Antibody formulation in histidine-acetate buffer

THE REPUBLIC OF POLAND (12) TRANSLATION OF THE EUROPEAN PATENT (19) PL (11) PL / EP 1802344

<img file="PL1802344T3_D0001.tif" />

Patent Office of the Republic of Poland (96) Date and number of the European patent application: October 19, 2005 05815641.5 (97) The grant of the European patent was announced:

2012-08-15 European Patent Bulletin 2012/33 EP 1802344 B1 (13) T3 (51) Int.CI.

A61K 39/395 (2006.01) (54) Title of the invention:

Formulation of the antibody in a histidine-acetate buffer (3 °) r,

Priority:

20.10.2004 US 620413 P (43) Application announced:

On July 4, 2007 in the European Patent Bulletin No. 2007/27 (45) The following was announced about the submission of the translation of the patent:

31.01.2013 Patent Office News 2013/01 (73) Patent holder:

Genentech, Inc., South San Francisco, US (72) Inventor (s):

JAMES D. ANDYA, Millbrae, US SHIANG C. GWEE, Pacifica, US JUN LIU, Pacifica, US

YE SHEN, San Francisco, US (74) Agent:

thing, pat. Piotr Godlewski JAN WIERZCHOŃ &amp; PARTNERS OFFICE OF PATENTS AND ZNAKÓW TOWAROWYCH SP. J. ul. Żurawia 47/49

00-680 Warsaw

Attention:

Within nine months of the publication of the information on the grant of the European patent, any person may file an objection to the European Patent Office against the European patent granted. The objection must be submitted in the form of a written statement of reasons. It is considered brought only when the opposition fee has been paid (Art. 99 (1) of the Convention on the Grant of European Patents).

14966/12 / P-RO / PG / KM

EP 1 802 344

Formulation of the antibody in a histidine-acetate buffer

[0001] The invention relates to antibody formulations, including monoclonal antibodies formulated in a histidine-acetate buffer, as well as a formulation comprising an antibody that binds to an IIHER2 domain (for example, Pertuzumab).

[0002] Over the past ten years, advances in biotechnology have made it possible to produce various proteins for pharmaceutical applications using recombinant DNA techniques. Since proteins are larger and more complex than traditional organic and inorganic drugs (i.e. possess numerous functional groups in addition to the complex three-dimensional structure), the formulation of such proteins presents special problems. In order for a protein to remain biologically active, the formulation must maintain intact the conformational integrity of at least the core amino acid sequence of the protein while at the same time protecting multiple functional groups of the protein from degradation. Degradation pathways for proteins can include chemical instability (i.e., any process involving the modification of a protein by bond formation or cleavage, resulting in a new chemical molecule) or physical instability (i.e. changes in higher-order protein structures). Chemical instability may result from deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Physical instability can result from, for example, denaturation, aggregation, precipitation, and adsorption. The three most common pathways of protein degradation are protein aggregation, deamidation and oxidation. Cleland et al. Critical Reviews in Therapeutic Drug Carrier Systems 10 (4): 307-377 (1993).

Antibody Formulation

[0003] Proteins used in pharmaceutical applications include antibodies. An example of an antibody useful for therapy is an antibody that binds to the HER2 antigen, such as Pertuzumab.

[0004] US Patent No. 6,339,142 describes a HER2 antibody composition comprising an anti-HER2 antibody mixture and one or more acidic variants thereof, wherein the amount of the acidic variant (s) is less than about 25%. Trastuzumab is an example of a HER2 antibody.

[0005] US Patent Nos. 6,267,958 and 6,685,940 (Andya et al.) Describe lyophilized antibody formulations, including HER2 and IgE antibody formulations. WO97 / 04807 and USA 2004 / 0197326A1 (Fick et al.) Describe methods of treating allergic asthma using an IgE antibody. WO99 / ​​01556 (Lowman et al.) Relates to an IgE antibody with isomerizable aspartyl residues and improved variants thereof. USA 2002/0045571 (Liu et al.) Provides a concentrated, low-viscosity protein formulation, illustrated with humanized IgE antibody formulations, rhuMAb E25 and E26. WO 02/096457 and US 2004/0170623 (Arvinte et al.) Describe a stable, liquid formulation containing an anti-IgE E25 antibody.

-2See also US 2004/0197324 Al (Liu and Shire) for a high concentration anti-IgE formulation.

[0006] US Patent No. 6,171,586 (Lam et al.) Describes stable, liquid antibody formulations. F (ab ') antibody₂ rhuMAb CD18 was formulated in sodium acetate and histidine-HCl buffers. A preferred formulation for rhuMAb CD 18 was 10mM sodium acetate, 8% trehalose, 0.01% TWEEN 20D, pH 5.0. Acetate formulations (pH 5.0) of rhuMAb CD20, stored at 40 ° for one month, showed greater stability than those samples formulated in histidine (pH 5.0 or 6.0).

[0007] US 2003/0190316 (Kakuta et al.) Relates to a formulated hPM-1 antibody, a humanized IL-6 receptor antibody. Loss of monomerism was greatest in sodium citrate (pH 6.7), then in sodium phosphate (pH 6.8), Tris-HCl (pH 7.2), histidine-HCl (pH 7.2) and glycine (pH 7, 6), in descending order. The influence 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 the stability of hPM-1 was assessed .

[0008] WO2004 / 071439 (Burkę et al.) States that impurities appeared in the formulation of natalizumab (humanized anti-integrin alpha4 monoclonal antibody) resulting from the degradation of polysorbate 80, apparently by oxidation reaction involving metal ions and histidine. For this reason, a phosphate buffer was chosen.

[0009] WO 2000/066160 (English equivalent EP 1 174 148A1) (Okada et al.) Relates to a formulation of a humanized C4G1 antibody that binds to a fibrinogen receptor from a platelet membrane glycoprotein GPIIb / IIIa, in a sodium phosphate buffer or sodium citrate.

[0010] WO2004 / 019861 (Johnson et al.) Relates to CDP870, a pegylated anti-TNFα Fab fragment, formulated at 200mg / ml in 50mM sodium acetate (pH 5.5) and 125mM sodium chloride.

[0011] WO2004 / 004639 (Nesta, P.) relates to a formulation for huC242-DMl, a tumor activated immunotoxin, in 50mM succinic acid (pH 6.0) and sucrose (5% w / v) buffer.

[0012] 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 rapidly lost its potency in histidine as the buffer oxidized.

[0013] WO 2004/001007 relates to a CD80 monoclonal antibody in a histidine-HCl buffer, sodium acetate or sodium citrate.

[0014] US Patent No. 6,252,055 (Relton, J.) relates to anti-CD4 and anti-CD23 antibodies formulated in maleate, amber, sodium acetate, or phosphate buffers, with phosphate specified as the preferred buffer.

[0015] US Patent No. 5,608,038 (Eibl et al.) Relates to highly concentrated preparations of polyclonal antibodies, with immunoglobulin, glucose, sucrose and sodium chloride.

[0016] WO03 / 015894 (Oliver et al.) Relates to aqueous formulations with 100mg / mL SYNAGIS®, 25mM Histidine-HCl, 1.6mM glycine, pH 6.0 and lyophilized SYNAGIS® which when formulated (before lyophilization ), contains 25mM histidine, 1.6M glycine and 3% w / v mannitol at pH 6.0.

[0017] US 2004/0191243 A1 (Chen et al.) Reports the formulation of ABX-IL8 of the human IgG2 antibody.

[0018] US 2003/0113316 Al (Kaisheva et al.) Relates to a lyophilized formulation of an anti-IL2 receptor antibody.

HER2 antibodies

[0019] The HER family of receptor tyrosine kinases is an important mediator of cell growth, differentiation and survival. The receptor family has four different members, including the epidermal growth factor receptor (EGFR, ErbBl, or HER1), HER2 (ErbB2 or pl85^neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).

[0020] EGFR, encoded by the erbB1 gene, is causally related to human tumors. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancers, as well as in gliomas. Increased expression of the EGFR receptor is often associated with increased production by the same tumor cells of the EGFR ligand transforming growth factor alpha (TGF-α), resulting in activation of the receptor through autocrine stimulation. Baselga and Mendelsohn Pharmac. Ther. 64: 127154 (1994). Monoclonal antibodies directed against EGFR or its ligands, TGFα and EGF, have been evaluated as therapeutic agents in the treatment of such cancers. See, e.g., Baselga and Mendelsohn., Supra, Masui et al. Cancer Research 44: 1002-1007 (1984); and Wu et al. J. Clin. Invest. 95: 1897-1905 (1995).

[0021] A second member of the HER family, p185^neu, was initially identified as the transforming gene product of chemically treated rat neuroma. The activated form of the neu proto-oncogene will arise from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human neu homolog is seen in breast and ovarian tumors and correlates with 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). To date, no point mutations analogous to those in the neu proto-oncogene have been reported for human tumors. HER2 overexpression (common, but not uniformly due to amplification), has been observed in other cancers, including cancers of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas, and bladder. See, inter alia, 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); Weiner et al., Cancer Res., 50: 421-425 (1990); Kem et al., Cancer Res., 50: 5184 (1990); Parka 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.,

-4 Cancer, 65: 88-92 (1990). HER2 may be overexpressed in prostate cancer (Gu et al. Cancer 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. Uroi. 150: 126-31 (1993)).

