIL, RANK ligand (also referred to as TRANCE), and TALL-1 (also called BlyS, BAFF or THANK). Examples of receptors in the TNF receptor superfamily include: Type 1 Tumor Necrosis Factor receptor (TNFR1), Type 2 Tumor Necrosis Factor receptor (TNFR2), P75 nerve growth factor receptor (NGFR), surface antigen CD40 B cell, OX-40 T cell antigen, Apo-1 receptor (also called Fas or CD95), Apo-3 receptor (also called DR3, swl-1, TRAMP and LARD), the receptor called "Transmembrane Activator" Interactor CAML "or" TACI ", protein BCMA, DR, DR5 (alternatively called Apo-2, TRAIL-R2, TR6, Tango-63, hAP08, TRICK2 or KILLER), DR6, DcRl (also called TRID, LIT or TRAIL- R3), DcR2 (also referred to as TRAIL-R4 or TRUNDD), OPG, DcR3 (also referred to as TR6 or M68), CARI, HVEM (also referred to as ATAR or TR2), GITR, ZTNFR-5, NTR-1, TNFL1, CD30, beta receptor of lymphotoxin (LTBr), receptor 4-1BB and TR9 (EP988, 371A1). The terms "ligand Apo-2", "Apo-2L", "Apo2L", Apo-2 ligand / TRAIL "and" TRAIL "are used here interchangeably to refer to a polypeptide sequence that includes amino acid residues 114-281 inclusive, -281 inclusive, residues 92-281 inclusive, residues 91-281 inclusive, residues 41-281 inclusive, residues 39-281 inclusive, residues 15-281 inclusive, or residues 1-281 inclusive of the amino acid sequence shown in Fig. 24 (SEQ ID No. 46), as well as biologically active fragments, deletional, insertional, and / or substitutional variants of the above sequences In one embodiment, the polypeptide sequence comprises residues 114-281 of Fig. 24 (SEQ ID No. 46) Optionally, the polypeptide sequence comprises residues 92-281 or residues 91-281 of Fig. 24 (SEQ ID No. 46) Apo-2L polypeptides can be encoded by the sequence natural nucleotide shown in Fig. 24 (SEQ ID No. 45) Optionally, the codon encoding the Proll9 residue (Fig. 24; SEQ ID No. 45) can be "CCT" or "CCG". Optionally the fragments or variants are biologically active and have at least about 80% amino acid sequence identity, or at least about 90% sequence identity, or at least 95%, 96%, 97%, 98% , or 99% sequence identity with any of the above sequences. The definition encompasses the substitutional variants of the Apo-2 ligand in which at least one of its natural bonds are substituted with other amino acids such as an alanine residue. The definition also encompasses a natural sequence Apo-2 ligand isolated from a source of Apo-2 ligand or prepared by recombinant and / or synthetic methods. The Apo-2 ligand of the invention includes the polypeptides defined as Apo-2 or TRAIL ligand described in WO97 / 01633 published on January 16, 1997, W097 / 25428 published July 17, 1997, 099/36535 published on 22 of 1999, WO 01/00832 published January 4, 2001, WO02 / 09755 published February 7, 2002, WO 00/75191 published December 14, 2000, and US Patent No. 6,030,945 issued February 29, 2000. The terms are used to refer generally to Apo-2 ligand forms that include monomer, dimer, trimer, hexamer or higher oligomer polypeptide forms. . All numbering of the amino acid residues for the Apo-2L sequence use the numbering according to Fig. 24 (SEQ ID No. 46), unless specifically stated otherwise. "Receptor ligand Apo-2" includes the receptors mentioned in the art as "DR4" and "DR5." Pan et al. have described the member of the TNF receptor family designated "DR4" (Pan et al., Science, 276: 111-113 (1997)); see also W098 / 32856 published July 30, 1998; WO 99/37684 published July 29, 1999; WO 00/73349 published December 7, 2000; U.S. Patent 6,433,147 granted August 13, 2002; US Patent 6,461,823 issued October 8, 2002, and US Patent 6,342,383 issued January 29, 2002).

Sheridan et al., Science, 277: 818-821 (1997) and Pan et al., Science, 277: 815-818 (1997) described another receptor for Apo2L / TRAIL (see also, 098/51793 published on November 19). 1998, W098 / 41629 published September 24, 1998). This receptor is designated DR5 (the receptor has also been alternatively termed Apo-2; TRAIL-R, TR6, Tango-63, hAP08, TRICK2 or KILLER; Screaton et al., Curr. Biol., 7: 693-696 (1997 ), Walczak et al., EMBO J., 16: 5386-5387 (1997), Wu et al., Nature Genetics, 17: 141-143 (1997), W098 / 35986 published August 20, 1998; EP870, 827 published October 14, 1998; W098 / 46643 published October 22, 1998; WO99 / 02653 published January 21, 1999; O99 / 09165 published on 25, 1999; W099 / 11791 published on November 11, 1999; North American 2002/0072091 published August 13, 2002; North American Patent 2002/0098550 published December 7, 2001; North American Patent 06,313,269 granted December 6, 2001; North American Patent 2001/0010924 published August 2, 2001; North American Patent 2003/01255540 published July 3, 2003; North American Patent 2002/0160446 published October 31, 2002, US Patent 2002 / 0048785 published on April 25, 2002; US Patent 6,569,642 issued May 27, 2003, US Patent 6,072,047 issued June 6, 2000, US Patent 6,642,358 issued November 4, 2003). As described above, other receptors for Apo-2L include DcRl, DcR2, and OPG. The term "Apo-2L Receptor" when used herein encompasses the receptor and the natural sequence receptor variants. These terms encompass the Apo-2L receptor expressed in a variety of mammals, including humans. The Apo-2L receptor can be endogenously expressed and occurs in a natural form in a variety of human tissue lines, or it can be expressed by recombinant or synthetic methods. A "Natural sequence Apo-2L receptor" comprises a polypeptide having the same amino acid sequence as an Apo-2L receptor derived from nature. Therefore, the natural sequence Apo-2L receptor can have the natural amino acid sequence of the native Apo-2L receptor of any mammal, including humans. Said natural sequence Apo-2L receptor can be isolated from nature or it can be produced by recombinant or synthetic means. The term "natural sequence Apo-2L receptor" specifically encompasses truncated or secreted natural forms of the receptor (eg, a soluble form containing, for example, an extracellular domain sequence), natural variant forms (eg, alternatively spliced forms) and allelic variants that occur naturally. The receptor variants may include deletion fragments or mutants of the Apo-2L receptor. The figures. 25A-C show the 411 amino acid sequence of the DR5 receptor, together with its nucleotide sequence (SEQ ID Nos. 47 and 48) published in WO 98/51793 on November 19, 1998. A variant of transcriptional splice of the DR5 receptor Human is known in art. This splicing variant encodes the 440 amino acid sequence of the human DR5 receptor shown in Figures 26A-C, along with its nucleotide sequence (SEQ ID Nos. 49 and 50), and published in WO 98/35986 on 20 1998. "Deadly receptor antibody" as used herein to refer generally to an antibody or antibodies directed to a receptor in the tumor necrosis factor receptor superfamily and containing a lethal domain capable of signal apoptosis, and said antibodies include the DR5 antibody and the DR4 antibody. "DR5 receptor antibody", "DR5 antibody", or "anti-DR5 antibody" is used in a broad sense to refer to antibodies that bind to at least one form of a DR5 receptor or an extracellular domain thereof. Optionally the DR5 antibody is fused or ligated with a consequence or heterologous molecule. Preferably the heterologous sequence allows or aids the antibody to form a higher order or oligomeric complexes. Optionally the DR5 antibody binds to the DR5 receptor but does not bind or cross-react with any additional Apo-2L receptor (eg DR4), DcRl, or DcR2). Optionally the antibody is an agonist of the DR5 signal activity. Optionally, the DR5 antibody of the invention binds to a DR5 receptor in a concentration range of about 0.1 nM to about 20 mM measured in a BIAnucleus binding assay. Optionally, the DR5 antibodies of the invention exhibit an IC50 value of about 0.6 nM to about 18 mM measured in a BIAnucleus binding assay. Simply for the present purpose, the term "Apomab" refers to an agonist antibody which binds to DR5 and which comprises the variable heavy and light variable amino acid sequences of SEQ ID NOS. 55 and 56. Preferably, Apomab comprises the light and heavy chains of SECs. ID Nros. 51 and 52, respectively.

II. Production of Antibodies The following are techniques for producing antibodies that can be formulated in accordance with the present invention. (i) Selection and preparation of antigens Preferably, the antigen to which the antibody binds is a biologically important glycoprotein and administration of antibody to a mammal suffering from a disease or disorder can provide a therapeutic benefit to the mammal. However, antibodies directed against non-polypeptide antigens (such as glycolipid antigens associated with tumors; see US Pat. No. 5,091,178) are also contemplated. When the antigen is a polypeptide, it can be a transmembrane molecule (for example a receptor) or a ligand such as a growth factor. Examples of antigens include molecules such as renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone release factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-l-antitrypsin; insulin A chain; B chain of insulin; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor (TF), and Willebrands factor; anti-coagulant factors such as Protein C; atrial natriuretic factor; pulmonary surfactant; a plasminogen activator, such as urokinase or a tissue-type plasminogen activator or human urine (t-PA); bombesin; thrombin; hemopoietic growth factor; alpha and beta factor of tumor necrosis; enkephalinase; RANTES (regulated by activation normally of expressed and secreted T cells); inflammatory protein of human macrophages (?? - 1-alpha); a serum albumin such as human serum albumin; inhibitory substance of Muellerian; A chain of relaxin; relaxin chain; prorelaxin; peptide associated with mouse gonadotropin; a microbial protein such as beta-lactamase; DNase; IgE; an antigen associated with cytotoxic T lymphocyte (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor such as a neurotrophic factor derived from bone (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or factor of nerve growth such as NGF-b; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-bl, TGF-b2, TGF-b3, TGF-b4, or TGF-b5; a tumor necrosis factor (TNF) such as TNF-alpha or TNF-beta; insulin-like growth factor-I and -II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor-binding proteins; Insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22 and CD40; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), eg, M-CSF, GM-CSF, and G-CSF; interleukins (ILs), for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 and IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; old age accelerator factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; messenger receivers; adresinas; regulatory proteins; integrins such as CDlla, CDllb, CDllc, CD18, ICAM, VLA-4 and VCAM; a tumor-associated antigen such as the HER2, HER3 or HER4 receptor; and fragments of any of the polypeptides previously listed. Examples of molecular targets for antibodies encompassed by the present invention include CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22, CD34 and CD40; members of the ErbB receptor family such as the EGF receptor, the HER2 receptor, HER3 or HER4; B cell surface antigens, such as CD20 or BR3; a member of the tumor necrosis receptor superfamily, which includes DR5; antigen of prostate cell (PSCA) cells; cell adhesion molecules such as LFA-1, Macl, pl50.95, VLA-4, ICAM-1, VCAM, alpha4 / beta7 integrin; and including the integrin, and alphav / beta3 which includes the alpha or beta subunits thereof (eg, anti-CDlla, anti-CD18 or anti-CDllb antibodies); growth factors such as VEGF as well as the receptors therefor; tissue factor (TF); a tumor necrosis factor (TNF) such as TNF-alpha or TNF-beta, alpha interferon (alpha-IFN); an interleukin, such as IL-8; IgE; antigens of the blood group; flk2 / flt3 receiver; Obesity receptor (OB) receiver mpl; CTLA-4; C protein etc. Soluble antigens or fragments thereof, optionally conjugated with other molecules, can be used as immunogens to generate antibodies. For transmembrane molecules such as receptors, fragments of these (for example, the extracellular domain of a receptor) can be used as an immunogen. Alternatively, cells expressing the transmembrane molecule can be used as an immunogen. Said cells can be derived from a natural source (for example cancer cell lines) or can be cells that have been transformed by recombinant techniques to express the transmembrane molecule. Other antigens and forms thereof useful for preparing antibodies will be apparent to those skilled in the art. For the production of HER2 antibodies, the HER2 antigen that will be used for the production thereof may for example be a soluble form of the extracellular domain of HER2 or a portion thereof, which contains the desired epitope. Alternatively, cells expressing HER2 on its cell surface (eg, NIH-3T3 cells transformed to hyperepress HER2; or a carcinoma cell line such as SK-BR-3 cells, see Stancovski et al., PNAS (USA) 88 : 8691-8695 (1991)) can be used to generate antibodies. (ii) Monoclonal Antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that may arise during production of the antibody. monoclonal antibody. Therefore, the "monoclonal" modifier indicates the character of the antibody that is not a mixture of discrete antibodies. For example, monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or they can be prepared by recombinant DNA methods (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to obtain lymphocytes that produce or are capable of producing antibodies that will bind specifically to the protein used for immunization. Alternatively, the lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using an appropriate fusing agent, such as polyethylene glycol, to form a hybridoma cell 1 (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Hybridoma cells prepared in this manner are seeded and cultured in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused, parental myeloma cells. For example, if the parental myeloma cells lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the culture medium for the hybridoma will typically include hypoxanthine, aminopter ina, and thymidine (HAT medium), which are substances that prevent the growth of cells deficient in HGPRT. Preferred myeloma cells are those that are efficiently fused, that support the production of high stable level of antibody by the selected antibody producing cells, and are sensitive to a medium such as the HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from mouse tumors MOPC-21 and MPC-11 obtainable at the Cell Distribution Center of the Salk Institute, San Diego, California USA, and SP-2 or X63-Ag8-653 cells obtainable from American Type Culture Collection, Rockville, Maryland USA. Human-mouse and human myeloma heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984)).; and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The culture medium in which the hybridoma cells are grown was tested for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells was determined by immunoprecipitation by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can be determined for example by the Scatchard analysis of Munson et al., Anal. Biochem. , 107: 220 (1980). After identifying the hybridoma cells that produce antibodies of the desired specificity and / or activity, the clones can be subcloned by limiting dilution procedures and culturing by conventional methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM medium or RPMI-1640. In addition, the hybridoma cells can be cultured in vivo as ascitic tumors in an animal. The monoclonal antibodies secreted by the subclones are conveniently separated from the culture medium, ascitic fluid or serum by conventional antibody purification methods, such as, for example, protein A-Sepharose, by hydroxylapatite chromatography by gel electrophoresis, dialysis, or affinity chromatography. The DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional methods (for example using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as the preferred source of said DNA. Once isolated, the DNA can be placed within expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster's ovary (CHO) cells, or myeloma cells that on the other hand they do not produce antibody proteins, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in DNA bacteria encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Plückthun, Immunol. Revs. , 130: 151-188 (1992). In another embodiment, monoclonal antibodies or antibody fragments can be isolated from collections of phage antibodies using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage collections. Subsequent publications describe the production of high affinity human antibodies (nM range) by chain transfer (Marks et al., Bio / Technology, 10: 779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategy to build very large phage collections (Waterhouse et al., Nuc Acids, Res., 21: 2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies. The DNA can be modified, also for example, by replacing the coding sequence with the heavy chain and light chain constant domains instead of the homologous murine sequences (U.S. Patent No. 4,816,567; and Morrison, et al., Proc. Nati Acad. Sci. USA, 81: 6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Typically said non-immunoglobulin polypeptides are substituted with the constant domains of an antibodyor are substituted with the variable domains of an antigen combiner site of an antibody to create a chimeric divalent antibody comprising an antigen combining site having specificity for an antigen and another antigen combining site having specificity for an antigen different. (iii) Humanized Antibodies Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "imported" residues, which are typically taken from an "imported" variable domain. Humanization can be effected essentially following the method of Winter et al. (Jones et al., Nature, 321: 522-525 (1986)).; Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), substituting the hypervariable region sequences with the corresponding sequences of a human antibody. Accordingly, said "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted with residues from analogous sites in rodent antibodies. The choice of human variable domains, both light and heavy, to be used in the preparation of humanized antibodies is very important to reduce antigenicity. According to what is called the "best fit" method, the variable domain sequence of a rodent antibody is screened against a whole collection of known human variable domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human structural region (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a particular structural region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same structure can be used for different humanized antibodies (Cárter et al., Proc. Nati. Acad.

Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)). It is also important that the antibodies are humanized with high affinity retention for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a method of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the humanized parental sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and exhibit probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. The inspection of these exhibits allows the analysis of the probable role of residues in the functioning of the candidate immunoglobulin sequence, that is, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this manner, FR residues can be selected which can be combined from the receptor and import sequences in order to achieve the desired antibody characteristic, such as an affinity increase for the target antigens. In general, hypervariable region residues are directly and more substantially involved in the influence of antigen binding. WO01 / 00245 describes the production of examples of humanized HER2 antibodies that bind to HER2 and block the activation of the ligand of a HER receptor. The humanized antibody of particular interest mentioned here blocks the activation of APK intermediated by EGF, TGF-a and / or HRG essentially as efficiently as the murine monoclonal antibody 2C4 (or a Fab fragment thereof) and / or binds HER2 essentially as murine monoclonal antibody 2C4 (or a Fab fragment thereof). The present humanized antibody can comprise, for example, incorporated non-human hypervariable region residues. The present humanized antibody can comprise, for example, non-human hypervariable region residues incorporated into a human variable heavy domain and can further comprise a structural region (FR) substitution in a selected position of the group consisting of 69H, 71H and 73H using the variable domain numbering system specified in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ( 1991). In one embodiment, the humanized antibody comprises FR substitutions at positions two or all positions 69H, 71H and 73H.

An example of a humanized antibody of interest given herein comprises variable heavy domain complementarity determining residues GFTFTDYT X, where X is preferably D or S (SEQ ID NO: 7); DVNPNSGGSIYNQRFKG (SEQ ID No. 8); and / or NLGPSFYFDY (SEQ ID No. 9), optionally comprises amino acid modifications of those CDR residues, for example, wherein the modifications essentially maintain or improve the affinity of the antibody. For example, the antibody variant of interest may have from about one to about seven or about five amino acid substitutions in the preceding variable heavy CDR sequences. Said antibody variants can be prepared by affinity maturation, for example, as described below. The most preferred humanized antibody comprises the variable heavy domain amino acid sequence in SEQ ID No. 4. The humanized antibody may comprise variable light domain complementaryity residues KASQDVSIGVA (SEQ ID NO: 10); SASYXXX, where X as position 5 is preferably R or L, where X in position 6 is preferably Y or E, and X as position 7 is preferably T or S (SEQ ID No. 11); and / or QQYYIYPYT (SEQ ID No. 12), for example in addition to those CDR residues of variable heavy domain of the preceding paragraph. Such humanized antibodies optionally comprise amino acid modifications of the above CDR residues, for example, where the modifications essentially maintain or improve the affinity of the antibody. For example, the antibody variant of interest may have from about one to about seven or about five amino acid substitutions in the above light variable CDR sequences. Said antibody variants can be prepared by affinity maturation, for example, as described below. The most preferred humanized antibody comprises the variable light domain amino acid sequence in SEQ ID No. 3. The present application also contemplates antibodies matured by affinity that bind to HER2 and block the activation of the ligand of a HER receptor. The main antibody can be a human antibody or a humanized antibody, for example one comprising the variable light and / or heavy sequences of SEQ ID Nos. 3 and 4, respectively (ie, variant 574). The affinity-matured antibody is preferably bound to the HER2 receptor with an affinity greater than that of murine 2C4 or variant 574 (eg from about two or about four times to about one hundred times or about one thousand fold of improved affinity, for example, according to to what was verified using an extracellular domain HER2 (ECD) ELISA). Examples of variable heavy CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or combinations of two or more (eg, two, three, four, five, six, or seven of these residues). Examples of variable light CDR residues for alteration include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97, or combinations of two or more (e.g., two to three, four, five, or up to about ten of these residues). Various forms of humanized antibody or antibody matured by affinity are contemplated. For example, the humanized antibody or the affinity matured antibody can be an antibody fragment. such as a Fab that is optionally conjugated with one or more cytotoxic agents to generate an immunoconjugate. Alternatively, the humanized antibody or the affinity matured antibody may be a full-length antibody such as a full-length IgGl antibody. (iv) Human Antibodies As an alternative for humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, by immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the gene (JH) of the heavy chain binding region of the antibody in germline and chimeric mutant mice results in a complete inhibition of the production of endogenous antibodies. The transfer of the human germline immunoglobulin gene collection in said germline mutant mice will result in the production of human antibodies by challenge with antigen. See, for example, Jakobovits et al., Proc. Nati Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno. , 7:33 (1993); and U.S. Patent Nos. 5,591,669, 5,589,369 and 5,545,807. Alternatively, phage display technology (McCafferty et al., Nature 348: 552-553 (1990)) can be used to produce human antibodies and in vitro antibody fragments, from repertoires of variable domain (V) genes of immunoglobulin to from non-immunized donors. According to this technique, the antibody domain V genes are cloned in frame within a coat protein gene which may be higher or lower than a filamentous bacteriophage such as MI3 or fd, and are displayed as fragments of functional antibodies on the surface of the phage particle. Because the filamentous particle contains a single strand DNA copy of the phage genome, selections based on the functional properties of the antibody will also result in the selection of the gene encoding the antibody that exhibits those properties. Therefore, the phage mimic some of the properties of the B cell. The phage display can be carried out in a variety of formats; for review see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a diverse collection of anti-oxa zolone antibodies from a small random combinatorial library of V genes derived from spleens of immunized mice. A repertoire of V genes from non-immunized human donors can be constructed and antibodies can be isolated for a diverse collection of antigens (including auto-antigens), essentially following the techniques described by Marks et al., J. Mol. Biol. 222: 581-597 (1991), or Griffith et al., EMBO J. 12: 725-734 (1993). See, also, US Patent Nos. 5,565,332 and 5,573,905. As discussed above, human antibodies can also be generated by B cells activated in vitro (see U.S. Patent Nos. 5,567,610 and 5,229,275).

Human HER2 antibodies have been described in U.S. Patent No. 5,772,997 issued June 30, 1998, and WO 97/00271 published January 3, 1997. (v) Fragments of Antibodies Several techniques have been developed for the production of antibody fragments. Traditionally these fragments were derived through proteolytic digestion of full-length antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229 : 81 (1985)). However, these fragments can now be produced directly with recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be recovered directly from E. coli and can be chemically coupled to form F (ab ') 2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another embodiment, F (ab ') 2 fragments can be isolated directly from the culture of recombinant host cells. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In one embodiment, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. The antibody fragment can also be a "linear antibody", for example, that described in US Patent 5,641,870. Such linear antibody fragments may be monospecific or bispecific. (vi) Bispecific Antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Examples of bispecific antibodies can bind to two different epitopes of the HER2 protein. Other such antibodies may combine a HER2 binding site with the binding sites for EGFR, HER3 and / or HER4. Alternatively, an HER2 arm can be combined with an arm that binds to a trigger molecule in a leukocyte such as a T cell receptor molecule (e.g., CD2 or CD3), or Fe receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) in order to focus the cellular defense mechanisms on the cell expressing HER2. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing HER2. These antibodies possess a HER2 binding arm and an arm that binds the cytotoxic agent (e.g., saporin, anti-interferon-, vinca alkaloid, castor A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or as antibody fragments (for example bispecific antibodies F (ab '(2) · O 96/16673 describes a bispecific HER2 / FCYRIII antibody and US Patent No. 5,837,234 discloses a bispecific HER2 / FcyRI antibody and IDM1 (Osidem) A bispecific HER2 / FCOI antibody is shown in WO98 / 02463 US Patent No. 5,821,337 teaches a bispecific HER2 / CD3 antibody The MDX-210 is a HER2 -BY bispecific Ab-CFYRIII Methods for preparing bispecific antibodies are known in the art The traditional production of full-length bispecific antibodies is based on the coexpression of two light chain-immunoglobulin heavy chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305-537-539 (1983).) Due to the random provision of light and heavy immunoglobulin chains, these hybridomas (quadromas) they produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually carried out by affinity chromatography steps, is quite cumbersome, and the product yield is low. Similar procedures have been described in WO 93/08829, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

According to a different embodiment, the variable domains of antibodies with the desired binding specificity (antibody-antigen combining sites) are fused to the immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site necessary for the light chain linkage present in at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain fusions, and if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into an appropriate host organism. This provides greater flexibility for adjusting the mutual proportions of the three polypeptide fragments in the embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. However, it is possible to insert the coding sequences for two or all three polypeptide chains into an expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the relationships have no particular significance.

In a preferred form of this embodiment, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a heavy chain-light chain pair of hybrid immunoglobulin (which provides a second binding specificity). ) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from the undesirable immunoglobulin chain combinations, since the presence of a light chain of immunoglobulin in only one half of the bispecific molecule provides an easy form of separation. This embodiment was described in WO 94/04690. For further details to generate bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986). According to another embodiment described in the North American Patent N °. No. 5,731,168, the interface between a pair of antibody molecules can be manipulated to bring to a peak the percentage of heterodimers that are recovered from the culture of recombinant cells. The preferred interface comprises at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid backbones of the interface of the first antibody molecule are replaced with longer secondary chains (for example tyrosine or tryptophan). At the interface of the second antibody molecule compensating "cavities" of identical or similar size to that of the long side chains are created by replacing long amino acid secondary chains with smaller ones (for example alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer with respect to other undesirable end products such as homodimers. The bispecific antibodies include cross-linked antibodies or "heteroconjugates". For example, one of the antibodies in the heteroconjugate can be coupled to avidin, and the other to biotin. Such antibodies have been proposed, for example, for cells of the immune system to target undesirable cells (US Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360, WO 92/200373 , and EP 03089). Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art, and have been described in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques. The techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, cannabis antibodies can be prepared using chemical bonds. Brennan et al., Science, 229: 81 (1985) describe a method in which full-length antibodies are proteolytically dissociated to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the complexing agent dithiol, sodium arsenite, to stabilize the neighboring dithiols and prevent the formation of intermolecular disulfide. The generated Fab 'fragments are then converted to thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The produced bispecific antibodies can be used as agents for the selective immobilization of enzymes. Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form blister-like antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe the production of an F (ab ') 2-molecule of fully humanized bispecific antibody. Each Fab 'fragment was secreted separately from E. coli and subjected to directed chemical coupling, in vitro, to form the bispecific antibody. The bispecific antibody formed in this manner was able to bind to cells that overexpress the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against the targets of human breast tumors. Several techniques have also been described for preparing and isolating wild-type bispecific antibody fragments directly from recombinant cell cultures. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. , 148 (5): 15 7-1553 (1992). The leucine zipper peptides of the Fos and Jun proteins were ligated to the Fab 'portions of two different antibodies by gene fusion. The antibody homodimers were reduced in the hinge region to form monomers and then re-oxidized to form the antibody heterodimer. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Nati Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for preparing bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains in 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 for preparing bispecific antibody fragments by the use of single chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immuno1., 152: 5368 (1994). Antibodies with more than two valencies have been contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991). (vii) Sequence modifications of other amino acids The amino acid sequence modifications of the antibodies described herein have been contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the Antibody were prepared by introducing appropriate nucleotide changes within the nucleic acid of the Antibody or by peptide synthesis. Such modifications include, for example, deletions of, and / or insertions within and / or substitutions of residues within the amino acid sequence within the antibody. Any combination of deletion, insertion, and substitution is prepared to reach the final construction, with the condition that the final construction possesses the desired characteristics. Amino acid changes can alter the post-translational processes of the Antibody, such as changing the number or position of the glycosylation sites. A useful method for the identification of certain residues or regions of the Antibody that are preferred locations for mutagenesis is termed "alanine scanning mutagenesis" as described by Cunningham and Wells Science, 244: 1081-1085 (1989). Here, a residue or group of target residues is identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced with a negatively charged or neutral amino acid (more preferably alanine or polyalanine) to effect the interaction of the amino acids with the antigen. Those amino acid locations that demonstrate functional sensitivity for substitutions are then refined by introducing more or other variants into, or for, the substitution sites. Therefore, although the site for the introduction of an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, random scanning or mutagenesis is carried out at the target region or codon, and the expressed Antibody variants are screened and detected for the desired activity.