[0022] Antibodies directed against rat p185 have been described^neu and human HER2 protein products. Drebin and colleagues raised antibodies against the rat neu gene product, p185neu. See, for example, Drebin et al., Cell 41: 695706 (1985); Myers et al., Meth. Enzyme. 198: 277-290 (1991); and WO94 / 22478 Drebin et al. Oncogene 2: 273-277 (1988) reports that mixtures of antibodies interacting with two different pl85 regions^neu result in a synergistic anti-tumor effect on neu-transformed NIH-3T3 cells implanted into athymic mice. See also U.S. Patent 5,824,311 issued October 20, 1998.

Hudziak et al., Mol. Cell. Biol. 9 (3): 1165-1172 (1989) describes the production of a panel of HER2 antibodies characterized using the human breast cancer cell line SK-BR-3. The relative proliferation of SK-BR-3 cells after antibody treatment was assessed by staining the monolayers with crystal violet after 72 hours. Using this assay, maximum inhibition was obtained with the antibody called 4D5 which inhibited cell proliferation by 56%. Other antibodies in the panel reduced cellular proliferation to a lesser extent in this study. Additionally, the 4D5 antibody was found to sensitize breast cancer cell lines expressing HER2 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. further 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. Moth. 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): 1466114665 (1994); Scott et al. J. Biol. Chem. 266: 14300-5 (1991); D'souza et al. Proc. Natl. Acad. Sci. 91: 7202-7206 (1994); Lewis et al. Cancer Research 56: 1457-1465 (1996); and Schaefer et al. Oncogene 15: 1385-1394 (1997).

[0024] A recombinant humanized version of the murine HER2 antibody, 4D5 (huMAb4D5-8, rhuMAb HER2, Trastuzumab or HERCEPTIN®; US Patent No. 5,821,337) is clinically active in patients with HER2 overexpressing metastatic breast tumors who have previously received extensive therapy. antitumor (Baselga et al., J. Clin. Oncol. 14: 737-744 (1996)). 25 September 1998 Trastuzumab obtained a market authorization from the US Food and Drug Administration for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2.

[0025] Other HER2 antibodies with different properties are 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

- 5,2589 (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).

[0026] Homology searches have resulted in the identification of two other members of the HER receptor family; HER3 (US Patent Nos. 5,183,884 and 5,480,968, as well as Kraus et al. PNAS (USA) 86: 9193-9197 (1989)) and HER4 (EP Patent Application No. 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA, 90: 1746-1750 (1993); and Plowman et al., Nature, 366: 473-475 (1993)). Both of these receptors show increased expression on at least some breast cancer cell lines.

[0027] HER receptors are generally found in a variety of combinations in cells and heterodimerization is believed to increase the diversity of cellular responses to various HER ligands (Earp et al. Breast Cancer Research and Treatment 35: 115-132 (1995)). EGFR is bound by six different ligands; epithelial growth factor (EGF), transforming growth factor alpha (TGF-α), amphiregulin, heparin binding epithelial growth factor (HB-EGF), betacellulin, and epiregulin (Groenen et al. Growth Factors 11: 235-257 (1994)). The proteins of the heregulin family, arising from single-gene alternative splicing, are ligands for HER3 and HER4. The heregulin family includes the 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 (NDF); glial growth factors (GGF); Acetylcholine receptor stimulating activity (ARIA); and a factor derived from sensory and motor neurons (SMDF). For reviews, see Groenen et al. Growth Factors 11: 235-257 (1994); Lemke, G. Molec. &amp; 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) reported to bind to 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 neuregulin-4, which binds HER4 (Harari et al. Oncogene 18: 2681-89 (1999)), HB-EGF, betacellulin and epiregulin also bind to HER4.

[0028] While EGF and TGFa do not bind HER2, EGF stimulates EGFR and HER2 to form a heterodimer that activates EGFR and results in HER2 transphosphorylation in the heterodimer. Dimerization and / or transphosphorylation appears to activate HER2 tyrosine kinase. See Earp et al., Supra. Similarly, when HER3 is co-expressed with HER2, an active signaling complex is formed and anti-HER2 antibodies are capable of destroying this complex (Sliwkowski et al., J. Biol. Chem., 269 (20): 1466114665 (1994)). Additionally, the affinity of HER3 for heregulin (HRG) is increased to a higher affinity state when it is co-expressed with HER2. See also, Levi et al., Journal of Neuroscience 15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al., Cancer Res., 56: 1457-1465 (1996) with reference

-6 to the HER2-HER3 protein complex. HER4, like HER3, forms an active signaling complex with HER2 (Carraway and Cantley, Cell 78: 5-8 (1994)).

[0029] To target the HER signaling pathway, rhuMAb 2C4 (Pertuzumab, OMNITARG ™) was developed as a humanized antibody that inhibits HER2 dimerization with other HER receptors, thereby inhibiting ligand-induced phosphorylation and activation and activation of the following RAS and AKT pathways. In Phase I trials of Pertuzumab as a single agent for the treatment of solid tumors, Pertuzumab was used to treat 3 subjects with advanced ovarian cancer. One had a sustained partial response and an additional subject had stabilized disease for 15 weeks Agus et al. Proc Am Soc Clin Oncol 22: 192, Abstract 771 (2003).

DR5 antibodies

[0030] Various ligands and receptors have been identified in the art, belonging to the tumor necrosis factor (TNF) superfamily. Such ligands include tumor necrosis factor alpha (TNF-alpha), tumor necrosis factor beta (TNF-beta or lymphotoxin-alpha), lymphotoxin-beta (LT-beta), CD30 ligand, CD27 ligand, CD40 ligand, ΟΧ- ligand 40, 4-1BB ligand, LIGHT, Apo-1 ligand (also referred to as Fas ligand or CD95 ligand), Apo-2 ligand (also referred to as Apo2L or TRAIL), Apo-3 ligand (also referred to as TWEAK), APRIL, OPG ligand (also referred to as RANK ligand, ODF or TRANCE) and TALL-1 (also referred to as BlyS, BAFF or THANK) (See, e.g., 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 Preference, Academic Press, 2000, pp. 377- 411; Locksley et al., Cell, 104: 487-501 (2001); Gruss and Dower, Blood, 85: 3378-3404 (1995); Schmid et al., Proc. Natl. Acad. Sci., 83: 1881 (1986); Dealtry et al., Eur. J. Immunol., 17: 689 (1987); Pitti et al., J. BioL Chem., 271: 12687-12690 (1996); Wiley et al., 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); WO98 / 28426 published Jul 2, 1998; WO98 / 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)).

[0031] Induction of various cellular responses dependent on such ligands of the TNF family is usually initiated by their binding to specific cell receptors. Some, but not all, ligands of the TNF family bind to and induce various biological actions through cell surface death receptors to activate caspases or enzymes that carry out cell death or apoptotic pathways (Salvesen et aL, Cell, 91: 443-446 (1997)) . The TNF receptor superfamily identified to date include TNFR1, TNFR2, TACI, GITR, CD27, ΟΧ-40, CD30, CD40, HVEM, Fas (also referred to as Apo-1 or CD95), DR4 (also referred to as TRAIL-R1), DR5

-7 (also referred to as Apo-2 or TRAIL-R2), DcRl, DcR2, osteoprotegerin (OPG), RANK and Apo-3 (also referred to as DR3 or TRAMP).

[0032] Most of these TNF receptor family members share the typical structure of cell surface receptors, including the extracellular, transmembrane and intracellular regions, while others are found naturally as soluble proteins lacking a transmembrane and intracellular domain. The extracellular portion of a typical TNFR contains repeating patterns of the amino acid sequences of multiple cysteine-rich domains (CRDs) starting at the NH2 terminus.

[0033] The ligand, referred to as Apo-2L or TRAIL, was identified several years ago as a member of the TNF family of cytokines. (see, e.g., Wiley et al. Immunity, 3: 673-682 (1995); Pitti et al., J. Biol. Chem., 271: 12697-12690 (1996); WO 97/01633; WO 97/25428 ; U.S. Patent 5,763,223 granted June 9, 1998; U.S. Patent 6,284,236 granted September 4, 2001). The full-length natural sequence of the human Apo2L / TRAIL polypeptide is a Type II transmembrane protein, 281 amino acids long. Some cells can produce a natural, soluble form of the polypeptide by enzymatic cleavage of the extracellular region of the peptide (Mariani et al., J. Cell. Biol., 137: 221229 (1997)). Crystallographic studies of soluble forms of Apo2L / TRAIL revealed a homotrimeric structure similar to that of TNF and other related proteins (Hymowitz et al., Molec-Cell, 4: 563-571 (1999); Cha et al., Immunity, 11: 253-261. (1999); Mongkolsapaya et al., Nature Structural Biology, 6: 1048 (1999); Hymowitz et al., Biochemistry, 39: 633-644 (2000)). However, it has been found that Apo2L / TRAIL, unlike other members of the TNF family, has the unique structural feature of three cysteine ​​residues (at position 230 of each homotrimer subunit) coordinating together a zinc atom, and that zinc bonding is important for the stability and biological activity of the trimer. (Hymowitz et al., Supra, Bodmer et al., J. Biol. Chem., 275: 20632-20637 (2000)).