The amino acid sequence inserts include amino- and / or carboxyl-terminal fusion which is comprised within a length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal inserts include an Antibody with an N-terminal methionyl residue of the antibody fused to a cytotoxic polypeptide. Other insertion variants of the antibody molecule include fusion with the N- or C- terminus of the Antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the half-life of the antibody in the serum. Another type of variant is a variant amino acid substitution. These variants have at least one amino acid residue in the Antibody molecule replaced with a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but alterations of the FR or Fe region have also been contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes may be introduced, termed "exemplary substitutions" in Table 1, or as further described below with reference to the amino acid classes, may be introduced and the Product can be tracked and detected. Table 1 Substantial modifications in the biological properties of the antibody are achieved by selecting substitutions that differ significantly in their effect or by maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, in the form of a sheet conformation or helical conformation, ( b) the charge or hydrophobicity of the molecule at the target site, or (c) the mass of the secondary chain. The amino acids can be added according to the similarities of properties of their secondary chains (in AL Lehninger, in Biochemistry, second edition, pages 73-75, orth Publishers, New York (1975)): (1) non-polar: Ala ( A), Val (V), Leu (L), Lie (I), Pro (P), Phe (F), Trp (W), Met (M) (2) Polar by charged: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acid: Asp (D), Glu (E) (4) Basic: Lys (K) ), Arg (R), His (H) Alternatively, residues that occur naturally can be divided into groups based on common secondary chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acid: Asp, Glu; (4) basic: His, Lys, Arg; (5) the residues that influence the chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions involve an exchange of a member of one of these classes with another class. Any cysteine residue that is not involved in maintaining the proper conformation of the antibody can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds can be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment). A particularly preferred type of substitutional variants involves replacing one or more hypervariable region residues of a major antibody (eg, a humanized or human antibody). Generally, the resulting variants selected for further development will have improved biological properties related to the primary antibody from which they are generated. A convenient way to generate said substitutional variants involves affinity maturation using phage display. In summary, several hypervariable region sites (eg, sites 6-7) are mutated to generate all possible amino substitutions at each site. Antibody variants generated in this way are exhibited monovalently from filamentous phage particles in the form of fusions with the gene product III of M13 loaded into each particle. The variants exhibited by phages are then traced to determine their biological activity (for example, binding affinity) as described herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen complex and the antibody to identify the points of contact between the antibody and its antigen. These contact residues and nearby residues are candidates for substitution according to the techniques developed here. Once such variants are generated, the panel of variants is screened as described herein and antibodies with superior properties in one or more relevant assays can be selected for further development. Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By alteration is meant deletion of one or more carbohydrate moieties that are in the antibody and / or aggregate of one or more glycosylation sites that are not present in the antibody. The glycosylation of antibodies is typically linked to N- or linked to 0-. Linked to N- refers to the binding of the carbohydrate portion to the secondary chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for the enzymatic binding of the carbohydrate moiety to the secondary chain asparagine. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. "O-linked glycosylation" refers to the binding of one of the sugars N-acetylgalactosamine, galactose, or xylose with a hydroxyamino acid, more commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

The addition of the glycosylation sites to the antibody was conveniently effected by altering the amino acid sequence so as to contain one or more of the tripeptide sequences described above (for the N-linked glycosylation sites). The alteration may also be effected by addition, or substitution, of one or more serine or threonine residues to the original antibody sequence (for glycosylation sites linked to 0). When the antibody comprises an Fe region, the carbohydrate bound thereto can be altered. For example, antibodies with a mature carbohydrate structure lacking fucose attached to an Fe region of the antibody have been described in US Patent Application No. US 2003. / 0157108 Al, Presta, L. See also U.S. Patent No. 2004/0093621 Al (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate bound to an Fe region of the antibody are given as reference in WO03 / 011878, Jean-Mairet et al. and U.S. Patent No. 6,602,684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide bound to an Fe region of the antibody have been reported in WO97 / 30087, Patel et al. See, also, W098 / 58964 (Raju, S.) and W099 / 22764 (Raju, S.) referring to the antibodies with altered carbohydrate attached to the Fe region thereof. Antibody compositions comprising antibodies comprising antibodies of major species with said carbohydrate structures attached to the Fe region are contemplated herein. The nucleic acid molecules encoding the amino acid sequence variants of the Antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of variants of amino acid sequences occurring naturally), or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, such as mutagenesis of PCR, and cassette mutagenesis, of a version of the variant or non-variant antibody prepared above. (viii) Screening for antibodies with the desired properties The techniques for generating antiperspirants have been described above. Antibodies with certain biological characteristics can be selected, if desired. To identify an antibody that blocks the activation of ligands of a HER receptor, the ability of the antibody to block the HER ligand that binds to cells that express the HER receptor (for example, in conjugation with another HER receptor with which said HER receptor of interest forms a hetero-oligomer HER) can be determined. For example, cells that express naturally, or transfected to express the HER receptors of the HER hetero-oligomer can be incubated with the antibody and can then be exposed to the labeled HER ligand. The ability of the HER2 antibody to block ligand binding to the HER receptor in the hetero-oligomer HER can then be evaluated. For example, the inhibition of HRG binding to MCF7 breast tumor cell lines by HER2 antibodies can be carried out using MCF7 cultures of monolayers on ice in a 24-well plate format, essentially as described in FIG. WO01 / 00245. The HER2 monoclonal antibodies can be added to each receptacle and can be incubated for 30 minutes. Then 125I-labeled G? 6ß1? 77-224 (25 pm) can be added, and the incubation can be continued for 4 to 16 hours. Dose response curves can be prepared and the IC5o value for the antibody of interest can be calculated. In one embodiment, the antibody that blocks the activation of the ligand of a HER receptor will have an IC50 to inhibit the binding of HRG to the MCF7 cells in this assay, of about 50nM or less, more preferably ???? or less. When the antibody is an antibody fragment such as a Fab fragment, the IC50 to inhibit the binding of HRG to the MCF7 cells in this assay, can be, for example, approximately ????? or less, more preferably 50nM or less.

Alternatively, or additionally, the ability of the HER2 antibody to block tyrosine phosphorylation stimulated by HER ligand, of a HER receptor present in the hetero-oligomer HER, can be verified. For example, cells that endogenously express HER receptors or that are transfected to express them can be incubated with the antibody, and then assayed for tyrosine phosphorylation activity dependent on the HER ligand, using monoclonal anti-phosphotyrosine (which is optionally conjugated with a detectable label). The kinase receptor activation assay described in U.S. Patent No. 5,766,863 is also available to determine the activation of the HER receptor and to block this activity by an antibody. In one embodiment, an antibody that inhibits HRG stimulation of the tyrosine phosphorylation pl80 in MCF7 cells can be screened, essentially as described in WO01 / 00245. For example, MCF7 cells can be deposited in 24-well plates, and monoclonal antibodies can be added to HER2 in each of the wells, and incubated for 30 minutes at room temperature; then rHRGßll77-244 can be added to each receptacle to a final concentration of 0.2 nM, and the incubation can be continued for 8 minutes. The media can be aspirated from each receptacle, and the reactions can be interrupted by addition of 100 μ? Sample buffer SDSr (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8). Each sample (25 μ?) Can be electrophoresed in a gradient gel of 4-12% (Novex) and then can be electrophoretically transferred to a polyvinylidene difluoride membrane. Antiphosphotyrosine immunoborons (at 1 pg / ml) can be developed, and the intensity of the predominantly reactive band at Mr¾¾180,000 can be quantified by reflectance densitometry. The selected antibody will preferably significantly inhibit the stimulation of HRG from the tyrosine phosphorylation pl80 to about 0-35% control in this assay. A dose response curve for the inhibition of HRG stimulation of the tyrosine phosphorylation pl80 determined by the reflectance densitometry can be prepared, the IC50 can be calculated for the antibody of interest. In one embodiment, the antibody that blocks activation of the ligand of a HER receptor will have an IC50 to inhibit the HRG stimulation of tyrosine phosphorylation of pl80 in this assay, of about 50nM or less, more preferably ???? or less. When the antibody is an antibody fragment such as a Fab fragment, the IC50 to inhibit the stimulation of HRG from the tyrosine phosphorylation pl80 in this assay can be, for example, approximately ????? or less, more preferably 50n or less. The inhibitory effects of growth of the antibody in MDA-B-175 cells can also be verified, for example, essentially as described in Schaefer et al. Oncogene 15: 1385-1394 (1997). According to this assay, MDA-MB-175 cells can be treated with a monoclonal antibody HER2 (10 g / mL) for 4 days and can be stained with methylrosaniline chloride. Incubation with an HER2 antibody may show a growth inhibitory effect in this cell line similar to that exhibited by monoclonal antibody 2C. In another embodiment, the exogenous HRG will not significantly reverse this inhibition. Preferably, the antibody will be able to inhibit cell proliferation of MDA-MB-175 cells to a greater degree than monoclonal antibody 4D5 (and optionally to a greater extent than monoclonal antibody 7F3), both in the presence and in the absence of hexogen HRG . In one embodiment, the HER2 antibody of interest can block the heregulin-dependent association of HER2 with HER3 in both MCF7 and SK-BR-3 cells, determined in a co-immunoprecipitation experiment as described in WO01 / 00245 substantially more efficiently than monoclonal antibody 4D5, and preferably substantially more efficiently than monoclonal antibody 7F3. To identify growth inhibitory HER2 antibodies, antibodies that inhibit the growth of cancer cells that overexpress HER2 can be screened. In one embodiment, the growth inhibitory antibody of choice is capable of inhibiting the growth of SK-BR-3 cells in cell cultures by about 20-100% and preferably about 50-100% at an antibody concentration of about 0. , 5 to 30 and g / ml. To identify these antibodies, the SK-BR-3 assay described in US Patent No. 5,677,171 can be performed. According to this assay, SK-BR-3 cells are cultured in a 1: 1 mixture of F12 and supplemental DMEM medium with 10% fetal bovine serum, glutamine and penicillin streptomycin. The SK-BR-3 cells are deposited in 20,000 cells in a 35mm cell culture plate (2mls / 35mm plate). 0.5 to 30 pg / ml of the HER2 antibody is added per plate. After six days, the number of cells compared to untreated cells is counted using a COULTER ™ electronic cell counter. Those antibodies that inhibit the growth of SK-BR-3 cells by approximately 20-100% or approximately 50-100% can be selected as growth inhibitory antibodies. See U.S. Patent No. 5,677,171 for assays for screening for growth inhibitory antibodies, such as 4D5 and 3E8. To select HER2 antibodies that induce apoptosis, an annexin binding assay is available using BT474 cells. BT474 cells are cultured and seeded in plates as discussed in the preceding paragraph. The medium is then removed and replaced by fresh medium alone or by a medium containing 10 g / ml of the monoclonal antibody. After a three-day incubation period, the monolayers are washed with PBS and detached by trypsinization. The cells are then centrifuged, resuspended in Ca 2+ binding buffer, and aliquots are introduced into tubes as discussed above for the cell death assay. The tubes then receive the labeled annexin (eg, annexin V-FTIC) (lpg / ml). Samples can be analyzed using a FACSCAN ™ flow cytometer and FACSCONVERT ™ CellQuest software (Becton Dickinson). Those antibodies that induce statistically significant levels of annexin binding in relation to the control, are selected as apoptotic inducing antibodies. In addition to the annexin binding assay, a DNA staining assay is available using the BT474 cells. To carry out this assay, the BT474 cells that have been treated with the antibody of interest described in the preceding two paragraphs are incubated with 9yg / ml of HOECHST 33342 ™ for 2 hours at 37 ° C, and then analyzed in a EPICS ELITE ™ flow cytometer (Coulter Corporation) using MODFIT LT ™ software (Verity Software House). Antibodies that induce a change in the percentage of apoptotic cells, which is 2 or more (and preferably 3 times or more) than untreated cells (up to 100% of apoptotic cells) can be selected as pro-apoptotic antibodies using this assay . See W098 / 17797 for screening assays for apoptosis-inducing HER2 antibodies, such as 7C2 and 7F3. To screen for antibodies that bind to an epitope on HER2 linked with an antibody of interest, a routine cross-blockade assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be performed. ), to verify if the antibody blocks the binding of an antibody, such as 2C4 or Pertuzumab, to HER2. Alternatively, or additionally, an epitope mapping can be performed by methods known in the art and / or the structure of antibody-HER2 (Franklin et al. Cancer Cell 5: 317-328 (2004)) can be studied to verify which domains of HER2 are bound to the antibody. (ix) Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent., a toxin (e.g., a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof), or a radioactive isotope (i.e., a radioconjugate). Chemotherapeutic agents useful in the generation of said immunoconjugates have been described above. Conjugates of an antibody of one or more small molecule toxins such as a calicheamicin, an maytansine (US Pat. No. 5,208,020), a trichotene, and CC1065 have also been contemplated herein. In a preferred embodiment of the invention, the antibody is conjugated with one or more maytansine molecules (for example about 1 to about 10 molecules of maytansine per antibody molecules). Maytansine can be converted to, for example, May-SS-Me which can be reduced to May-SH3 and can react with modified antibodies (Chari et al, Cancer Research 52: 127-131 (1992)) to generate the maytansinoid-antibody immunoconjugate. Another immunoconjugate of interest comprises an antibody HER2 conjugated with one or more calicheamicin molecules. The family of calicheamicin antibiotics is capable of producing double-stranded DNA openings at sub-picomolar concentrations. The structural analogs of calicheamicin that may be used include but are not limited to, 1, or 1, (X31, N-acetyl-1, PSAG and T1! (Hinman et al., Cancer Research 53: 3336-3342. (1993) and Lode et al., Cancer Research 58: 2925-2928 (1998).) See also, US Patents Nos. 5,714,586; 5,712,374; 5,264,586; and 5,773,001 which are expressly incorporated herein by reference. reference Enzymatically active toxins and fragments thereof that may be used include the diphtheria A chain, the non-adherent active fragments of diphtheria toxin, the A chain of exotoxin (from Pseudomonas aeruginosa), ricin A chain, A of abrin, A chain of modeccina, alpha-sarcina, proteins of Aleurites fordii, proteins of diantins, Phytolaca americana (PAPI, PAPII, and PAP-S), inhibitor of momordica charantia, curcina, crotina, inhibitor of saponaria oficinalis, gelonina , mitogeline, restrictocin, phenomycin, enomycin, and trichothecenes, see for example WO 93/21232 published October 28, 1993. The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (for example a ribonuclease or a DNA endonuclease such as deoxyribonuclease; DNase). A variety of radioactive isotopes are available for the production of radioconjugated HER2 antibodies. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and the radioactive isotopes of Lu. The conjugates of the antibody and the cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), succinimidyl-4- (N-) 1-carboxylate. maleimidomethyl) cyclohexane, iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldeido), bis-azido compounds (such as bis hexane diamine) (p-azidobenzoyl), bis-diazonium derivatives (such as p-bis-diazoniumbenzoyl-ethylenediamine), diisocyanates (such as 2,6-tolienium diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoromethyl). 2,4-dinitrobenzene) For example, a castorium immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). 1-isothiocyanatobenzyl-3-methyldiethylene triaminopentaacetic acid labeled with carbon 14 (MX- DTPA) e s an exemplary chelating agent for the conjugation of radionucleotides to the antibody. See WO94 / 11026. The linker can be a "dissociable linker" that facilitates the release of the cytotoxic drug into the cell. For example, a labila-acid linker, a peptidase-sensitive linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al, Cancer Research 52: 127-131 (1992)). Alternatively, a fusion protein comprising the HER2 antibody and the cytotoxic agent can be prepared for example by recombinant techniques or peptide synthesis. In another embodiment the antibody can be conjugated to a "receptor" (such as streptavidin) for the use of the pre-targeting of a tumor where the antibody-receptor conjugate is administered to the patient, followed by removal of the non-adherent conjugate circulation. , using a clarifying agent and then administration of a "ligand" (for example avidin) which is conjugated with a cytotoxic agent (for example a radionucleotide). (x) Other modifications of antibodies Other modifications of the antibody are contemplated here. For example, the antibody can be ligated to one of a variety of non-proteinaceous polymers, for example polyethylene glycol, polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol. The antibody can also be entrapped in microcapsules prepared for example by coacervation or interfacial polymerization techniques (e.g. hydroxymethylcellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules respectively), in colloidal drug release systems (for example liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques have been described in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). It may be convenient to modify the antibody of the invention with respect to effector function for example to improve antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in an Fe region of the antibody. Alternatively or additionally, cysteine residues can be introduced into the Fe region, which allows the formation of the interchain disulfide bond in this region. The homodimeric antibody thus generated may have improved internalization capacity and / or may increase cell death mediated by supplements and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity can also be prepared using cross-linked heterobifunctional linkers such as those described in Wolff et al. Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody having dual Fe regions can be manipulated and thus can have improved complement lysis, and ADCC capabilities. See Stevenson et al. Anti- Cancer Drug Desígn 3: 219-230 (1989). WO00 / 42072 (Presta, L.) discloses antibodies with enhanced ADCC function in the presence of human effector cells where the antibodies comprise amino acid substitutions in the Fe region thereof. Preferably, the improved ADCC antibody comprises substitutions at positions 298, 333, and / or 334 of the Fe region. Preferably the altered Fe region is a Fe region of human IgGl comprising or consisting of one, two or three substitutions. of these positions. Antibodies with altered Clq binding and / or complement dependent cytotoxicity (CDC), have been described in W099 / 51642, US Patent No. 6,194,551B1, US Patent No. 6,242,195B1, US Patent No. 6,528,624B1 and Patent. North American No. 6,538,124 (Idusogie et al.). The antibodies comprise an amino acid substitution at one or more of the amino acid positions 270, 322, 326, 327, 329, 313, 333 and / or 334 of the Fe region thereof. To increase the half-life of the antibody in the serum, a recovery receptor binding epitope can be incorporated into the antibody (especially an antibody fragment) as described for example in US Patent No. 5,739,277. As used herein, the term "recovery receptor binding epitope" refers to an epitope of the Fe region of an IgG molecule (eg, IgGi, IgG2, IgG3, or IgG4) that is responsible for increasing life mean serum in vivo of the IgG molecule. Antibodies with substitutions in a Fe region thereof and with an increase in serum half-lives have also been described in WO00 / 42072 (Presta, L.). Antibodies genetically engineered with three or more (preferably four) functional antigen binding sites have also been contemplated. North American Application No. US2002 / 0004587 Al, Miller et al.). The HER2 antibodies described herein can also be formulated immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art such as those described in Epstein et al., Proc. Nati Acad. Sci. USA, 82: 3688 (1985); H ang et al., Proc. Nati Acad. Sci. USA, 77: 4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545; and W097 / 38731 published October 23, 1997. Liposomes with improved circulation time have been described in the US Pat.

No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and phosphatidylethanolamine derivatized with PEG (PEG-PE). The liposomes are extruded through filters of defined pore size to provide liposomes with the desired diameter. The Fab 'fragments of the antibody of the present invention can be conjugated with the liposomes described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) through a disulfide exchange reaction. Optionally, a chemotherapeutic agent is contained within the liposome. See Gabizon et al. J. National Cancer Inst.81 (19) 1484 (1989). (ix) Exemplary Antibodies Examples of antibodies that can be formulated in accordance with the present invention include but are not limited to the following: anti-ErbB antibodies including anti-HER2 antibodies, such as those described in more detail herein; antibodies that bind to a surface marker, of B cells such as CD19, CD20 (for example Rituximab (RITUXAN®) and humanized 2H7), CD22, CD40 or BR3; antibodies that bind to IgE, including Omalizumab (XOLAIR®) commercially available in Genentech, E26 (Figs 17A-B given here), HAE1 (Figs 17A-B given here), IgE antibodies with a substitution amino acid at position 265 of a Fe region thereof (U.S. Patent 2004/0191244 Al), Hu-901 (Fig. 17A-B given here), an IgE antibody such as that of O2004 / 070011, or a antibody (including antibody fragments and full-length antibodies) comprising the variable domains of any of those IgE antibodies. See also, Presta et al., J. Immunol. 151: 2623-2632 (1993); International Publication No. WO 95/19181; U.S. Patent No. 5,714,338, issued February 3, 1998; U.S. Patent No. 5,091,313, issued February 25, 1992; WO 93/04173 published March 4, 1993; WO 99/01556 published January 14, 1999; and U.S. Patent No. 5,714,338; antibodies that bind to vascular endothelial growth factor (VEGF), or a receptor thereof that include Bevacizumab (AVASTIN ™), commercially available from Genentech, and Ranibizumab (LUCENTIS ™); anti-IL-8 antibodies (St John et al., Chest, 103: 932 (1993), and International Publication No. WO 95/23865); anti-PSCA antibodies (WO01 / 40309); anti-CD40 antibodies, including S2C6 and the humanized variants thereof (WO00 / 75348); anti-CDlla antibodies, including efalizumab (RAPTIVA®) (U.S. Patent No. 5,622,700, WO 98/23761, Steppe et al., Transplant Intl. 4: 3-7 (1991), and Hourmant et al., Transplantation 58 : 377-380 (1994)); anti-CD18 antibodies (U.S. Patent No. 5,622,700, issued April 22, 1997, or as in O 97/26912, published July 31, 1997); anti-Apo-2 receptor antibody (WO 98/51793 published November 19, 1998); anti-TNF-alpha antibodies including cA2 (REMICADE®), CDP571 and MAK-195 (See, U.S. Patent No. 5,672,347 issued September 30, 1997, Lorenz et al., J. Immunol. 156 (): 16 6 -1653 (1996), and Dhainaut et al., Crit. Care Med. 23 (9): 1461-1469 (1995)); Anti-Tissue Factor (TF) (European Patent No. 0 420 937 Bl granted on November 9, 1994); anti-human integrin a4ß? (WO 98/06248 published February 19, 1998); anti-EGFR antibodies, including humanized or chimerized 225 antibody, such as in WO 96/40210 published on December 19, 1996; anti-CD3 antibodies, such as OKT3 (U.S. Patent No. 4,515,893 issued May 7, 1985); anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT®) and (ZENAPAX®) (See U.S. Patent No. 5,693,762 issued December 2, 1997); anti-CD4 antibodies such as the antibody cM-7412 (Choy et al., Arthritis Rheum 39 (1): 52-56 (1996)); anti-CD52 antibodies such as CAMPATH-1H (Riechmann et al., Nature 332: 323-337 (1988); anti-Fc receptor antibodies such as the M22 antibody directed against FCYRI such as in Graziano et al., J. Immunol. 10): 4996-5002 (1995); anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkey et al .. Cancer Res. 55 (23Suppl): 5935s-5945s (1995); antibodies directed against epithelial cells of breast including huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al. Cancer Res. 55 (23): 5852s-5856s (1995); and Richman et al. Cancer Res. 55 (23 Supp): 5916s-5920s (1995)); antibodies that bind to colon carcinoma cells such as C242 (Litton et al., Eur J. Immunol. 26 (1): 1-9 (nineteen ninety six) ); anti-CD38 antibodies, for example AT 13/5 (Ellis et al., J. Immunol 155 (2): 925-937 (1995)); anti-CD33 antibodies such as Hu M195 (Jurcic et al .. Cancer Res 55 (23 Suppl): 5908s-5910s (1995) and CMA-676 or CDP771; anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al. 55 (23 Suppl): 5899s-5907s (1995); anti-EpCA antibodies such as 17-1A (PANOREX®); anti-GpIIb / IIIa antibodies such as abciximab or c7E3 Fab (REOPRO ©); anti-RSV antibodies such as EDI-493 (SYNAGIS®); anti-CMV antibodies such as PROTOVIR®; anti-HIV antibodies such as PR0542; anti-hepatitis antibodies such as the anti-Hep B antibody OSTAVIR ©; anti-CA 125 OvaRex antibody; BEC2 antibody from the anti-idiotypic GD3 epitope, anti-c ^ 3 antibody VITAXIN®; anti-human renal cell carcinoma antibody such as ch-G250; ING-1; anti-human 17-1A antibody (3622W94); anti-human colorectal tumor antibody (A33); R24 antibody of anti-human melanoma directed against the GD3 ganglioside; squamous cell carcinoma anti-human (SF-25); and anti-human leukocyte antigen antibodies (HLA) such as the Smart ID10 antibody and the anti-HLA DR antibody Oncolym (Lym-1). (xi) Compositions of antibody variants The present invention, in at least one aspect, refers to formulations comprising a composition comprising a mixture of a major species antibody, and one or more variants thereof. When the main species antibody binds to HER2, preferably the HER2 antibody (either one or both major HER2 antibody species and antibody variants thereof), is that which binds to the Domino II of HER2, inhibits HER dimerization in more efficiently than Trastuzumab, and / or binds to a heterodimeric HER2 binding site. The present preferred embodiment of the main species antibody is one comprising the variable light and variable heavy amino acid sequences in SEQ ID Nos. 3 and 4, and more preferably comprising the light chain amino acid sequence selected from SEQ ID NO. 15 and 23, and a heavy chain amino acid sequence selected from SEQ ID No. 16 and 24. In one embodiment, the formulated HER2 antibody composition comprises a mixture of the main species HER2 antibody, and an amino acid sequence variant of the same which comprises an amino-terminal amino acid extension. Preferably, the amino-terminal leader extension is on a light chain of the antibody variant (for example on one or two light chains of the antibody variant). The main species antibody HER2 or the antibody variant can be a full length antibody or an antibody fragment (for example Fab fragments of F (ab ') 2), but preferably both are full length antibodies, the present variant of The antibody can comprise an amino-terminal leader extension over one or more of the heavy or light chains thereof. Preferably, the amino-terminal leader extension is on one or two light chains of the antibody. The amino-terminal leader extension preferably comprises or consists of HSV-. The presence of the amino-terminal leader extension in the composition can be detected by various analytical techniques, but not limited to, N-terminal sequence analysis, assay for charge heterogeneity (e.g., cation exchange chromatography or capillary zone electrophoresis), mass spectrometry, etc. The amount of antibody variant in the composition is generally in a range from and from an amount that constitutes the limit of detection of any assay (preferably N-terminal sequence analysis) used to detect the variant to an amount less than the amount of a major species antibody. In general, about 20% or less (for example from about 1% to about 15%, for example from 5% to about 15%) of the antibody molecules in the composition, comprise an amino-terminal leader extension. Said percentage amounts are preferably determined using quantitative N-terminal sequence analysis or cation exchange analysis (preferably using a weak, high resolution cation exchange column, such as a PROPAC WCX-10 ™ cation exchange column). Apart from the amino-terminal leader extension variant, other alterations of the amino acid sequence, the main species antibody and / or the variant, including but not limited to an antibody comprising a lysine C residue, have been contemplated -terminal in one or both heavy chains thereof, a variant of deamidated antibody, etc. In addition, the main species antibody or variant may further comprise variations of glycosylation, non-limiting examples of which include the HER2 antibody comprising an oligosaccharide structure Gl or G2 linked to the Fe region, an HER2 antibody comprising a carbohydrate moiety attached to a light chain (e.g., one or two carbohydrate moieties attached to one or two light chains of the antibody), HER2 antibody comprising a non-glycosylated heavy chain.

III. Preparation of the formulation The present invention provides, in a first aspect, a pharmaceutical formulation of salts comprising the monoclonal antibody, preferably a full length human antibody or humanized IgGl, in histidine acetate buffer, at pH 5.5 to 6. , 5, preferably pH 5.8 to 6.2. However, the antibody in the formulation can be an antibody fragment comprising an antigen binding region such as a Fab or F (ab ') 2 fragment. In another embodiment, the invention relates to a pharmaceutical formulation comprising, or consisting essentially of a full-length IgGl antibody, susceptible to deamidation or aggregation in an amount of from about 10mg / mL to about 250mg / mL; histidine acetate buffer, pH 5.5 to 6.5; a saccharide selected from the group consisting of trehalose and sucrose, in an amount of from about 60mM to about 250mM; and polysorbate 20 in an amount of from about 0.01% to about 0.1%. In a further embodiment, the invention provides a pharmaceutical formulation comprising an antibody that binds to domain II of HER2 in a histidine buffer at a pH of from about 5.5 to about 6.5, a saccharide and a surfactant. For example, the formulation may comprise Pertuzumab in an amount of from about 20 mg / mL to about 40 mg / mL, histidine acetate buffer, sucrose, and polysorbate 20, where the pH of the formulation is from about 5.5 to about 6.5.