[0034] It has been reported in the literature that Apo2L / TRAIL may play a role in the regulation of the immune system, including autoimmune diseases such as rheumatoid arthritis (see, e.g., 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)).

[0035] Soluble forms of Apo2L / TRAIL have also been reported to induce apoptosis in a variety of cancer cells, including colon, lung, breast, prostate, bladder, kidney, ovarian and brain tumors, as well as melanoma, leukemia and multiple myeloma (see e.g. Wiley et al., Supra, Pitti et al., Supra, U.S. Patent 6,030,945 issued Feb.29, 2000; U.S. Patent 6,746,668 issued June 8, 2004; Rieger et al., FEBS Letters, 427: 124128 (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); Mizutani 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. Natl. Acad. Sci., 97: 1754-1759 (2000)). In vivo studies in mouse models further suggest that Apo2L / TRAIL, alone or in combination with chemotherapy or radiation therapy, may have significant anti-inflammatory effects.

- 8 tumor (see, e.g., 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). Unlike many types of cancer cells, most normal human cell types appear resistant to induction of apoptosis by some recombinant forms of Apo2L / TRAIL (Ashkenazi et al., Supra, Walzcak et al., Supra). Jo et al reported that the soluble polyhistidine-labeled form of Apo2L / TRAIL induces apoptosis in vitro in normal isolated human, but not 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 some recombinant Apo2L / TRAIL preparations may differ in their biochemical properties and biological effects on diseased and healthy cells depending, for example, on the presence or absence of the marker molecule, zinc content and% trimer content (See Lawrence et al. ., Nature Med., Letter to the Editor, a7: 383-385 (2001); Qin et al., Nature Med., Letter to the Editor, 7: 385-386 (2001)).

[0036] Apo2L / TRAIL has been found to bind at least five different receptors. At least two of the Apo2L / TRAIL binding receptors contain a functional cytoplasmic death domain. One of these receptors is designated DR4 (and alternatively as TR4 or TRAIL-R1) (Pan et al., Science, 276: 111-113 (1997); see also WO98 / 32856 published Jul 30, 1998; WO99 / ​​37684 published Jul 29 1999; WO 00/73349 issued December 7, 2000; US 6,433,147 issued August 13, 2002; US 6,461,823 issued October 8, 2002 and US 6,342,383 issued January 29, 2002).

[0037] Another such receptor for Apo2L / TRAIL was designated DR5 (also referred to alternatively as Apo-2; TRAIL-R or TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER) (see e.g. Sheridan et al. al., Science, 277: 818-821 (1997), Pan et al., Science, 277: 815-818 (1997), WO98 / 51793 published November 19, 1998; WO98 / 41629 published September 24, 1998; 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); WO98 / 35986 published August 20, 1998; EP870.827 published Oct. 14, 1998; WO98 / 46643 published October 22, 1998; WO99 / ​​02653 published January 21, 1999; WO99 / ​​09165 published February 25, 1999; WO99 / ​​11791 published March 11, 1999; US 2002/0072091 published August 13, 2002; 2002/0098550 published December 7, 2001; 6,313,269 issued December 6, 2001; 2001/0010924 published August 2, 2001; US 2003/01255540 published Jul 3, 2003; US 2002/0160446 published October 31, 2002, US 2002/0048785 published April 25, 2002; US 6,342,369 issued Feb 2002; 6,569,642 issued May 27, 2003, 6,072,047 issued June 6, 2000, issued 6,642,358 issued November 4, 2003; IS 6.743.625 issued June 1, 2004). DR5, like DR4, has been reported to contain a cytoplasmic death domain and is capable of apoptotic signaling by ligand binding (or by binding a molecule such as an antibody

-9 agonistic, which mimics the action of a ligand). The crystal structure of the complex formed between Apo-2L / TRAIL and DR5 is described in Hymowitz et aL, Molecular Cell, 4: 563-571 (1999).

[0038] Upon ligand binding, both DR4 and DR5 can induce apoptosis independently by uptake and activation of the apoptosis initiator caspase-8 by an adapter molecule containing a death domain termed 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)).

[0039] Apo2L / TRAIL has also been reported to bind receptors, termed DcR1, DcR2, and OPG, which are believed to function as inhibitors rather than mediators of signaling (see, e.g., DcR1 (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); DegliEsposti et al., J. Exp. Med., 186: 1165-1170 (1997); and Mongkolsapaya et al., J. Immunol., 160: 3-6 (1998)); DcR2 (also called 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. Unlike DR4 and DR5, the DcR1 and DcR2 receptors do not signal apoptosis.

[0040] Certain antibodies that bind to DR4 and / or DR5 receptors have been reported in the literature. For example, an anti-DR4 antibody that targets the DR4 receptor and has agonist or apoptotic activity in certain mammalian cells is described in, e.g., WO 99/37684 published Jul 20, 1999; WO 00/73349 published Jul 12, 2000; WO 03/066661 published August 14, 2003. See also, e.g., 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; WO 03/038043 published May 8, 2003; WO 03/037913 published May 8, 2003. Some anti-DR5 antibodies are similarly described, see, e.g., WO 98/51793 published November 8, 1998; Griffith et al., J. Immunol., 162: 2597-2605 (1999); Ichikawa et al., Nature Med., 7: 954960 (2001); Hylander et al., An Antibody to DR5 (TRAIL-Receptor 2) Suppresses the Growth of Patient Derived Gastrointestinal Tumors Grown in SCID mice, Abstract, 2nd International Congress on Monoclonal Antibodies in Cancers, August 29 - September 1, 2002, Banff, Alberta, Canada; WO 03/038043 published May 8, 2003; W0 03/037913 published May 8, 2003. In addition, some antibodies with cross-reactivity to DR4 and DR5 receptors have been described (see, e.g., US Patent 6,252,050 issued June 26, 2001).

The invention relates, at least in part, to the identification of a histidine-acetate buffer, pH 5.5 to 6.5, as being particularly useful for formulating monoclonal antibodies, especially full-length IgG1 antibodies, which are prone to deamidation and / aggregation. The formulation delays the degradation of the antibody product therein.

[0042] Therefore, in a first aspect, the invention relates to a stable pharmaceutical formulation as defined in claim 1.

[0043] A pharmaceutical formulation is disclosed comprising: (a) a full length IgG1 antibody prone to deamidation and aggregation in an amount from about 10mg / mL to about 250mg / mL; (b) histidine acetate buffer, pH 5.5 to 6.5; (c) a saccharide selected from the group consisting of trehalose and sucrose in an amount from about 60mM to about 250mM; and (d) polysorbate 20 in an amount from about 0.01% to about 0.1%.

[0044] A method of reducing deamidation and aggregation of a therapeutic monoclonal antibody is provided, comprising formulating the antibody in a histidine acetate buffer, pH 5.5 to 6.5.

[0045] A pharmaceutical formulation is disclosed comprising an antibody that binds to domain II of HER2 in a histidine buffer at a pH of about 5.5 to about 6.5, a saccharide, and a surfactant.

[0046] The invention relates to a pharmaceutical formulation comprising Pertuzumab in an amount from about 20mg / mL to about 40mg / mL, histidine acetate buffer, sucrose and polysorbate 20, wherein the pH of the formulation is about 5.5 to about 6.5.

[0047] A vial with a stopper pierceable with a stainless steel syringe or reservoir is disclosed containing the formulation inside the vial or reservoir, optionally in a frozen form.

[0048] A method of making a pharmaceutical formulation is disclosed, comprising: (a) producing a monoclonal antibody formulation; and (b) assessing the physical stability, chemical stability, or biological activity of the monoclonal antibody in the formulation.

Brief Description of Drawings

[0049]

Figure 1 shows Domains I-IV (SEQ ID Nos. 19-22, respectively) of the extracellular domain of HER2.

Figures 2A and 2B show the alignment of the amino acid sequences of the light (Vl) variable [chain] (Fig. 2A) and heavy (Vh) variable [chain] (Fig. 2B) domains of the murine monoclonal antibody 2C4 (SEQ ID Nos. 1 and 2, respectively) ; Vl and Vh domains of humanized version 2C4, 574 (SEQ ID Nos. 3 and 4, respectively), and human shared Vl and Vh framework sequences (kappa light [chain] subgroup I, hum κΐ; heavy [chain] subgroup III, humlll,) (SEQ ID Nos. 5 and 6, respectively). Asterisks indicate the differences between the humanized version of 2C4, 574 and the murine monoclonal antibody 2C4 or between the humanized version of 2C4, 574 and the human framework sequence. Match Determining Regions (CDRs) are given in parentheses.

Figures 3A and 3B show the amino acid sequences of Pertuzumab light chain and heavy chain (SEQ ID Nos. 15 and 16, respectively). CDRs in bold. The theoretical molecular weights of the light chain and heavy chain are 23,526.22 Da and 49,216.56 Da (cysteine ​​in reduced form). The carbohydrate moiety is attached to Asn 299 of the heavy chain.