In another aspect, the invention provides a pharmaceutical formulation comprising a DR5 antibody in a histidine buffer at a pH of from about 5.5 to about 6.5, a saccharide and a surfactant. Said formulation can comprise, for example, Apomab in an amount of from approximately 10 mg / mL to 30 mg / mL, histidine acetate buffer, trehalose, and polysorbate 20, where the pH of the formulation is from approximately 5.5 to approximately 6, 5. The formulation is especially useful for antibodies that are susceptible to deamination and / or aggregation and / or fragmentation, because the buffer retards the deamidation and / or aggregation and / or fragmentation of the antibody formulated therein. In addition, unlike other histidine buffers prepared using HC1, the histidine acetate buffer lacks the chloride ion which is beneficial because this buffer, when combined with the saccharide had the same protective effect on the antibody as the polysorbate. , and was stable and compatible with storage in stainless steel tanks. Therefore, in addition to the formulation per se comprising the antibody susceptible to deamidation, aggregation and / or fragmentation, the invention provides a method for reducing the deamidation, aggregation and / or fragmentation of a therapeutic monoclonal antibody (e.g. to a composition at a different pH or in a different buffer), which comprises formulating the antibody in a histidine acetate buffer, at pH 5.5 to 6.5. In this embodiment, it is possible to determine or measure the deamidation, aggregation and / or fragmentation before and after formulating the antibody, said formulated antibody demonstrating acceptable deamidation, aggregation and / or fragmentation in the formulation upon storage thereof. The antibody in the formulation can be linked to an antigen including but not limited to: HER2, CD20, IgE, DR5, BR3 and VEGF. When the formulated antibody binds HER2, it is preferably one that binds to Domain II of HER2, more effectively inhibits HER dimerization than Trastuzumab, and / or binds to a heterodimeric binding site of HER2. In the preferred embodiment herein, of a formulated HER2 antibody, it is one which comprises the varying variable and heavy light amino acid sequences in SEQ ID Nos. 3 and 4, and more preferably comprising the amino acid sequences of light chain and heavy chain in SEQ ID Nos. 15 and 16 (Pertuzumab). Examples of CD20 antibodies that can be formulated herein include: "C2B8" which is currently referred to as "Rituximab" ("RITUXAN®") and is commercially available from Genentech (see also US Patent No. 5,736,137, which was expressly incorporated as reference); the murine antibody 2B8 labeled with yttrium- [90] -, designated "Y2B8" or "Ibritumomab Tiuxetan" ZEVALIN® commercially available from Biogen-Idec (see also U.S. Patent No. 5,736,137, which is expressly incorporated herein by reference); Murine IgG2a "Bl", also referred to as "Tositumomab," optionally labeled with 131I to generate the "131I-B1" antibody (iodo tosurumomab 1131, BEXXAR ™), (U.S. Patent No. 5,595,721, expressly incorporated herein by reference ); murine monoclonal antibody "1F5" (Press et al., Blood 69 (2): 584-591 (1987) and variants thereof including 1F5"framework patched" or humanized (WO03 / 002607, Leung, S.); ATCC deposit HB -96450); murine 2H7 antibody and chimeric 2H7 (Clark et al., PNAS 82: 1766-1770 (1985); US Patent No. 5,500,362, expressly incorporated herein by reference); 2H7 humanized; huMax-CD20 (WO 04/035607, Genmab, Denmark); AME-133 (Applied Molecular Evolution); A20 antibody or variants thereof such as chimeric or humanized A20 antibody (cA20, hA20, respectively) (US Patent 2003/0219433, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, B-Cl or NU-B2 available from International Leukocyte Typing Workshop (Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p.440, Oxford University Press (1987).) In the preferred embodiment of a formulated CD20 antibody, the CD20 antibody is a humanized 2H7 antibody The preferred humanized 2H7 antibodies are 2H7v16 and 2H7v511 The humanized 2H7v16 can be an intact antibody or an antibody fragment comprising the variable light and variable heavy sequences in Figures 18A-B (SEQ ID Nos. 26 and 29) When the humanized 2H7vl6 antibody is a full-length antibody, it preferably comprises the light and heavy chain amino acid sequences with SEQ ID NOS. 63 and 65. When the antibody binds to VEGF, it preferably comprises the variable domain sequence described in Fig. 19. The most preferred anti-VEGF antibody is the full-length humanized IgGl antibody, Bevaci. zumab (AV7ASTIN ™), commercially available from Genentech. When the formulated antibody binds to IgE, it is preferably selected from the group consisting of: E25, Omalizumab (XOLAIR®), commercially available from Genentech (see also Figures 17A-B), E26 (Figures 17A-B) HAE1 (Figures 17A-B given here), IgE antibody with an amino acid substitution at position 265 of a Fe region thereof (U.S. Patent 2004/0191244 Al), Hu-901 (Figures 17A -B are given here), an IgE antibody such as in WO2004 / 070011, or an antibody (including antibody fragments and full-length antibodies) comprising the variable domain of any of those IgE antibodies. When the antibody binds to a receptor and to the super family of tumor necrosis factor (TNF) or a mortality receptor, it binds preferably to DR5, and preferably is an agonist antibody. Publications in this area include Sheridan et al., Science, 277: 818-821 (1997), Pan et al., Science, 277: 815-818 (1997), W098 / 51793 published November 19, 1998; W098 / 41629 published September 24, 1998; Screaton et al., Curr. Biol., 7: 693-696 (1997); alczak et al., EMBO J., 16: 5386-5387 (1997); Wu et al., Nature Genetics, 17: 141-143 (1997); W098 / 35986 published August 20, 1998; EP870,827 published October 14, 1998; W098 / 46643 published October 22, 1998; O99 / 02653 published January 21, 1999; WO99 / 09165 published February 25, 1999; W099 / 11791 published March 11, 1999; North American Patent 2002/0072091 published August 13, 2002; Patents North American 2002/0098550 published December 7, 2001; U.S. Patent 6,313,269, issued December 6, 2001; North American Patent 2001/0010924 published August 2, 2001; U.S. Patent 2003/01255540 published July 3, 2003; North American Patent 2002/0160446 published October 31, 2002, North American Patent 2002/0048785 published April 25, 2002; US Patent 6,342,369 granted in February 2002; US Patent 6,569,642 granted May 27, 2003, US Patent 6,072,047 issued June 6, 2000, US Patent 6,642,358 issued November 4, 2003; U.S. Patent 6,743,625 issued June 1, 2004. The most preferred DR5 antibody is Apomab. Each of the formulations indicated above comprises a buffer, preferably a histidine buffer, and more preferably a histidine acetate buffer with a pH of 5.5 to 6.5, preferably from 5.8 to 6.2, for example about 6.0. The concentration of buffer is dictated, at least in part, by the desired pH. Examples of concentrations for the buffer are within the range of from about lmM to about 200mM, preferably from about 10mM to about 40mM, and more preferably about 20mM. The concentration of antibody in the formulation is preferably in the range of from about 10mg / mL to about 250mg / mL. The concentration of antibody can be determined based on the use and proposed and mode of administration of the formulation. For example, when the formulation is for IV administration (for example an HER2 antibody), the concentration of antibody in the formulation is preferably from about 20 mg / mL to about 40 mg / mL. In the exemplified Pertuzumab formulation intended for intravenous (IV) administration, the antibody concentration was from about 20mg / mL to about 40mg / mL, more preferably about 30mg / mL. When the antibody is for SQ or IM administration (for example for an anti-IgE antibody) higher antibody concentrations may be desired. Such substantially high antibody concentrations may be from about 50mg / mL to about 250mg / mL, or from about 80mg / mL to about 250mg / mL, or from about 100mg / mL to about 200mg / mL. When the formulation comprises a DR5 antibody such as Apomab, the antibody concentrations given as an example are from about 10mg / mL to about 30mg / mL, for example about 20mg / mL of DR5 antibody; said formulation is useful for intravenous administration. The formulation for administration is preferably an aqueous formulation (not freeze-dried) and has not been subjected to prior lyophilization. Although the formulation may be lyophilized, preferably it is not. However, the freezing of the aqueous formulation, without the simultaneous drying that occurs during freeze drying, is specifically contemplated here, which facilitates a longer storage thereof, for example in a stainless steel tank. The formulation preferably further comprises a saccharide, more preferably a di-saccharide, such as trehalose or sucrose. The saccharide is generally included in an amount that reduces the formation of soluble aggregate, such as that which occurs in freezing / thawing. Examples of saccharide concentrations are those within the range of from about 10mM to about 1mL, for example from about 60mM to about 250mM, and even more preferably about 120mM for an HR2 antibody formulation, and about 240mM for a formulation of DR5 antibody. Although it was discovered here that a formulation comprising histidine acetate buffer and saccharide was stable, the formulation optionally further comprises a surfactant, such as polysorbate, more preferably polysorbate 20. The surfactant is generally included in an amount that reduces aggregate formation insoluble (such as that which occurs by agitation or transport). The concentration of surfctant is preferably from about 0, 0001% to about 1.0%, more preferably from about 0.01% to about 0.1%, for example about 0.02%. Optionally the formulation does not contain a tonifying amount of a salt such as sodium chloride. The formulation is generally sterile, and this can be achieved by following procedures known to a person skilled in the art, to generate sterile pharmaceutical formulations suitable for administration to human subjects, including filtration through sterile filtration membranes, before, or after the preparation of the formulation. In addition, the formulation is conveniently a formulation that has been shown to be storage stable. Various stability tests are available to those skilled in the art to confirm the stability of the formulation. For example, the formulation can be a formulation that is stable in storage: at about 40 ° C for at least 4 weeks; at about 5 ° C or about 15 ° C for at least 3 months or at least one year; and / or approximately -20 ° C for at least 3 months. The stability can be tested by evaluating the physical stability, chemical stability and / or biological activity of the antibody in the formulation at the time of formulation as well as after storage at the indicated temperatures. The physical and / or chemical stability can be evaluated qualitatively and / or quantitatively in a variety of different ways including the evaluation of aggregate formation (for example using size exclusion chromatography, turbidity measurement and / or visual inspection); verifying the heterogeneity of the charge using cation exchange chromatography or capillary zone electrophores; analysis of the amino-terminal or carboxy-terminal sequence; mass spectrometric analysis; SDS-PAGE analysis to compare the intact and reduced antibody; analysis of the peptide map (for example tryptic or LYS-C); evaluating the biological activity or the antigen-binding function of the antibody; etc. The instability can result in aggregation, deamidation (e.g., deamidation Asn), oxidation (e.g., Met oxidation), isomerization (e.g. Asp isomerization), clipping / hydrolysis / fragmentation (e.g. fragmentation of the hinge region). , formation of succinimide, unpaired cysteine (s), N-terminal extension, C-terminal processing, glycosylation differences, etc. The biological activity or the antigen-binding function can be evaluated using various techniques available to the person skilled in the art. As indicated above, the freezing of the formulation has been specifically contemplated here. Therefore, the formulation can be evaluated to determine the stability during freezing and thawing. Accordingly, the invention also provides a method for preparing a pharmaceutical formulation comprising preparing the formulation as described herein and evaluating the physical stability, chemical stability or biological activity of the monoclonal antibody in a formulation. In the preferred embodiment, the formulation is provided inside a bottle with a plug pierceable by a syringe, preferably in aqueous form. The bottle is conveniently stored at approximately 2-8 ° C until it is administered to a subject in need. The bottle can be for example a bottle of 20 cc (for example for a dose of 420 mg) or a bottle of 50 cc (for example for a dose of 1050 mg). For a DR5 antibody such as Apomab, the formulation can be provided in a 5-ce glass bottle (for example, filled with 5.5 ml). In another embodiment, the formulation is provided inside a stainless steel tank. The formulation in the stainless steel tank is optionally frozen and not freeze-dried. One or more other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980), may be included in the formulation, provided they do not affect adversely the desirable characteristics of the formulation. Acceptable carriers, excipients or stabilizers are non-toxic to those who receive them at the doses and concentrations employed, and include additional buffering agents; co-solvents; antioxidants that include ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (for example, Zn-protein complexes); biodegradable polymers such as polyesters; preservatives and / or salt-forming counterions such as sodium.

IV. Treatment with the Antibody Formulation In one embodiment, the invention provides a method for the treatment of a disease or disorder in a subject which comprises administering the formulation described herein to a subject in an amount effective to treat the disease or disorder. When the antibody of the formulation binds to HER2, it is preferably used to treat cancer. The cancer will generally comprise cells that express HER2, such that the present HER2 antibody is capable of binding to the cancer cells. Therefore, the invention in this embodiment relates to a method for the treatment of cancer expressing HER2, in a subject, which comprises administering the pharmaceutical formulation of the HER2 antibody to the subject, in an amount effective to treat cancer. Several cancers that can be treated with the composition are listed in the definitions section above. It has also been contemplated that the formulation of the HER2 antibody can be used to treat various diseases or non-malignant disorders including an autoimmune disease (eg, psoriasis); endometriosis; scleroderma; restenosis; polyps such as colon polyps, nasal polyps or gastrointestinal polyps; fibroadenoma; respiratory disease (see previous definition); cholecystitis; neurofibromatosis; polycystic kidney disease; inflammatory diseases; skin disorders including psoriasis and dermatitis; vascular disease (see previous definition); disorders involving abnormal proliferation of vascular epithelial cells; gastrointestinal ulcers; Menetrier's disease, secretory adenomas or protein loss syndrome; kidney disorders; angiogenic disorders; ocular disease such as age-related macular degeneration, presumed ocular histoplasmosis syndrome; retinal neovascularization from proliferative diabetic retinopathy, retinal vascularization, diabetic retinopathy or age-related macular degeneration; pathologies associated with bones such as osteoarthritis, rickets and osteoporosis; damage followed by a cerebral ischemic event; fibrotic or edemas diseases such as liver cirrhosis, pulmonary fibrosis, carcoidosis, thyroiditis, systemic hyperviscosity syndrome, Osler eber-Rendu disease, chronic occlusive pulmonary disease, or edema after burns, trauma, radiation, stroke, hypoxia or ischemia; reaction to skin hypersensitivity, diabetic retinopathy and diabetic nephropathy; Guillain Barre syndrome; graft versus host disease or transplant rejection; Paget's disease; inflammation of the bones or joints; photoenvironment (caused by UV radiation in human skin); benign prosthetic hypertrophy; some microbial infections that include microbial pathogens selected from adenovirus, antavirus, Borrelia burgdorferi, Yersinia spp, and Bordetella pertussis; thrombi caused by platelet aggregation; reproductive disorders such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding or menometrorrhagia; synovitis; atheroma; acute and chronic nephropathies (including proliferative glomerulonephritis and diabetes-induced kidney disease); eczema; hypertrophic scar formation; endotoxic shock and fungal infection; familial polyposis adenomatosis; neurodegenerative diseases (for example Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration); myelodysplastic syndromes; aplastic anemia; ischemic injury; fibrosis of the lung, kidney or liver; T-cell mediated hypersensitivity disease; pyloric hypertrophic infantile stenosis; urinary obstruction syndrome; psoriatic arthritis; and Hasimoto's thyroiditis. Indications for preferred non-malignant therapy include psoriasis, endometriosis, scleroderma, vascular disease (eg restenosis, atherosclerosis, coronary artery disease or hypertension), colon polyps, fibroadenoma or respiratory diseases (eg asthma, chronic bronchitis, bronchiectasis or cystic fibrosis). When the antibody in the formulation binds to a B-cell surface marker such as CD20 or BR3, the formulation can be used to treat a malignant B-cell disease, such as NHL or CLL, an autoimmune disease, rejection of the grafts or to block an immune response to a foreign antigen, such as an antibody, a toxin, a viral gene therapy vector, a graft, an infectious agent or an alloantigen (see WO 01/03734, Grillo-Lopez et al.). When the antibody in the formulation is an IgE antibody, it can be used to treat an IgE-mediated disorder (USSN 2004/0197324 Al, Liu and Shire), such as allergic asthma, allergic rhinitis, atopic dermatitis, allergic gastroenteropathy, hypersensitivity, eczema, urticaria, allergic bronchopulmonary aspergillosis, parasitic disease, hyper-IgE syndrome, ataxia-telangiectasia, iskott-Aldrich syndrome, thymic alimfoplasia, IgE myeloma and graft versus host reaction. Antibodies that bind to a receptor in the TNF superfamily (eg, bind to DR5) or bind to VEGF (or a receptor thereof) can be used to treat cancer, several forms of which have been described in the section of definitions above. Preferably, the cancer treated with a DR5 antibody formulation is a solid tumor or NHL. When the disorder is cancer, the patient can be treated with a combination of the antibody formulation and a chemotherapeutic people. The combined administration includes co-administration or concurrent administration, using separate formulations or a single pharmaceutical formulation and consecutive administration in any order, where preferably there is a period of time in which both (or all) of the active agents simultaneously exert their biological activities. Therefore, the chemotherapeutic agent can be administered before or after administration of the composition. In this embodiment, the time between at least one administration of the chemotherapeutic agent and at least one administration of the composition is preferably about 1 month or less, and more preferably is about 2 weeks or less. Alternatively, the chemotherapeutic agent and the composition are administered concurrently to the patient, in a single formulation or in separate formulations. Treatment with the formulation will result in an improvement in the signs or symptoms of cancer or disease. For example, when the disease being treated is cancer, such therapy may result in improved survival (overall survival and / or progression-free survival) and / or may result in an objective clinical response (partial or complete). ). In addition, treatment with the combination of the chemotherapeutic agent and the antibody formulation can result in a therapeutic, synergistic or more than additive benefit for the patient. Preferably, the antibody of the formulation administered is a naked antibody. However, the administered antibody can be conjugated with a cytotoxic agent. Preferably, the immunoconjugate and / or the antigen to which it is linked is, is internalized by the cell, which results in an increase in the therapeutic efficacy of the immunoconjugate to kill the cancer cell to which it binds. In a preferred embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the cancer cell. Examples of such cytotoxic agents include maytansinoids, calicheamicins, ribonucleases and DNA endonucleases. The formulation is administered to a human patient according to known methods, such as intravenous administration, for example in the form of a bolus or by continuous infusion for a period of time, by intramuscular, intraperitoneal, intracerebroespinal, subcutaneous, intra-articular, intrasynovial, intrathecal, topical oral or inhalation routes. Intravenous, intramuscular or subcutaneous administration of the antibody composition is preferred, intravenous administration being most preferred. For subcutaneous release, the formulation can be administered through a syringe; the device for injection (for example, the INJECT-EASE ™ and GENJECT ™ device); pocket injector (such as GENPEN ™); Needleless device (for example, MEDIJECTOR ™ and BIOJECTOR ™); or patch release system. For the prevention or treatment of the disease, the appropriate antibody dose will depend on the type of disease to be treated, as defined above, of the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes. , of the previous therapy, of the patient's clinical history and of the response to the antibody, and the discretion of the attending physician. The antibody is conveniently administered to the patient at one time or in a series of treatments. Depending on the type and severity of the disease, approximately 1 pg / kg to 50 mg / kg (for example, 0.1-20 mg / kg) of antibody HER2 or DR5 is an initial experimental dose for administration to the patient, in, for example, one or more separate administrations, or by continuous infusion. The antibody dose will generally be in the range of about 0.05 mg / kg to about 10 mg / kg. If a chemotherapeutic agent is administered, it will usually be administered at the doses known therefor, or optionally decreased due to the combined action of the drugs or the negative side effects attributable to the administration of the chemotherapeutic agent. The preparation and dosing schedules for such chemotherapeutic agents can be used according to the manufacturer's instructions or can be empirically determined by the person skilled in the art. The preparation and dosing programs for such chemotherapy have also been described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). Other therapeutic regimens may be combined with the antibody, including but not limited to: a second (third, fourth, etc.) chemotherapeutic agent (ie "cocktails" of different chemotherapeutic agents); another monoclonal antibody; a growth inhibitory agent; a cytotoxic agent; a chemotherapeutic agent; a drug directed to EGFR; a tyrosine kinase inhibitor; an anti-angiogenic agent; and / or cytokine; etc. In addition to the therapeutic regimens, the patient may undergo surgical removal of cancer cells and / or radiation therapy.

V. Articles of Manufacture In another embodiment of the invention, an article of manufacture containing the pharmaceutical formulation of the present invention and providing instructions for its use is provided. The article of manufacture comprises a container. Suitable containers include, for example, bottles, flasks (e.g., dual chamber bottles), syringes (such as dual chamber syringes), and test tubes. The container may be formed of a variety of materials such as glass or plastic. The container contains the formulation and the label on, or associated with the container can indicate the instructions for use. The container containing the formulation can be a multipurpose vial, which allows repeated administrations (eg, 2-6 administrations) of the reconstituted formulation. The article of manufacture may additionally include other desirable materials from a commercial and user's point of view, including buffers, diluents, filters, needles, syringes and pack inserts, with instructions for use as indicated in the previous section. The invention will be better understood with reference to the following examples. However, they should not be considered as limiting the scope of the invention. All patent literature and citations are incorporated herein by reference.

EXAMPLES Liquid Pertuzumab Stable Formulation These examples describe the development and stability test of stable liquid formulations comprising Pertuzumab at protein concentrations within the range of from about 10 mg / mL to 180 mg / mL. The selected formulations had low turbidity, and were physically and chemically stable. A chloride ion was extracted from the formulation to reduce the risk of corrosion. The formulation was isotonic, and suitable for subcutaneous or intramuscular administration. The formation of insoluble aggregates by the aggression of the agitation was avoided, using a formulation of histidine acetate and sucrose without the need to include polysorbate 20.

Analytical Methods Color, Appearance and Clarity (CAC) Color, appearance and clarity were determined by visual inspection of the bottles against a white and black base under a white fluorescent light, at room temperature.

Measurements of the UV Concentration An aliquot of the liquid product was first diluted with the formulation buffer-, so that the Araax near 278 nm was within the absorbance unit of 0.5-1.0. The UV absorbance of the diluted samples was measured in a quartz cuvette with a path length of 1 cm on an HP 8453 spectrometer. Absorbance was measured at 278 nm and 320 nm. The absorbance from 320 nm was used to correct the scattering of the base light due to larger aggregates, bubbles and particles. The measurements were bleached against the formulation buffer. The protein concentration was determined using the absorptivity of 1.50 (mg / mLJ ^ cm "1.

PH Measurements The pH was measured at room temperature using a pH meter RADIOMETER COPENHAGEN PHM82 ™. The probe used was a reference / glass electrode combined with a radiometric connector (Sigma, Cat # E-5759). Standard solutions of pH 4.01 and pH 7.00 (EM Science) were used.

Ion Exchange Interchange Chromatography (IEX) Cation exchange chromatography was used to measure changes in charge variants. This assay uses a DIONEX PROPAC WCX-10 ™ column on an HP 1100 ™ HPLC system. Samples were diluted to 1 mg / mL with mobile phase A containing 20 mM MES at pH 6.0. 50 mL of diluted samples that were kept at room temperature were then loaded onto the column. The peaks were eluted with a shallow NaCl gradient using mobile phase B containing 20 mM of MES, 250 mM of NaCl, pH 6.0. The eluent was monitored at 280 nm. The data analyzed using HP CHEMSTATION ™ software (Rev A08.03).

Capillary Zone Electrophoresis (CZE) The purity of the Fab and F (ab ') 2 fragments was determined by CZE. This test was carried out on a BIORAD BIOFOCUS ™ 3000 ™ capillary electrophoresis system with a BIOCAP XL ™ capillary, 50 μp? I.D., 44.6 cm of total length and 40 cm, to the detector.

Size Exclusion Chromatography (SEC) Size exclusion chromatography was used to quantify aggregates and fragments. This assay uses a TSK G3000 S XL ™ column, 7.8 x 300 mm and the assays are on an HP 1100 ™ HPLC system. The samples were diluted at 10 mg / mL with the mobile phase and the injection volume was 20 μL. The mobile phase was 100 mM K2HP04 at pH 6.8 and the protein was eluted with an isocratic gradient at 0.5 mL / min for 45 minutes. The eluent absorbance was monitored at 280 nm. The integration was made using HP CHEMSTATION ™ software (Rev A08.03).

Biological Activity The biological activity of Pertuzumab was determined by measuring its ability to inhibit the proliferation of the human breast cancer cell line MDA-MB-175-VII.

EXAMPLE 1 Antibody fragments of Fab and F (ab ') 2 of Pertuzumab were formulated at a protein concentration of 1.0 mg / mL under the following buffer conditions: 10 mM citrate, 140 mM NaCl, pH 4.0; 10 mM succinate, 140 mM NaCl, pH 5.0; 10 mM succinate, 140 mM NaCl, pH 6.0; 10 mM histidine, 140 mM NaCl, pH 7.0; and 10 mM glycylglycine, 140 mM NaCl, pH 8.0.

Each formulation was filtered and then aliquots were placed in glass flasks of 3ce WHEATON ™ USP Type I, sealed with gray butyl stoppers coated with TEFLON ™. The samples were stored at 40 ± 2 ° C. Pharmacological product stability analyzes showed that Fab and F (ab ') 2 were more stable between pH 5.0 and 6.0.

Table 2. Effect of pH on the degradation of Fab or F (ab '> 2 stored at 40 ° C EXAMPLE 2 Pertuzumab was formulated in 20 mM histidine acetate buffer with 120 mM sucrose and 0.02% polysorbate 20. The pHs of the formulations were adjusted with acetic acid to a final pH between 5.0 and 7, 0 The protein concentration was 30 mg / mL. Each formulation was placed in glass bottles of 3 ce Type I, USP, and stored at 40 ° C for stability analysis. The results showed that Pertuzumab was more stable around pH 6.0.

Table 3. Effect of pH on the degradation of Pertuzumab stored at 40 ° C EXAMPLE 3 Formulations of Pertuzumab at protein concentrations of 100 mg / mL were prepared in the following excipients: (1) 10 mM histidine-HCl, 240 m sucrose, 0.02% polysorbate 20, pH 6.0; (2) 10 mM histidine-acetate, 240 mM sucrose, 0.02% polysorbate 20, pH 6.0; (3) 10 mM histidine phosphate, 240 mM sucrose, 0.02% polysorbate 20, pH 6.0; (4) 10 mM histidine sulfate, 240 mM sucrose, 0.02% polysorbate 20 at pH 6.0.

Each formulation was placed in a 3-ply Type I USP glass vial VITRUM ™ FORM sealed with butyl rubber stoppers covered with FLUROTEC ™. The samples were stored at 30 ° C and at 40 ° C and stability for quality (CAC) and purity (SEC, IEC) were evaluated. The stability results showed that the Pertuzumab in the histidine phosphate buffer degraded much more rapidly than in other histidine buffer by storage at 40 ° C (Fig. 8 and Fig. 9).

EXAMPLE 4 Pertuzumab was concentrated by ultrafiltration / diafiltration at various concentrations in the following buffers: (1) 20 mM histidine-acetate, pH 6.0; (2) 10 mM histidine HCl, pH 6.0, and (3) 10 mM histidine sulfate, pH 6.0.

The turbidity of each formulation was measured before filtering. The results, as shown in Fig. , showed that Pertuzumab samples formulated in histidine acetate and histidine HC1 had less amounts of insoluble aggregates than histidine sulfate buffer.

EXAMPLE 5 Pertuzumab was formulated at 30 mg / mL in 20 mM histidine acetate, 120 mM sucrose, 0.02% polysorbate 20, at pH 6.0. Pertuzumab was introduced into HASTELLOY ™ miniature stainless steel tanks and 316 L. All samples were stored at -20 ° C and 5 ° C and evaluated for quality (CAC), purity (SEC, IEC) and the resistance (UV-Vis). Stability analyzes showed that Pertuzumab was stable in this formulation in storage at -20 ° C and at 5 ° C for at least 3 months. The chloride free formulation is compatible with the 316L stainless steel tank and HASTELLOY ™.

Table 4. Pertuzumab Stability in Stainless Steel Tanks a Step for color, appearance and clarity: clear to slightly opalescent solution, colorless to pale yellow.

EXAMPLE 6 Pertuzumab was formulated using tangential flow filtration (TFF). The final formulation contains 20 m of histidine acetate, 120 mM of sucrose, 0.02% of polysorbate 20, at pH 6.0 at a protein concentration of 30 mg / mL. Samples were placed in Type I T-glass bottles, at 20 MI VITRUM ™ USP SHAPE capped with 20 mm butyl rubber stoppers, lined with FLUROTEC ™, and sealed with aluminum flip-top covers. All samples were stored at -70 ° C, 5 ° C, 15 ° C, and stability was evaluated to determine quality (CAC), purity (SEC, IEC), resistance (UV-Vis), and power (Bioassay). The results showed that Pertuzumab is stable in this formulation by storage at 5 ° C and 15 ° C for at least 3 months.

Table 5. Pertuzumab Stability in Glass Jars EXAMPLE 7 Pertuzumab was formulated at 100 mg / mL, under the following buffer conditions: (1) 10 mM histidine-HCl, pH 6.0; (2) 10 mM histidine-HCl, 240 mM sucrose, at pH 6.0; (3) 20 mM succinate at pH 6.0; and (4) 20 mM succinate, 240 mM sucrose at pH 6.0. Each formulation was added with a different concentration of polysorbate 20. All the samples were placed in Type I USP 3-ounce glass bottles, and were shaken horizontally at 70 rpm at room temperature for up to 7 days. The stability of each sample was evaluated at 7 days to observe the turbidity. The results showed that the use of polysorbate 20 in the final formulation effectively prevented the formation of insoluble aggregates. See Figure 11.

EXAMPLE 8 Pertuzumab was prepared in the following formulations: (1) 25 mg / mL Pertuzumab, 10 mM Histidine-HCl, 240 mM sucrose, a? 6.0; (2) 50 mg / mL of Pertuzumab, 10 mM of histidine-HCl, 240 mM of sucrose, at pH 6.0; (3) 60 mg / mL of Pertuzumab, 20 mM of histidine acetate, 120 mM of sucrose, at pH 6.0. Various amounts of polysorbate 20 were added to each formulation. All samples were placed in Type I USP 3-ounce glass bottles and agitated horizontally at 70 rpm at room temperature for up to 7 days. The physical stability of each sample was evaluated at 7 days to determine the turbidity. The results showed that the use of polysorbate 20 in the formulation of sucrose and histidine-HCl effectively prevented the formation of insoluble particles. The formulation containing histidine acetate and sucrose seemed to have the best protective effect on the protein as polysorbate 20. See Figure 12.

EXAMPLE 9 Pertuzumab was formulated in the following manner: (1) 100 mg / mL protein, 10 mM Histidine-HCl, at pH 6.0; (2) 100 mg / mL protein, 20 mM succinate, at pH 6.0; (3) 60 mg / mL protein, 20 mM histidine acetate at pH 6.0.

Each formulation was mixed with different amounts of sucrose. All samples were sterilized into 3-C Type I USP glass bottles. Then they were frozen at -70 ° C and thawed at 5 ° C three times. The physical stability of each sample was determined after freeze and thaw cycles. The results showed that sucrose prevents the formation of soluble aggregates during the freezing and thawing process. See Figure 13.