Figures 4A and 4B show the Pertuzumab light chain and heavy chain amino acid sequences, each containing the intact amino-terminal signal peptide sequence (SEQ ID Nos. 17 and 18, respectively).

Figure 5 shows schematically the binding of 2C4 at a heterodimeric binding site in HER2, thereby preventing heterodimerization with activated EGFR or HER3.

Figure 6 shows HER2 / HER3 association with the MAPK and Akt pathways.

Figure 7 compares the activities of Trastuzumab and Pertuzumab.

Figure 8 shows the stability of Pertuzumab formulations based on analyzes

Ion exchange [chromatography] (ΙΕΧ).

Figure 9 shows the stability of Pertuzumab formulations based on steric exclusion chromatography (SEC) analyzes.

Figure 10 reflects the physical stability of Pertuzumab in various formulations.

Figure 11 is from a Pertuzumab mixing study in liquid formulations.

Figure 12 is from another Pertuzumab mixing study in liquid formulations.

Figure 13 is from a freeze-thaw study of Pertuzumab formulation.

Figures 14A and 14B show the amino acid sequences of the light chain (SEQ ID No. 13) and heavy chain (SEQ ID No. 14) of Trastuzumab.

Figures 15A and 15B show the Pertuzumab light chain sequence variant (SEQ ID No. 23) and the Pertuzumab heavy chain sequence variant (SEQ ID No. 24).

Figures 16A and 16B show the structures of the oligosaccharides frequently observed in IgG antibodies.

Figures 17A and 17B show the light and heavy chain sequence (SEQ ID No. 3744) of specific anti-IgE antibodies, E25, E26, HAE1, and Hu-901. In Fig. 17A, the light [chain] variable domain ends with VEIK residues 111. In Fig. 17B, the heavy [chain] variable domain ends with VTVSS residues about residue 120.

Figure 18A is a sequence alignment comparing the amino acid sequences of the variable [chain] light domain (VL) for each of murine 2H7 (SEQ ID No. 25), humanized 2H7v16 variant (SEQ ID No. 26) and human kappa light chain subgroup I (SEQ ID No. 27) . The CDRs from V1 2H7 and hu2H7v16 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 comparing the amino acid sequences of the heavy (Vh) variable domain of each of murine 2H7 (SEQ ID No. 28), humanized 2H7v16 variant (SEQ ID No. 29), and human heavy chain shared sequence subgroup III (SEQ ID No. No. 30). The CDRs from Vh 2H7 and hu2H7v16 are as follows: CDRI (SEQ ID No. 60), CDR2 (SEQ ID No. 61) and CDR3 (SEQ ID No. 62).

[0050] In Fig. 18A and Fig. 18B, the CDRI, CDR2 and CDR3 in each chain are appended within parentheses flanked by the FR1-FR4 framework regions as indicated. 2H7 refers to the murine 2H7 antibody. Asterisks between the two sequence rows indicate positions that differ between the two sequences. The residues are numbered according to Kabat et al. Sequences of 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 shows the variable domain sequences of three different VEGF antibodies having the sequences of SEQ ID Nos. 31-36.

Figure 20 shows the elution profile of steric exclusion chromatography (SEC) of the following Apomab samples: (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 prior to analysis.

Figure 21 shows the pH ratio profile for Apomab monomer loss during storage. The monomer kinetics of the SEC were monitored during storage at 30 ° C and 40 ° C and a first order rate constant was calculated.

Figure 22 provides the elution profile from Ion Exchange Chromatography (IEC) of the following Apomab samples: (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 prior to analysis.

Figure 23 shows the pH ratio profile for the loss of IEC major peak during storage. The IEC major peak kinetics was monitored during storage at 30 ° C and 40 ° C and a first order rate constant was 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 nucleotide position 447 (in SEQ ID No. 45) was used to indicate that the nucleotide base may be T or G.

Figures 25A and 25B show the 411 amino acid sequence of the human DR5 receptor (SEQ ID No. 47) as published November 19, 1998 in WO 98/51793 and the nucleotide coding sequence (SEQ ID No. 48).

Figures 26A and 26B show the 440 amino acid sequence of the human DR5 receptor (SEQ ID No. 49) and the nucleotide sequence encoding it (SEQ ID No. 50), as also published August 20, 1998 in WO 98/35986.

Figure 27 shows the amino acid sequence of the heavy chain of Apomab 7.3 (SEQ ID No. 51).

Figure 28 shows the amino acid sequence of the light chain of Apomab 7.3 (SEQ ID No. 52).

Figure 29 shows the alignment of the amino acid sequences of the 16E2 heavy chain (SEQ ID No. 53) and the heavy chain of Apomab 7.3 (SEQ ID No. 51).

Figure 30 shows the alignment of the amino acid sequences of the 16E2 light chain (SEQ ID No. 54) and the light chain of Apomab 7.3 (SEQ ID No. 52).

Figures 31A and 3IB show the heavy [chain] variable [domain] sequence (Figure 31 A; SEQ ID No. 55) and the light [chain] variable [domain] amino acid sequence (Figure 3 IB; SEQ ID No. 56) of Apomab 7.3. CDR residues are in bold.

Figure 32 shows the alignment of the mature 2H7vl6 and 2H7v511 light chains (SEQ ID Nos. 63 and 64, respectively). The sequence is shown with Kabat numbering for the variable domain residues and Eu numbering for the constant domain residues.

Figure 33 shows the alignment of the mature 2H7vl6 and 2H7v511 heavy chains (SEQ ID Nos. 65 and 66, respectively). The sequence is shown with Kabat numbering for the variable domain residues and Eu numbering for the constant domain residues.

Detailed Description of the Preferred Embodiments

I. Definitions

[0051] The expression pharmaceutical formulation refers to a preparation which is in a form which enables the active ingredient to be biologically effective and which does not contain additional components which are unacceptably toxic to the subject to which the formulation would be administered. Such formulations are sterile.

[0052] The sterile formulation is aseptic or free of viable microorganisms and their spores.

[0053] The frozen formulation is that at a temperature below 0 ° C. Generally, the frozen formulation is not freeze-dried, nor is it subjected to prior or subsequent lyophilization. Preferably the frozen formulation comprises a frozen storage drug substance (in stainless steel tanks) or a frozen drug substance (vial end shape).

[0054] A stable formulation is one in which the protein substantially retains its physical and / or chemical stability and / or biological activity during storage. Preferably, the formulation substantially retains its physical stability as well as its biological activity during storage. The shelf life is generally selected based on the intended shelf life of the formulation. Various analytical techniques are available in the art to measure protein stability, as reviewed 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:

- 14 29-90 (1993). Stability can be measured 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 stable at about 5 ° C and / or 15 ° C for at least 3 months and / or stable at about 20 ° C for at least 3 months or at least 1 year. Moreover, the formulation is preferably stable after freezing (to e.g. -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 ways, including assessment of aggregate formation (e.g. using size exclusion chromatography, turbidity measurements, and / or by visual inspection); estimating charge heterogeneity using cation exchange chromatography or zone capillary electrophoresis; amino terminal or carboxyl terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare intact and reduced antibodies; peptide map analysis (e.g., tryptic or LYS-C); evaluating the biological activity or antigen-binding function of the antibody; etc. Instability can include one or more of: aggregation, deamidation (e.g., deamidation of Asn), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), cleavage / hydrolysis / fragmentation (e.g. fragmentation of the linker region), succinate formation, unpaired cysteine ​​(cysteines), N-terminal extension, C-terminal processing, differences in glycosylation, etc.

[0055] A deamidated monoclonal antibody is one in which one or more of its asparagine residues have been derivatized, eg, to aspartic acid or iso-aspartic acid.

[0056] An antibody prone to deamidation is one that contains one or more residues which have been shown to be prone to deamidation.

[0057] An antibody prone to aggregation is one that has been shown to aggregate with other antibody molecule (s), especially upon freezing and / or mixing.

[0058] An antibody prone to fragmentation is one which has been shown to be cleaved into two or more fragments, for example, in its linker region.

[0059] The reduction of deamidation, aggregation, or fragmentation is intended to prevent or reduce the amount of deamidation, aggregation, or fragmentation relative to an antibody formulated at a different pH or buffer.

[0060] The biological activity of a monoclonal antibody herein refers to the ability of the antibody to bind to an antigen and results in a measurable biological response that can be measured. vitro or in vivo. Such activity may be antagonistic (for example, where the antibody is a HER2 antibody) or agonist (for example, where the antibody binds DR5). In the case of Pertuzumab, in one embodiment, the biological activity relates to the ability of the formulated antibody to inhibit proliferation of the human breast cancer cell line, MDA-MB-175-VII. Where the antibody is Apomab, the biological activity can refer to, for example, the ability of the formulated antibody to kill colon cancer cells, Colo205.

[0061] Isotonic means that the formulation of interest has substantially the same osmotic pressure as human blood. Osmotic formulations will generally have an osmotic pressure of from about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or cryiometric type osmometer.

[0062] The buffer as used herein relates to a buffered solution which is resistant to pH changes by the action of its conjugate acid-base components. The buffer of the invention has a pH preferably ranging 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 most preferably has a pH of about 6 , 0. Examples of buffers controlling the pH in this range include acetate, succinate, succinate, gluconate, histidine, citrate, glycylglycine, and other organic acid buffers. A preferred buffer here is the histidine buffer.