EXAMPLE 10 The preferred Pertuzumab formulation for therapeutic use consists essentially of 30mg / mL of Pertuzumab in 20mM histidine acetate, 120mM sucrose histidine, 0.02% polysorbate 20, at pH 6.0.

PM: Molecular weight.

Configuration of the bottle of a dose of 420 Vial: Formal Type I Glass Vitrum of 20 cc Cap: 20 mm DAIKYO GRAY ™, laminated with fluor-resin Cover: aluminum flip top of 20 mm Filling Volume: 14,50 mL Delivery: 14.0 mL of Pertuzumab in a normal saline IV bag.

Bottle configuration in 1050 mg dose: Bottle: Vitrum Formal Type I Glass, 50 ce Cap: 20 mm DAIKYO GRAY ™, fluor-resin laminate Cover: 20 mm aluminum flip top Filling Volume: 36.0 mL Delivery: 35.0 mL of Pertuzumab in a normal saline IV bag.

EXAMPLE 11 This example refers to another formulation of Pertuzumab that was used in clinical trials in Phase I and Phase II. The composition consists of 25 mg / ml Pertuzumab, 10 mM histidine HC1 buffer, 240 mM sucrose, 0.02% polysorbate 20, at pH 6.0.

Polysorbate 20 0.2 mg / ml (0.02%) EXAMPLE 12 Cell apoptosis is mediated by intrinsic and extrinsic pathways. Chemotherapy can cause cell deterioration and can trigger apoptosis through the intrinsic pathway in response to cell deterioration. However, cancer cells often develop resistance to chemotherapy through mutations in the p53 tumor suppressor gene (Ashkenazi A. Targeting Death and Decoy Receptors of the Tumor-Necrosis Factor Superfamily, Nature Reviews 2: 420-430 (2002 )). Mortality receptors, such as DR4 and DR5, located on the surface of cells trigger apoptosis through the extrinsic pathway that does not involve p53. Agonist molecules, such as Apo2L, bind to DR4 and DR5 receptors, and activate caspases 8 and 10 through the mortality domain associated with Fas. Caspase 8 and 10 then activate caspases 3, 6, and 7 to induce apoptosis. The molecular signal of the mortality receptors on tumor cells has therapeutic potential for the elimination of cancer cells that are resistant to conventional therapies, and molecules such as Apo2L, are currently under clinical evaluation. "Apomab" is a humanized IgGl derived from CHO full length, built with a light chain lamda. It is an agonist antibody against DR5 has been shown to induce apoptosis of several cancer cell lines. Preclinical studies using a murine tumor implant model have shown that Apomab has a similar or improved tumor reduction compared to Apo2L. Apomab is being evaluated as an anti-cancer agent in the indications of advanced solid tumors and in Non-Hodgkin's Lymphoma (NHL). The heavy and light chain amino acid sequences of Apomab used in these experiments are shown in Figures 27 and 28.

Preparation of Antibody Formulations The recombinantly produced Apomab has a very diluted concentration of protein and a high pH. The material was concentrated to approximately 20 mg / mL and exchanged in 20 mM sodium acetate, pH 5.0 buffer using a tangential flow filtration system (TFF) from Milipore Labscale, with a 50 cm membrane MILLIPORE PELLICON ™ XL, PLCGC10. Apomab samples were formulated in several buffer systems covering a pH range of 4.0 to 7.0 using sodium acetate, histidine acetate, and sodium phosphate without trehalose and TWEEN 20® using dialysis with a membrane cut-off. a molecular weight of 10,000 Da (Pierce, Inc). Trehalose was added at 240 mM in the last dialysis. After dialysis, 0.02% TWEEN 20 ™ was added to the formulation and the samples were filtered with 0.22 μm filters (Millipore, Inc.). A volume of 0.5 mL of Apomab was introduced into sterile 3-ce glass bottles (Form Vitrum, Inc.) and sealed with 13 mm stoppers (Daikyo, Inc.). The stability of the protein was evaluated at -70 ° C, 5 ° C, 30 ° C, and 40 ° C with storage for up to 3 months.

Stability of the Formulation of Apomab For the stability tests of the pharmacological product, Apomab formulated in mass, it was introduced in VITRUM® glass bottles of 5 ce. The bottles were filled with 5.5 mL of the formulated antibody, provided with 20 mm DAIKYO® plugs, and stored at -70 ° C, 5 ° C, 30 ° C, and 40 ° C in the upright position. For the stability test of the drug substance, the mass-formulated Apomab was sterilized through a 0.22 mm filter and 10 mL were introduced into 316L stainless steel mini-tanks in a 20cc autoclave. The tanks were placed upright at -20 ° C and 5 ° C. A 1 mL aliquot was removed aseptically from the mini-tanks at specified time intervals to verify the quality of the protein. The control vials were 1 mL aliquots in 3-ce glass bottles stored at -20 ° C.

Color, Appearance, and Clarity The clarity, appearance, and color of the samples were visually verified under a white fluorescent light using a light inspection station with a black and white base. For the analysis of the pharmacological substance, the samples from the mini-tanks were transferred to 3-cc glass jars for inspection. pH The pH was measured at room temperature with THERMO ORION SURE-FLO ROSS ™ semi-micro pH electrodes to measure the buffers or a THERMO ORION GLS ™ combination micro-pH electrode to measure the pH-tracking samples of the protein , a Beckman microelectrode probe, for stability samples of Toxicology. The METERLAB ™ pHM240 pH / Ion meter (Radiometer Analytical) was calibrated every day with buffer standards (EM Science) at pH 7 and pH 4.

Concentration The protein concentration was determined by ultraviolet absorption spectroscopy using an AGILENT 8453 ™ spectrophotometer. The samples were diluted with white buffer of appropriate formulation to provide an absorbance of 0.5 to 1.0. The instrument was bleached with diluting solution and the spectrum was scanned from 240 to 500 nm. The absorbance value at 320 nm was subtracted from the absorbance at 279 nm to correct for deviations and light scattering. Protein concentrations were calculated by the following equation: Conc. (Mg / mL) = (A279 - A320) Dilution factor absorption coefficient in cm "1 (mg / mL) - i The coefficient of absorptivity based on the sequence was initially determined as 1.32 cm "1 (mg / mL) -1 and this value was used for the pH tracking studies, a posterior value of 1.7 cm'1 ( mg / mL) _1 was determined by amino acid analysis and proteolysis methods and this value was used for the stability analysis of Apomab used in Toxicology studies.

Ion Exchange Interchange Chromatography Ion exchange chromatography was performed on 1100 series HPLC (Agilent Technologies, Inc.) equipped with diode array detector. Chromatography was carried out on a PROPAC WCX-10 ™ column (Dionex) (4 x 250 mm), at a flow rate of 0.5 mL / min and at a column temperature at 40 ° C. Mobile phase A was 25 mM sodium phosphate pH 6.5. Mobile phase B was 100 m sodium chloride in the same buffer as mobile phase A. The column was equilibrated with 100% mobile phase A. For the pH tracking samples, an amount of 20 mg of Apomate on the column, and the absorbance was monitored at 214 nm. The protein was eluted from the column with the following gradient: Time (min)% A% B 100 0 0 100 100 0 100 0 For the stability analysis of the material used in the Toxicology studies, an amount of 30 mg was charged Apomate on the column, and the absorbance was monitored at 280 nm. The protein was eluted from the column with the following gradient: Gradient: Time (min)% _ A% _B 0 100 0 40.0 40 60 41.0 0 100 45.0 0 100 45.1 100 0 60.0 100 0 Exclusion Chromatography Size Size exclusion chromatography was performed on 1100 series HPLC (Agilent Technologies, Inc.) equipped with a diode array detector .. An amount of 50 ug of Apomab was loaded onto a TSK Gel 3000SWXL ™ column (7 , 8 x 300 mm) and operated at a flow rate of 0.9 mL / min for 20 minutes to monitor the pH of the samples, and at 0.5 mL / min for 30 minutes for the Toxicology stability samples, with 0.20 M potassium phosphate, 0.25 M potassium chloride; at pH 6.2 as mobile phase. The absorbance was monitored at 280 nm. Power The purpose of the power bioassay was to measure the ability of Apomab to kill Colo205 cells using ALAMARBLUE ™. Colo205 is a colon carcinoma cell line, which expresses both mortality receptors DR5 and DR4. This assay incorporates a fluorometric / colorimetric growth indicator, based on the detection of metabolic activity. ALAMARBLUE ™ is a redox dye that is blue and non-fluorescent in the oxidized state. The intracellular metabolic reduction makes it a red color that is also fluorescent. The changes in color and fluorescence are proportional to the metabolic activity and the number of living cells. The signal decreases when the cells die. The Apomab is diluted in an anti-Fc medium and then Coló 205 cells are added to the Apomab samples, and incubated at 37 ° C for 48 hours. ALAMARBLUE ™ was added during the last 2-3 hours. The plate was read at 530 nm excitation and with an emission of 590 nm to obtain relative fluorescence units (RFU). The data was analyzed with KALE I DAGRAPH ™. A death dilution curve was generated.

RESULTS Formulation pH Tracking Study The effect of pH on the stability of the antibody was studied using the Apomab produced from an unamplified stable cell line. For this analysis, Apomab was formulated at 20 mg / mL of antibody in 20 m of sodium acetate buffer at pH 4.0, 4.5, 5.0, 5.5; and 20 mM histidine acetate buffer at pH 6.0 and 6.5; and 20 mM sodium phosphate buffer at pH 7.0. All formulations contained 240 mM trehalose and 0.02% TWEEN 20®. The formulations were stored for up to 3 months at temperatures of -70 ° C, 5 ° C, 30 ° C, and 40 ° C and the stability of the protein was determined by various analytical assays including CAC, pH, concentration, SEC and IEC. No significant changes were observed during the storage of the samples, in the concentration of CAC, pH or protein. The analysis of the samples by SEC showed that no significant changes occurred during storage at 5 ° C and -70 ° C. However, degradation was observed as the formation of antibody fragments and soluble aggregates occurred during storage at 30 ° C and 40 ° C (Fig. 20). To compare the formulations, the kinetics of the antibody monomer during storage was monitored, and the first-order regimen constants were calculated. The profile of the pH regime obtained for the loss of antibody monomer is shown in Figure 21. The optimal condition for the stability of the antibody monomer was obtained by formulation in histidine acetate buffer at pH 6.0. The heterogeneity of the Apomab load was monitored with IEC. No significant change occurred in the IEC profile during storage at 5 ° C and -70 ° C. However, degradation was observed as the formation of acidic or basic variants occurred depending on the formulation (Fig. 22). In general, the increase in basic variants was formed at a lower pH of formulation, and more acidic variants were formed at a higher pH of the formulation. To compare the formulations, the main peak IEC kinetics were monitored during storage and the first-order regimen constants were calculated. The profile of the pH regime obtained for the loss of IEC main peak is shown in Figure 23. The constants of the regime observed by IEC were approximately 10 times higher than those of SEC (Fig. 21). Therefore, the main peak loss of IEC was the primary degradation of the antibody that will ultimately limit the shelf life of the product. In addition, as observed for SEC, the optimal antibody stability to stabilize the main IEC peak was obtained by formulation in histidine acetate buffer at pH 6.0. After analysis of the pH screening data described above, an Apomab formulation comprising 20 mg / mL of antibody was selected in 20 mM histidine acetate, 240 mM trehalose, 0.02% polysorbate 20, at pH 6.0 For the pharmacological product, the configuration of the bottle consisted of 5.5 mL of filling, in a FORMA VITRUM ™ bottle of 5 ce, with a DAIKYO ™ West 20 mM stopper. The Apomab was stored in stainless steel tanks.

The stability of the Apomab drug product was evaluated in the 5cc glass bottle configuration described above. The bottles were stored at -70 ° C (controls), 5 ° C, 30 ° C, and 40 ° C. The bottles were taken at specific time intervals and analyzed for the following tests: color, appearance, clarity (CAC), pH, protein concentration, SEC, IEC and potency. The results of these tests were shown in Table 6 for the samples stored at -70 ° C and 5 ° C and in Table 7 for the samples stored at 30 ° C and 40 ° C.

Table 6. Stability Data for Apomab Stored at -70 ° C and at 5 ° C Power Concentrac SEC (ion {% IEC Activida Temp Time Clarity Color PH (mg / mL) monomer (% peak d (° C) Point) Main Specifi ca) Criteria of 6.0 60 - Appearance: Report Report +0.3 20 + 2 > 95% Report 140% NA T = 0 Clear Colorless 5, 9 20, 2 99, 8 63 94 -70 1 Clear Colorless 6, 0 20, 5 99, 8 63 86 month -70 2 Colorless Clear 6, 0 20, 4 99, 7 64 91 month -70 3 Clear Colorless 6, 0 20, 5 99, 7 63 83 month -70 6 Light colorless 6, 0 20, 4 99, 7 64 85 month -70 9 Light I ncoloro 6, 0 20, 4 99, 8 65 89 month -70 12 Light colorless 6, 0 20, 8 99, 7 63 107 month 5 1 Clear Colorless 6, 0 20, 5 99, 7 63 89 month 5 2 Clear Colorless 6.0 20, 4 99, 7 64 99 month 5 3 Clear Colorless 6, 0 20, 6 99, 7 63 84 month 5 6 Clear Colorless 6.0 20, 5 99, 7 64 93 month 5 9 Clear Colorless 6, 0 20, 6 99, 7 64 88 month 5 12 Clear Colorless 6.0 20.7 99, 6 64 106 month Table 7. Stability Data for Aporaab Stored at 30 ° C and 40 ° C Potency (% of activity SEC Temp Concentration Time (% IEC (% peak speci (° C) Clarity Point Color pH (mg / mL) main monomer) Eica) Criteria of 6.0 60 acceptance: Info me Report + 0.3 20 + 2 > 95% Report 140t 1 Clear Colorless 6.0 20, 6 98, 2 59 91 month 30 2 Clear Colorless 6.0 20.3 97, 4 54 80 month 30 3 Clear Colorless 6, 0 20, 6 97, 2 49 74 month 30 6 Clear Colorless 6.0 20.2 94, 1 37 51 month 30 9 Lightly Slightly 6.0 20.4 93, 2 31 55 month yellow 30 12 Lightly Slightly 6.0 20, 6 91.6 25 59 month yellow 40 1 Clear Colorless 6, 0 20.4 96, 6 44 79 month 40 2 Clear Colorless 6, 0 20.0 93.7 31 64 month 40 3 Clear Lightly 5, 9 20, 3 91, 5 22 53 month yellow 40 6 month Lightly Light 6, 0 20.2 83, 9 NT 26 yellow 40 9 Light Yellow 5, 9 20, 3 78.8 NT 25 month 40 12 Light Yellow 5, 9 20, 5 71, 4 NT 31 month NT = not quantified No change in the quality of the protein was observed after 12 months of storage at -70 ° C and 5 ° C. For example, the pH remained at 6.0 ± 0.3, the Apomab presented as a clear and colorless liquid, the protein concentration remained at 2.0 ± 2.0 mg / mL, and the percentage of monomer did not change . In addition, no significant change was observed in the percentage of IEC main peak, and in the percentage of specific activity determined by the cell death potency test, which was within the assay accuracy of 60% to 140% activity specify The results showed that the Apomab stored in glass bottles of 5 ce was stable for at least 12 months at 5 ° C. Table 7 shows that changes in protein quality occurred at 30 ° C and 40 ° C. The SEC showed a decrease in the percentage of monomer, with an elevation mainly in the fragment species. The aggregates increased as well as the higher temperature, but the regime was much slower. However, the aggregates increased significantly after 6 months at 40 ° C. The percentage of IEC of main peak decreased with a corresponding increase of the acidic variants. The basic peaks decreased slightly after two months at 40 ° C and 9 months at 30 ° C. After six months of storage at 40 ° C, degradation occurred to a degree where the main IEC peak could not be integrated. The cell death bioassay showed a percentage loss of specific activity at higher temperatures, with a shorter storage time. The protein concentration and the pH did not vary. The solution became slightly yellow after 3 months at 40 ° C and 9 months at 30 ° C, and became yellow after 9 months at 40 ° C.