[0063] The histidine buffer is a buffer that contains histidine ions. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, and histidine sulfate. The preferred histidine buffer as identified in the examples herein turned out to be histidine acetate. In a preferred embodiment, the histidine acetate buffer is produced by titrating 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 to 6.5, preferably a pH of 5.8 to 6.2.

[0064] The saccharide comprises the general composition (CH₂O) n and its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. Examples of saccharides include glucose, sucrose, trehalose, lactose, fructose, maltose, arabitolite, glerythol, silolite , sorbitol, mannitol, melibiosis, melesitose, raffinose, mannotriosis, stachyosis, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose etc. Preferred saccharides are non-reducing disaccharides such as trehalose or sucrose.

[0065] The surfactant relates to a surfactant, preferably a non-ionic surfactant. Examples of surfactants include polysorbate (e.g., polysorbate 20 and polysorbate 80); poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octylglycoside; lauryl-, myristyl-, linoleyl- or stearylsulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocami dopropyl o-, linolamidopropyl-, mirystami dopropyl o-, palmidopropyl- or isostearamidopropyl-betaine (e.g. lauroami dopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and the MONAQUAT ™ series (Mona Industries, Inc., Paterson, NJ); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene (e.g., Pluronics, PF68, etc.); etc. A preferred surfactant is polysorbate 20.

[0066] HER receptor is a receptor tyrosine kinase that belongs to the HER receptor family and includes EGFR, HER2, HER3 and HER4 receptors and other members of that family to be identified in the future. HER receptor will be generally

- 16 contained an extracellular domain that can bind HER ligand; a lipophilic transmembrane domain; an evolutionarily conserved intracellular tyrosine kinase domain; and a carboxy terminus signaling domain shielding several tyrosine residues that may be phosphorylated. Preferably the HER receptor is native sequence human HER receptor.

[0067] The extracellular domain of HER2 comprises four domains, Domain I (amino acid residues from about 1-195), Domain II (amino acid residues from about 196-320), Domain III (amino acid residues from about 321-488), and Domain IV (amino acid residues from about amino acids from about 489-632) (residue numbering without signal peptide). See Garrett et al. Moth. Cell. 11: 495-505 (2003), Cho et al. Nature 421: 756-760 (2003), Franklin et al. Cancer Cell 5: 317328 (2004) or Plowman et al. Proc. Natl. Acad. Sci. 90: 1746-1750 (1993). See also Fig. 1.

[0068] The expressions ErbBl, HER1, epidermal growth factor receptor, and EGFR are used interchangeably and refer to EGFR as disclosed, for example, in Carpenter et al. Ann. Rev. Biochem. 56: 881-914 (1987), including its naturally occurring mutational forms (e.g., EGFR deletion mutant as in Humphrey et al. PNAS (USA) 87: 42074211 (1990)). erbBl refers to the gene encoding the protein product, EGFR.

[0069] The terms' ErbB2 and HER2 are used interchangeably and refer 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) (Genebank accession number Χ03363). The expression erbB2 refers to the gene encoding the human ErbB2 and neu refers to the gene encoding the rat pl85neu. Preferred HER2 has the native sequence of human HER2.

[0070] ErbB3 and HER3 refer to a receptor polypeptide as disclosed in, for example, US Patent Nos. 5,183,884 and 5,480,968, as well as Kraus et al. PNAS (USA) 86: 91939197 (1989).

[0071] The expressions ErbB4 and HER4 refer to a receptor polypeptide as disclosed, for example, in EP Patent Application No. 599,274; Plowman et al., Proc. Natl. Acad Sci. USA, 90: 1746-1750 (1993); and Plowman et al., Nature, 366: 473-475 (1993), including isoforms thereof, e.g. as disclosed in WO99 / ​​19488, published April 22, 1999.

[0072] A HER ligand is a polypeptide that binds to and / or activates a HER receptor. A HER ligand of particular interest is a human HER ligand of a native sequence, such as epithelial growth factor (EGF) (Savage et al., J. Biol. Chem. 247: 7612-7621 (1972)); transforming growth factor alpha (TGF-α) (Marquardt et al., Science 223: 1079-1082 (1984)); amphiregulin, also known as autocrine schwannoma or keratinocyte growth factor (Shoyab et al. Science 243: 1074-1076 (1989); Kimura et al. Nature 348: 257-260 (1990); and Cook et al. Moth. 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: 28412848 (1997)); heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al., Nature

- 17,387: 512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc. Natl. Acad. Sci. 94: 95629567 (1997)); neuregulin-4 (NRG-4) (Harari et al. Oncogene 18: 2681-89 (1999)) or kripto (CR-1) (Kannan et al. J. Biol. Chem. 272 ​​(6): 3330-3335 ( 1997)). HER ligands which bind EGFR include EGF, TGF-α, amphiregulin, betacellulin, HB-EGF, and epiregulin. HER ligands that bind HER3 include heregulins. HER ligands capable of binding HER4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4, and heregulins.

[0073] Heregulin (HRG) as used herein refers to a polypeptide encoded by a heregulin gene product as disclosed in US Patent No. 5,641,869 or Marchionni et al., Nature, 362: 312-318 (1993). Examples of heregulin include heregulin-a, heregulin-βΐ, heregulin-β2, and heregulin-33 (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 (GGF) (Marchionni et al., Nature, 362: 312-318 (1993)); sensory and motor neuron-derived factor (SMDF) (Ho et al. J. Biol. Chem. 270: 14523-14532 (1995)); γ-heregulin (Schaefer et al. Oncogene 15: 1385-1394 (1997)). The expression includes biologically active fragments and / or native sequence variants of the native sequence HRG polypeptide, such as an EGF-like domain fragment thereof (e.g. ΗΚΘβ 1177-244)

[0074] A HER dimer is a non-covalently linked dimer containing at least two different HER receptors. Such complexes can form when a cell expressing two or more HER receptors is exposed to a HER ligand and can be isolated by immunoprecipitation and analyzed by SDS-PAGE as described, for example, in Sliwkowski et al., J. Biol. Chem., 269 (20): 14661-14665 (1994). Examples of such HER dimers include the EGFR-HER2, HER2-HER3 and HER3-HER4 heterodimers. Moreover, the HER dimer may contain two or more HER2 receptors linked to another HER receptor such as HER3, HER4, or EGFR. Other proteins such as a cytokine receptor subunit (e.g., gpl30) can be linked to the dimer.

[0075] The heterodimeric binding site on HER2 refers to a region in the extracellular domain of HER2 that contacts or interacts with a region in the extracellular domain of EGFR, HER3, or HER4 during dimer formation therewith. The region is in Domain II of HER2. Franklin et al. Cancer Cell 5: 317-328 (2004).

[0076] HER activation or HER2 activation refers to the activation or phosphorylation of any one or more HER receptors or HER2 receptors. In general, HER activation results in signal transduction (e.g., causing, through the intracellular kinase domain of the HER receptor, phosphorylation of tyrosine residues on the HER receptor or substrate polypeptide). HER activation may be dependent on HER ligand binding to a HER dimer containing the HER receptor of interest. Binding of a HER ligand to a HER dimer may activate the kinase domain of one or more HER receptors in the dimer, thereby resulting in phosphorylation of tyrosine residues at one or more HER receptors and / or

- phosphorylation of tyrosine residues in an additional substrate polypeptide (s) such as intracellular Akt or MAPK kinases.

[0077] The expression antibody is used in the broadest sense and specifically covers full-length antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two full-length antibodies, and antibody fragments as long as they exhibit the desired biological activity.

The expression monoclonal antibody used refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e. the individual antibodies making up the population are identical and / or bind to the same epitope, except for possible variants that may arise during the production of the monoclonal antibody, these generally present variants are in smaller amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies to different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are not contaminated with other immunoglobulins. A monoclonal dose is indicative of the nature of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be interpreted as requiring production of the antibody in any particular way. For example, monoclonal antibodies for use in the present invention may be produced by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be produced by recombinant DNA methods (see, e.g., US Patent No. 4,816). .567). "Monoclonal antibodies" can also be isolated from antibody phage libraries using the techniques described, for example, in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991).

[0079] Monoclonal antibodies in particular 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 given species or belonging to a particular class or subclass of antibodies. while the remainder of the chain (s) is identical or homologous to the corresponding sequences in antibodies derived from other species or belonging to a different class or subclass of antibodies, so are fragments of such antibodies as long as they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). Chimeric antibodies of interest include vascularized antibodies comprising variable antigen-binding variable domain sequences derived from non-human primates (e.g., Narrow-nosed Apes, Apes, etc.) and human constant region sequences.

[0080] The antibody fragments contain a portion of a full length antibody, preferably including an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; diabodies; linear antibodies; single chain

- 19 antibody molecules; and multispecific antibodies, formed from the antibody fragment (s).

A full-length antibody is one that contains an antigen binding variable region as well as a light chain constant domain (Cl) and heavy chain constant domains, Cul, Ch2, and Ch3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the full-length antibody has one or more effector functions.

[0082] The phrase antibody of main species refers to that antibody structure in a composition which is the quantitatively dominant antibody molecule in the composition. For example, the main species can be a HER2 antibody, such as an antibody that binds to the IIHER2 Domain, an antibody that inhibits HER dimerization more effectively than Trastuzumab, and / or an antibody that binds to a heterodimeric HER2 binding site. A HER2 antibody is disclosed which comprises the amino acid sequences of the variable [chain] light and variable [chain] heavy domains in SEQ ID Nos. 3 and 4, and most preferably comprising the light chain and heavy chain amino acid sequences in SEQ ID Nos. 15 and 16. (Pertuzumab).

[0083] An amino acid sequence variant of an antibody is an antibody with an amino acid sequence different from that of the main species. Typically, the amino acid sequence variants will have at least 70% homology with the main species antibody, and preferably at least 80%, more preferably at least about 90% homology with the main species antibody. The amino acid sequence variants have substitutions, deletions, and / or additions at certain positions within or adjacent to the amino acid sequence of the main species antibody. Examples of amino acid sequence variants include an acid variant (e.g., a deamidated antibody variant), a basic variant, an amino-terminal extension antibody in the form of a leader sequence (e.g. VHS-) on its one or two light chains, the antibody with a C-terminal lysine residue on its one or two heavy chains, etc., and includes combinations of heavy and / or light chain amino acid sequence variations. An antibody variant of particular interest is an antibody having an amino terminal extension as a leader sequence on one or two of its light chains, optionally further comprising a different amino acid sequence and / or glycosylation differences from the main species.

[0084] A therapeutic monoclonal antibody is an antibody used in the treatment of a human subject. The therapeutic monoclonal antibodies disclosed include: HER2 antibodies for the treatment of cancer and various non-cancerous diseases or disorders; CD20 or BR3 antibodies for the treatment of B cell tumors, autoimmune diseases, transplant rejection, or blocking an immune response to a foreign antigen; IgE antibodies for the treatment of IgE mediated disorders; DR5 or VEGF antibodies for the treatment of cancer.

[0085] A glycosylation variant of an antibody is an antibody with one or more carbohydrate molecules attached thereto that differ from the one or more carbohydrate molecules attached to the main species antibody. Examples of glycosylation variants include an antibody with a G1 or G2 oligosaccharide structure, instead of a GO oligosaccharide structure, attached to its Fc region, an antibody with one or two carbohydrate molecules attached to its one or two light chains, an antibody without a carbohydrate attached to one or two of the antibody heavy chains. , etc., and combinations of glycosylation changes.

[0086] Where the antibody has an Fc region, an oligosaccharide structure, such as that shown in Fig. 16, can be attached to one or two of the antibody heavy chains, eg, to residue 299 (298, Eu residue numbering). For Pertuzumab, GO was the predominant oligosaccharide structure, with other oligosaccharide structures such as G0-F, Gl, Man5, Man6, Gl-1, Gl (1-6), Gl (1-3) and G2 found in the Pertuzumab composition in smaller amounts.

[0087] Unless otherwise stated, the oligosaccharide structure of GI includes the structures ΟΙ, Gl-1, GI (1-6), and GI (1-3).

[0088] An amino-terminal extension leader sequence refers to one or more amino acid residues of the amino-terminal leader sequence that are present at the amino terminus of any one or more heavy or light chains of an antibody. Exemplary amino terminus extensions in the form of a leader sequence contain or consist of three amino acid residues, VHS, present on one or both light chains of the antibody variant.

[0089] Homology is defined as the percentage of the variant amino acid sequence residues which are identical when the sequences are aligned and gaps are inserted, if necessary, to obtain the maximum percent homology. Equation methods and computer programs are well known in the art. One such computer program is Align 2, written by Genentech, Inc., filed December 10, 1991 with user documentation at United States Copyright Office, Washington, DC 20559.

[0090] Antibody effector functions relate to those biological activities attributed to the Fc region of an antibody (the Fc region of the native sequence or the amino acid sequence variant of the Fc region). Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody dependent cellular cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g. B-cell receptors; BCR), etc.

[0091] Depending on the amino acid sequence of the constant domain of their heavy chains, full-length antibodies can be assigned to different "classes. There are five major classes of full-length antibodies: Ig A, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into "subclasses" (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called respectively:

-21 α, δ, ε, γ and μ. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0092] "Antibody-dependent cellular cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (eg, natural cytotoxic (NK) cells, neutrophils and macrophages) recognize an antibody bound to target cell and then lyse the target cell. The primary ADCC mediating cells, NK cells, express only FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). In order to assess ADCC activity of a given molecule, an in vitro ADCC assay can be performed, as described in US Patent No. 5,500,362 or 5,821,337. Effector cells useful for such studies include peripheral blood mononuclear cells (PBMCs) and natural cytotoxic (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0093] "Human effector cells" are leukocytes that express one or more FcRs and exhibit effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural cytotoxic (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred. Effector cells can be isolated from their native source, eg, blood or PBMCs as described herein.

[0094] The terms "Fc receptor" and "FcR" are used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is the native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the subclasses FcyRI, FcyRII, and FcyRIII, including allelic variants and alternatively folded forms of these receptors. FcyRII receptors include FcyRIIA ("activating receptor") and FcyRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily within their cytoplasmic domains. The activating receptor FcyRIIA contains in its cytoplasmic domain an activating tyrosine-based immunoreceptor motif (ITAM). The inhibitory receptor FcyRIIB contains in its cytoplasmic domain an inhibitory tyrosine-based immunoreceptor (ITIM) motif. (See the review by M. in Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). 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 to be identified in the future, are encompassed by the term "FcR." The expression also includes the neonatal Fc receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim etaL, J. Immunol. 24: 249 (1994)).

[0095] "Complement dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target molecule in the presence of complement. The complement activation pathway is initiated by the binding of the first complement component (Clq) to a molecule (e.g., an antibody) bound to an appropriate antigen. To assess complement activation, a CDC assay can be performed, e.g., as described in Gazzano-Santoro et aL, J. Immunol. Methods 202: 163 (1996).

[0096] Native antibodies are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has evenly spaced disulfide bridges inside the chain. 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 (V_L.) and a constant domain at its other end. The light chain constant domain is aligned with the first heavy chain constant domain and the light chain variable domain is aligned with the heavy chain variable domain. Individual amino acid residues are believed to form the interface between the light chain and heavy chain variable domains.

[0097] The expression "variable" refers to the fact that certain portions of the variable domains differ significantly in sequence between antibodies and are used in the binding and specificity of each individual antibody for its particular antigen. However, the variability is not evenly distributed across the variable domains of antibodies. The variability is concentrated in three segments, called hypervariable regions, within both the light chain and the heavy chain variable domains. The parts of the variable domains that are more evolutionarily conserved are called framework regions (FR). Each of the native heavy and light chain variable domains comprises four FR regions, largely taking the β sheet structure, connected by three hypervariable regions that form loops connecting and in some cases forming part of the β sheet structure. The hypervariable regions in each chain are kept in close proximity to each other by the FR and, along with the hypervariable regions of the other chain, contribute to the formation of an antigen-binding site in the antibody (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 involved directly in the binding of an antibody to an antigen, but exert various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

[0098] The expression hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable regions generally contain amino acid residues from the complementarity determining or CDR regions (e.g., residues 24-34 (LI), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and 31-35 (Hl).

-23 50-65 (Η2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, Ed. 5th, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from hypervariable loops (e.g., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53- 55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). "Framework Region" or "FR" residues are variable domain residues, other than hypervariable region residues as defined herein.

[0099] Papain digestion of antibodies aims for two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site and a residual Fc fragment, whose name reflects its ability to crystallize readily. Pepsin treatment targets the F (ab ') 2 fragment, which has two antigen binding sites and is still capable of cross-linking with the antigen.

[0100] Fv is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent relationship. It is in such a configuration that the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VHV1 dimer. In total, the six hypervariable regions confer antigen binding specificity to the antibody. However, even a single variable domain (or a half of an Fv containing only three antigen-specific hypervariable regions) has the ability to recognize and bind an antigen, albeit with a lower affinity than the entire binding site.

[0101] The Fab fragment also comprises a light chain constant domain and a heavy chain first constant domain (CHI). Fab 'fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab 'in which the cysteine ​​residue (s) of the constant domain have at least one free thiol group. F (ab ') 2 antibody fragments were initially produced as pairs of Fab' fragments that have linker cysteines between them. Other chemical couplings of antibody fragments are also known.

[0102] The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (λ), based on the amino acid sequences of their constant domains.

[0103] Single-chain Fv or scFv antibody fragments contain the antibody VH and VL domains, wherein the domains are present as 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 reviews of scFv, see Pliickthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Serlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are described in

WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0104] The diabody expression refers to small antibody fragments with two antigen-binding sites, which fragments contain a heavy [chain] variable domain (VH) joined to a light [chain] variable (VL) domain on the same polypeptide chain (V_H. - Vl). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create antigen binding sites. Diabodies are described in more detail in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad Sci. USA, 90: 6444-6448 (1993).