Stability of the Pharmacological Substance The freeze-thaw stability data for the drug substance are shown in Table 8.

Table 8. Freezing-Defrosting Stability Data for Apomab Introduced in Frozen Miniature Stainless Steel Tanks - Temp (° C) thawed Concent SEC (Frozen / thawed or ration (% side Cycle No. Clarity Color pH (mg / mL) Monomer) Acceptance of Criterion: Report Report 6.0 + 0 20, 0 + > _95% 2.0 Control (no 0 Clear Colorless 6, 0 20, 9 99, 6 frozen) -20/25 1 Clear Colorless 6, 0 20, 8 99, 6 -20/25 2 Clear Colorless 6, 0 20, 8 99, 6 -20/25 3 Clear Colorless 6, 0 20, 9 99, 6 No significant changes in the chemical characteristics of the protein were observed after freezing at -20 ° C for at least 15 hours, and thawing at room temperature three times. For example, the Apomab was shown as a clear and colorless liquid, the pH remained at 6.0 ± 0.3, and the peak percentage of the SEC monomer did not vary. The stability of the Apomab in stainless steel containers was evaluated at -20 ° C and 5 ° C (Table 9).

Samples were taken from Table 9 mini-tanks. Stability Data for the Apomab Introduced in Stainless Steel Tanks Miniature Power (% SEC (IEC (1 activity Temp Time Specific peak concentration (° C) Point Clarity Color pH - (mg / mL) main monomer)) 6, 0 Acceptance of + Criterion: Report Report 0.3 20 + __ 2 > 95% In orme t 60 - 140% NA T = 0 Clear Colorless 5.9 20.0 99.7 63 88 -20 1 Clear Colorless 6.0 20, 6 99.7 63 107 month -20 3 Colorless Cycle 6.0 20, 6 99.7 63 82 month -20 6 Clear Colorless 6.0 20, 3 99.7 64 92 month -20 9 Cycle Colorless 6.0 20.6 99.7 64 92 month -20 12 Cycle Colorless 6.0 21.2 99.7 65 94 month 5 1 Colorless clear 6.0 20.5 99, 7 62 95 month 5 3 Cla or Colorless 6.0 20.7 99, 6 62 71 month 5 6 Clear Colorless 6.0 20, 4 99.5 62 84 month 5 9 Clear Colorless 6.0 20.8 99.4 61 84 month 5 12 Clear Colorless 6.0 21, .3 99.2 59 82 month aseptically at specific intervals and analyzed.

The Apomab did not show any change in the quality of the protein at 5 ° C per concentration of pH, CAC, protein and the percentage of main peak for IEC but lost 0.1% of monomer by SEC every 3 months. A decreased potency was observed during storage at 5 ° C for 3 months. However, the power of the sample increased again at 6 and 9 months. Therefore, the power difference observed at the 3-month time point was attributed to a variation of the test. Apomab showed no change in protein quality at -20 ° C by pH, CAC, protein concentration, percentage of monomer by SEC, percentage of main peak by IEC, and no significant change in potency. The stability data show that the Apomab is stable for at least 1 year at -20 ° C and three months at 5 ° C.

CONCLUSION Formulation screening studies were conducted to select a formulation for Apomab. A pH screening covering the pH range of 4.0 to 7.0 using sodium acetate, histidine acetate, and sodium phosphate as buffers, with 240 mM of trehalose dihydrate and 0.02% of polysorbate 20 showed that the Apomab is the most stable in solution at pH 6.0. Therefore, a formulation consisting of 20 mM histidine acetate, 240 mM trehalose, 0.02% polysorbate 2, at pH 6.0 was developed and experimentally demonstrated to be stable. Using this formulation, the Apomab showed that it was stable for at least 12 months at 5 ° C. In addition, the Apomab showed that it was stable for at least 12 months at -20 ° C and three months at 5 ° C when the 316L stainless steel containers were stored. The Apomab also showed that it was stable when subjected to up to three freeze / thaw cycles.

Claims (79)

1. REIVINDICATION Having thus specially described and determined the nature of the present invention and the manner in which it is to be put into practice, it is claimed to claim as proprietary and exclusive right: 1. A stable pharmaceutical formulation comprising a monoclonal antibody in buffer of histidine acetate, at pH 5.5 to 6.5.
2. 2. The formulation of claim 1 wherein the pH is from 5.8 to 6.2.
3. 3. The formulation of claim 1 wherein the concentration of histidine acetate buffer is from about ImM to about 200mM.
4. 4. The formulation of claim 3 wherein the concentration of histidine acetate buffer is from about 10mM to about 40mM.
5. 5. The formulation of claim 1 wherein the antibody concentration is from about 10mg / mL to about 250mg / mL.
6. 6. The formulation of claim 5 wherein the concentration of monoclonal antibody is from about 20 mg / mL to about 40 mg / mL.
7. 7. The formulation of claim 5 wherein the concentration of monoclonal antibody is from about 80 mg / mL to about 250 mg / mL.
8. The formulation of claim 1 further comprising saccharide.
9. 9. The formulation of claim 8 wherein the saccharide is a disaccharide.
10. The formulation of claim 8 wherein the saccharide is trehalose.
11. The formulation of claim 8 wherein the saccharide is sucrose.
12. 12. The formulation of claim 8 wherein the concentration of saccharide is from about 10mM to about 1M.
13. The formulation of claim 12 wherein the concentration of saccharide is from about 60mM to about 250mM.
14. The formulation of claim 1 further comprising a surfactant.
15. 15. The formulation of claim 14 wherein the surfactant is polysorbate.
16. 16. The formulation of claim 15 wherein the surfactant is polysorbate 20.
17. 17. The formulation of claim 14 in which the concentration of surfactant is from about 0.0001% to about 1.0%.
18. 18. The formulation of claim 17 wherein the concentration of surfactant is from about 0.01% up to about 0.1%.
19. 19. The formulation of claim 1 wherein the monoclonal antibody is a full-length antibody.
20. The formulation of claim 19 wherein the monoclonal antibody is an IgG1 antibody.
21. The formulation of claim 1 wherein the monoclonal antibody is a humanized antibody.
22. 22. The formulation of claim 1 wherein the monoclonal antibody is an antibody fragment comprising an antigen binding region.
23. 23. The formulation of claim 22 wherein the antibody fragment is a Fab or F (ab ') 2 fragment.
24. 24. The formulation of claim 1 which is sterile.
25. 25. The formulation of claim 1 wherein the monoclonal antibody binds to an antigen selected from the group consisting of HER2, CD20, DR5, BR3, IgE, and VEGF.
26. 26. The formulation of claim 25 wherein the antigen is CD20 and the monoclonal antibody is humanized 2H7.
27. 27. The formulation of claim 25 in which the antigen is VEGF and the monoclonal antibody is Bevacizumab.
28. 28. The formulation of claim 1 wherein the monoclonal antibody is susceptible to deamidation or aggregation.
29. 29. The formulation of claim 1 which is stable in storage at about 40 ° C for at least 4 weeks.
30. 30. The formulation of claim 1 which is storage stable at about 5 ° C about 15 ° C for at least 3 months.
31. 31. The formulation of claim 1 which is storage stable at about -20 ° C for at least 3 months.
32. 32. The formulation of claim 1 which is stable in freezing and thawing.
33. 33. The formulation of claim 1 which is aqueous. 3 .
34. The formulation of claim 1 which is frozen.
35. 35. The formulation of claim 1 which is not lyophilized and which has not been subjected to prior lyophilization.
36. 36. The formulation of claim 35 which is aqueous and which is administered to a subject.
37. 37. The formulation of claim 36 wherein the formulation is for intravenous (IV), subcutaneous (SC) or intramuscular (IM) administration.
38. 38. The formulation of claim 37 which is for IV administration and the antibody concentration is from about 20mg / mL to about 40mg / mL.
39. 39. The formulation of claim 37 which is for SC administration and where the antibody concentration is from about 80mg / Ml to about 250mg / mL.
40. 40. A bottle with a stopper pierceable by means of a syringe, comprising the formulation of claim 1 inside the bottle.
41. 41. The bottle of claim 40 which is stored at about 2-8 ° C.
42. 42. The bottle of claim 40 which is a 20cc or 50cc bottle.
43. 43. A stainless steel tank comprising the formulation of claim 1 inside the tank.
44. 44. The tank of claim 43 in which the formulation that is therein is frozen.
45. 45. A pharmaceutical composition comprising the formulation of claim 1 for treating a disease or disorder in a subject, in an amount effective to treat the disease or disorder.
46. 46. A pharmaceutical formulation comprising: (a) a full-length IgGl antibody susceptible to deamidation or aggregation in an amount of from about 10mg / mL to about 250mg / mL; (b) histidine acetate buffer, at pH 5.5 to 6.5; (c) saccharide selected from the group consisting of trehalose and sucrose, in an amount of from about 60mM to about 250mM; and (d) polysorbate 20 in an amount of from about 0.01% to about 0.1%.
47. 47. A method for reducing the deamidation or aggregation of a therapeutic monoclonal antibody, which comprises formulating the antibody in a histidine buffer at pH 5.5 to 6.5.
48. 48. The method of claim 47 which comprises evaluating any antibody aggregation or deamidation before and after formulating the antibody.
49. 49. A pharmaceutical formulation comprising an antibody that binds to domain II of HER2 in a histidine buffer at a pH of from about 5.5 to about 6.5, a saccharide and a surfactant.
50. 50. The formulation of claim 49 wherein the buffer is histidine acetate.
51. 51. The formulation of claim 49 wherein the HER2 antibody comprises the light and variable variable chain amino acid sequences in SEQ ID NOS. 3 and 4, respectively.
52. 52. The formulation of claim 51 wherein the HER2 antibody comprises a light chain amino acid sequence selected from SEQ ID Nos. 15 and 23, of a heavy chain amino acid sequence selected from SEQ ID NOS. 16 and 24.
53. 53. The formulation of claim 49 wherein the pH of the formulation is from about 5.8 to about 6.2.
54. 54. The formulation of claim 49 wherein the antibody binds to the junction between domains I, II, and III of HER2.
55. 55. The formulation of claim 49 wherein the antibody is a full-length antibody.
56. 56. The formulation of claim 49 wherein the concentration of antibody is from about 20mg / mL to about 40mg / mL.
57. 57. A pharmaceutical formulation comprising Pertuzumab in an amount from about 20mg / mL to about 40mg / mL, histidine acetate buffer, sucrose, and polysorbate 20, where the pH of the formulation is from about 5.5 to about 6. ,5.
58. 58. The formulation of claim 57 comprising about 30mg / mL of Pertuzumab, approximately 20m of histidine acetate, about 120mM sucrose, and about 0.02% polysorbate 20, where the pH of the formulation is from about 6.0.
59. 59. A bottle with a plug pierceable by a syringe, comprising the formulation of claim 49.
60. 60. A stainless steel tank comprising the formulation of claim 49 in the tank.
61. 61. A pharmaceutical composition for the treatment of cancer expressing HER2 in a subject by administering the pharmaceutical formulation of claim 49 to the subject in an amount effective to treat cancer.
62. 62. The pharmaceutical composition of claim 61 wherein the formulation is administered to the subject intravenously, subcutaneously or intramuscularly.
63. 63. A method for preparing a pharmaceutical formulation comprising: (a) preparing the formulation of claim 1; and (b) evaluating the physical stability, chemical stability or biological activity of the monoclonal antibody in the formulation.
64. 64. A pharmaceutical formulation comprising an i DR5 antibody in a histidine buffer at a pH of from about 5.5 to about 6.5, a saccharide and a surfactant.
65. 65. The formulation of claim 64 wherein the buffer is histidine acetate.
66. 66. The formulation of claim 64 wherein the DR5 antibody is an agonist antibody.
67. 67. The formulation of claim 64 wherein the DR5 antibody is Apomab.
68. 68. The formulation of claim 67 wherein the DR5 antibody comprises the heavy chain amino acid sequence of SEQ ID No. 51, and the light chain amino acid sequence of SEQ ID No. 52.
69. 69. The formulation of claim 64 in which the pH of the formulation is from about 5.8 to about 6.2.
70. 70. The formulation of claim 64 wherein the antibody is a full-length antibody.
71. 71. The formulation of claim 64 wherein the antibody concentration is from about 10mg / mL to about 30mg / mL.
72. 72. A pharmaceutical formulation comprising Apomab in an amount of from about 10mg / mL to about 30mg / mL, histidine acetate buffer, trehalose, and polysorbate 20 wherein the pH of the formulation is from about 5.5 to about 6. ,5
73. 73. The formulation of claim 72 comprising about 20mg / mL of Apomab, about 20mM of histidine acetate, about 240mM of trehalose and about 0.02% of polysorbate 20 where the pH of the formulation is about 6.0.
74. 74. A bottle with a plug pierceable by a syringe, comprising the formulation of claim 64.
75. 75. A stainless steel tank comprising the formulation of claim 64 in the tank.
76. 76. A pharmaceutical composition for the treatment of cancer in a subject by administering the pharmaceutical formulation of claim 64 to the subject in an amount effective to treat cancer.
77. 77. The pharmaceutical composition of claim 76 wherein the cancer is a solid tumor.
78. 78. The pharmaceutical composition of claim 76 wherein the cancer is a non-Hodgkin's lymphoma.
79. 79. The pharmaceutical composition of claim 76 wherein the formulation is administered to the subject intravenously, subcutaneously or intramuscularly.