[0105] "Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (the recipient antibody) in which the recipient's hypervariable region residues have been replaced with hypervariable region residues from a non-human species (donor antibody), such as a mouse, rat, rabbit, or other non-human primate having the desired specificity. , affinity and capacity. In some instances, human immunoglobulin framework region (FR) residues have been replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not present in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will optionally include at least a portion of an immunoglobulin constant region (Fc), typically derived from a human immunoglobulin. For further 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).

[0106] Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or Trastuzumab 3 (as described in HERCEPTIN® 3) (as HERCEPTIN® 3). US Patent 5,821,337; humanized 520C9 (WO93 / 21319) and humanized 2C4 antibodies as described herein.

[0107] For their own purposes, Trastuzumab, HERCEPTIN®, and huMAb4D5-8 relate to antibodies having the light and heavy chain amino acid sequences in SEQ ID NOS. 13 and 14, respectively.

[0108] Pertuzumab, rhuMAb 2C4, and OMNITARG ™ refer to an antibody comprising light chain variable and chain variable amino acid sequences

-25 heavy in SEQ ID No. 3 and 4, respectively. Where 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.

[0109] A naked antibody is an antibody (as defined) that is not attached to a heterologous molecule, such as a cytotoxic particle or a radioactive label.

[OHO] An "affinity matured" antibody is an antibody with one or more changes in one or more hypervariable regions thereof, resulting in an improvement in the affinity of the antibody for the antigen, as compared to a parent antibody that does not have the change (s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio / Technology 10: 779-783 (1992) describes affinity maturation using VH and V1 domain shuffling. Random mutagenesis of CDRs and / or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci. USA 91: 3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al., J. Immunol 154 (7): 3310-9 (1995);

and Hawkins et al, J. Mol. Biol. 226: 889-896 (1992).

[0111] An agonist antibody is an antibody that binds to and activates a receptor. In general, the ability of the agonist antibody to activate the receptor will be at least qualitatively similar (and may be substantially similar quantitatively) to the native 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 in cells expressing the TNF receptor (e.g., DR5). An assay for assessing the induction of apoptosis is described in WO98 / 51793 and WO99 / ​​37684.

[0112] An "isolated" antibody is that which has been identified and separated and / or recovered from a component of its natural environment. Contaminating components from its natural environment are materials that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of the antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to a degree sufficient to yield at least 15 residues of the N-terminal or internal amino acid sequence. using a spinner vessel sequencer or (3) to homogeneity [assessed] by SDS-PAGE under reducing or non-reducing conditions, using Coomassie Blue or, preferably, staining with silver. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Typically, however, an isolated antibody will be produced by at least one purification step.

[0113] A HER2 antibody that inhibits HER dimerization more effectively than Trastuzumab is one that reduces or eliminates HER dimers more effectively (eg, at least 2 fold more effectively) than Trastuzumab. Preferably, such an antibody inhibits HER2 dimerization at least as effectively as an antibody selected from the group consisting of the murine monoclonal antibody 2C4, the Fab fragment of the murine monoclonal antibody 2C4, Pertuzumab, and the Fab fragment of Pertuzumab. Inhibition of HER dimerization can be assessed by direct testing of HER dimers or by assessing HER activation or downstream signaling that results from HER dimerization and / or by evaluating the antibody-HER2 binding site, etc. Screening for antibodies with the ability to inhibit HER dimerization is more effective than Trastuzumab is described in Agus et al. Cancer Cell 2: 127-137 (2002) and WO01 / 00245 (Adams et al.). By way of example only, inhibition of HER dimerization can be tested by assessing, for example, inhibition of HER dimer formation (see, e.g., Figs. 1A-B of Agus et al. Cancer Cell 2: 127-137 (2002); and WO01 / 00245); reduction of HER ligand activation of cells expressing HER dimers (WO01 / 00245 and Figs. 2A-B of Agus et al. Cancer Cell 2: 127-137 (2002), for example); blocking HER ligand binding to cells expressing HER dimers (WO01 / 00245 and Fig. 2E of Agus et al. Cancer Cell 2: 127-137 (2002), for example); inhibiting the cell growth of cancer cells (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 HER ligand (WO01 / 00245 and Fig-y. 3 AD from Agus et al. Cancer Cell 2: 127-137 (2002), for example); inhibition of downstream signaling (e.g., inhibition of HRG dependent AKT phosphorylation or inhibition of HRG or TGFa dependent MAPK phosphorylation) (see WO01 / 00245 and Fig. 2C-D from Agus et al. Cancer Cell 2: 127-137 (2002), for example). One can also assess whether the antibody inhibits HER dimerization by examining the antibody-HER2 binding site, for example, by evaluating the structure or model, such as the crystal structure of the antibody bound to HER2 (See, for example, Franklin et al. Cancer Cell 5: 317-328 (2004 )).

[0114] The HER2 antibody may inhibit HRG dependent AKT phosphorylation and / or inhibit HRG or TGFa dependent MAPK phosphorylation more effectively (e.g., at least 2-fold more effectively) than Trastuzumab (see Agus et al. Cancer Cell 2: 127-137 ( 2002) and WO01 / 00245, by way of example).

[0115] The HER2 antibody may be one that does not inhibit HER2 ectodomain cleavage (Molinaera /. Cancer Res. 61: 4744-4749 (2001).

[0116] The HER2 antibody that binds to the heterodimeric HER2 binding site binds to residues in domain II (and optionally also binds to residues in other domains of the extracellular domain of HER2 such as domains I and III) and may sterically interfere with what at least to some extent, in the formation of a HER2-EGFR, HER2-HER3 or HER2-HER4 heterodimer. Franklin et al. Cancer Cell 5: 317-328 (2004) characterizes the HER2-Pertuzumab crystal structure, deposited at the RCSB Protein Data Bank

-27 (ID Code IS78), illustrating an exemplary antibody that binds to a heterodimeric HER2 binding site.

[0117] An antibody that binds to the Π domain of HER2 binds to residues in domain II and optionally to residues in another domain (s) of HER2, such as domains I and III. Preferably, an antibody that binds to domain II binds to the junction between domains I, II and III of HER2.

[0118] "Growth inhibitory factor" refers to a compound or composition that inhibits the growth of cells, especially tumor cells, expressing HER, in vitro or in vivo. Thus, a growth inhibitory agent may be one that significantly reduces the percentage of S-phase HER expressing cells. Examples of growth inhibitory agents include agents that block cell cycle progression (at a site other than the S phase), such as agents that cause G1 and M phase arrest. . Classic phase M blockers include vinca alkaloids (vincristine and vinblastine), taxanes, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. Agents that stop G 1 also act on S-phase arrest, 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, Mendelsohn and Israel, eds., Chapter 1, titled Celi cycle regulation, oncogenes, and antineoplastic drugs by Murakami ego et al.

(WB Saunders: Philadelphia, 1995), especially p. 13.

[0119] Examples of growth inhibitory antibodies are those that bind to HER2 and inhibit the growth of cancer cells that overexpress HER2. Preferred HER2 antibodies inhibit the growth of breast tumor SK-BR-3 cells in cell culture by more than 20%, preferably more than 50% (e.g. from about 50% to about 100%) at an antibody concentration of about 0.5 to 30 pg / ml, where growth inhibition is determined six days after exposure of SK-BR-3 cells to the antibody (see U.S. Patent No. 5,677,171 issued October 14) 1997). The SK-BR-3 cell growth inhibition assay is described in more detail in this patent and below. A preferred growth inhibitory antibody is a humanized variant of the murine monoclonal antibody 4D5, e.g., Trastuzumab.

[0120] An antibody that induces apoptosis is one that induces programmed cell death, referred to as annexin V binding, DNA fragmentation, cell contraction, expansion of the endoplasmic reticulum, cell fragmentation, and / or formation of membrane vesicles (called apoptotic bodies). Typically the cell is one that expresses the antigen to which the antibody binds. Preferably the cell is a tumor cell. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by DNA ladder; and nuclear / chrornatin condensation including DNA fragmentation can be assessed by any increase in hypodiploid cells. Preferably, an apoptosis-inducing antibody is one that results in about 2- to 50-fold, preferably about 5- to 50-fold, and most preferably about 10- to 50-fold induction of annexin binding compared to untreated cells when compared to untreated cells. annexin binding study using cells

Expressing the antigen to which the antibody binds. Examples of antibodies that induce apoptosis are HER2 antibodies 7C2 and 7F3 and some DR5 antibodies.

[0121] The epitope 2C4 is the region in the extracellular domain of HER2 to which the antibody 2C4 binds. Routine cross-blocking studies can be performed to screen for antibodies binding to the 2C4 epitope, such as that described in Antibodies, A Laboratory Manual, Cold-Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping can be performed to assess whether the antibody binds to the 2C4 epitope of HER2. The 2C4 epitope comprises residues from 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).

[0122] The epitope 4D5 is the region in the extracellular domain of HER2 to which the antibody 4D5 (ATCC CRL 10463) and Trastuzumab bind. This epitope is located close to the transmembrane domain of HER2 and within the HER2 IV Domain. Routine cross-blocking studies can be performed to screen for antibodies binding to the 4D5 epitope, such as described in Antibodies, A Laboratory Manual, Cold-Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping may be performed to assess whether the antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues in the HER2 region from about residue 529 to about residue 625, inclusive). The 7C2 / 7F3 epitope is the region at the amino terminus within Domain I of the extracellular domain of HER2 to which the 7C2 and / or 7F3 antibodies (each deposited with the ATCC, see below) bind. Routine cross-blocking studies can be performed to screen for antibodies that bind to the 7C2 / 7F3 epitope, such as those described in Antibodies, A Laboratory Manual, Cold-Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping may be performed to assess whether the antibody binds to the 7C2 / 7F3 epitope on HER2 (e.g., any one or more residues in the HER2 region from about residue 22 to about residue 53, inclusive).

[0123] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those who are already sick as well as those who are preventable. Hence, the patient to be treated may be diagnosed as sick or predisposed or susceptible to disease.

[0124] The terms "cancer" or "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, cancer, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial sarcoma), neuroendocrine tumors (including carcinoid, gastric and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenoma, melanoma, and lymphoid leukemia or tumors. More specific examples of such cancers include squamous cell carcinoma (e.g., squamous cell carcinoma), lung carcinoma, including small cell lung carcinoma, non-small cell lung carcinoma, lung adenoma, and squamous cell carcinoma of the lung, peritoneal carcinoma, and

-29 liver cell cancer, gastric or stomach cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, cancer endometrial or uterine cancer, salivary gland cancer, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, as well as head and neck cancer.

[0125] The phrase "effective amount" refers to an amount of a drug that is effective for the disease in the patient. When the disease is cancer, an effective amount of the drug may reduce the number of cancer cells; reduce the size of the tumor; inhibit (ie, slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; brake (i.e. slow to some extent and preferably stop) tumor metastasis; inhibit to some extent tumor growth; and / or ameliorate to some extent one or more symptoms associated with cancer. To some extent, the drug may prevent growth and / or kill existing cancer cells, and may be cytostatic and / or cytotoxic. An effective amount can prolong progression-free survival, resulting in an objective response (including partial response, PR, or complete response, CR), increase overall survival, and / or ameliorate one or more symptoms of cancer.

[0126] A HER2-expressing cancer is one that contains cells having the HER2 protein present on their surface.

[0127] A cancer that overexpresses the HER receptor is one that has significantly higher levels of a HER receptor, such as HER2, on its cell surface as compared to a non-cancerous tissue of the same cell type. Such overexpression may be caused by gene amplification or increased transcription or translation. HER receptor overexpression may be determined in a diagnostic or prognostic test by assessing elevated levels of the HER protein present on the cell surface (e.g. by immunohistochemistry, IHC). Alternatively or additionally, levels of the HER-encoding nucleic acid can be measured in a cell, e.g., by fluorescent in situ hybridization (FISH; see WO98 / 45479, published October 1998), by Southern blot, or by polymerase chain reaction (PCR) techniques such as quantitative real-time PCR (RT-PCR). HER receptor overexpression may also be tested by measuring the shed antigen (e.g. extracellular domain of HER) in biological fluids such as plasma (see, e.g., U.S. Patent No. 4,933,294 issued June 12, 1990; WO91 / 05264 issued April 18, 1991; U.S. Patent 5,401,638 issued March 28, 1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990)). In addition to the above studies, it is possible for the expert to conduct various in vivo tests. For example, one may expose cells of the patient's body to an antibody that is optionally labeled with a detectable label, e.g., a radioactive isotope, and assess the binding of the antibody to cells in a patient, e.g., by scanning for external radioactivity or by analyzing a biopsy taken from a patient previously exposed to antibody.

[0128] Conversely, a cancer that does not overexpress HER2 is one that does not express the HER2 receptor at levels higher than normal compared to non-cancer cells of the same tissue type.

[0129] A cancer that overexpresses a HER ligand is one that produces this ligand at levels significantly higher compared to a cell of the same type of non-cancerous tissue. Such overexpression may be caused by gene amplification or increased transcription or translation. HER ligand overexpression can be determined diagnostically by assessing the levels of the ligand (or its encoding nucleic acid) in the patient, e.g. in tumor biopsy or various diagnostic tests such as IHC, FISH, Southern blot, PCR or in vivo tests as described above.

[0130] The term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes disintegration of cells. The phrase is intended to include radioactive isotopes (e.g., At²¹¹, And¹³¹, And¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes Lu), chemotherapeutic agents and toxins such as small molecule toxins or enzymatic toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof.

[0131] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa, ethyleneimines and methylamelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylol melamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); betalapachon; lapachol; col of chi tin; betulinic acid; a camptothecin (including the synthetic analog topotecan (HYCAMTIN®), CPT-11 (Irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9-aminocamptothecin); briostatin; callistatin; CC1065 (including its synthetic analogues adozelesin, dwarfresin, and bizelesin); podophyllotoxin; podophylic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmicin (including synthetic analogs, KW-2189 and CB1TM1); eleuterobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitroureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enedin antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemycin, including dynemycin A; Esperamycin; as well as neocurinostatin chromophore and related chromoprotein chromophores of enedin antibiotic), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, cerinophilin, chromoprotein, dactinomycin, daunorubiaubicin, 6-detorinomycin, daunorubia-lycin, 6-detorinubicin, 6-detorubicin, doxorubicin

-31 (including ADRIAMYCIN®, morpholine-doxorubicin, cyano-morpholine-doxorubicin, 2-pyrroline-doxorubicin, doxorubicin liposomal HCl injections (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET® lipicinos®), pegelylated lipicinosomal doxorubicin TLC (MYOCET®) and deoxidoxorubicin), epirubicin, eorubicin, idarubicin, marcel omy tin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodonigrubicin, streptorigrubicin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; anti-adrenalines such as aminoglutethimide, mitotane, trilostane; folic acid supplement such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; enyluracil; amsacrine; bestrabucyl; byzantrene; edatract; defofamine; demecolcin; diazykwon; elfornithine; elliptin acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and anzamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizophiran; spirogerman; tenuazonic acid; triazonone; 2,2 ', 2-trichlorotriethylamine; trichotecenes (especially T-2 toxin, veracurin A, roridin A and anguidine); urethane; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside (Ara-C); thiotepa; taxoid, e.g. paclitaxel (TAXOL®), an albumin-based nanoparticle paclitaxel formulation (ABRAXANE ™) ​​and docetaxel (TAXOTERE®); chloranbucyl; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin and carboplatin; periwinkle compounds that prevent microtubule formation during tubulin polymerization, including vinblastine (YELBAN®), vincristine (ONCOYIN®), vindestine (ELDISINE®, FILDESIN®) and vinorelbine (NAYELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leukovovin; nowantron; edatrexate; daunomycin; aminopterin; ibandronate; RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g. BONEFOS® or OSTAĆ®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELIDudronate) or risedronate (ACTONEL®); troxacitabine (cytosine nucleoside 1,3-dioxolate analogs); antisense oligonucleotides, particularly those that inhibit expression of signaling genes involved in aberrant cellular proliferation, such as, for example, PKC-alpha, Raf, Η-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOYECTIN® vaccine, LEUYECTIN® vaccine and VAXID® vaccine; a topoisomerase 1 inhibitor (e.g. LURTOTECAN ©);

-32rmRH (e.g. ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, a COX-2 inhibitor (e.g. celecoxib or etoricoxib), a proteosome inhibitor (e.g. PS341); bortezomib (YELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; a Bcl2 inhibitor such as sodium oblimersen (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids, or derivatives of any of the above, as well as combinations of one or more of the above, such as CHOP, an abbreviation for combination therapy with cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, an abbreviation for oxaliplatin (™) treatment regimen in combination with 5-FU and leucovine.

[0132] Also included in this definition are antihormonal agents that regulate or inhibit the action of hormones, such as anti-estrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxy tamoxifen, toremifene (FARESTON® ), idoxifene, droloxifene, raloxifene (VISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®) and EM800 (such an agent can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover and / or decrease ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and non-steroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutothimide, 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; sex steroids including progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilobestrol and premarin, and androgens / retinoids such as fluoxymesterone, all transretinoid and fenretinoid acids; onapriston; anti-progesterones; estrogen receptor (ERD) down regulators; anti-androgens such as flutamide, nilutamide, and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.

[0133] The phrase EGFR-targeting drug refers to a therapeutic agent that binds EGFR and, optionally, inhibits EGFR activation. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind 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 US Patent No. 4,943,533, Mendelsohn et al.) And variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTIX®) and transformed human 225 (H225) (see WO 96/40210, Imclone Systems Inc.); type II EGFR mutant binding antibodies (US Patent No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in US Patent No. 5,891,996; and human EGFR binding antibodies such as ΑΒΧ-EGF (see WO98 / 50433, Abgenix). An anti-EGFR antibody may be combined with a cytotoxic agent, thereby forming an immunoconjugate (see, e.g., EP659,439A2, Merck GmbH Patent). Examples of small molecules that bind to

EGFRs include ZD1839 or Gefitinib (IRESSA ™; Astra Zeneca), CP-358774 or Erlotinib HCL (TARCEVA ™; Genentech / OSI) and AG1478, AG1571 (SU 5271; Sugen).

[0134] A tyrosine kinase inhibitor is a molecule that inhibits to some extent the activity of a tyrosine kinase tyrosine kinase such as the HER receptor. Examples of such inhibitors include the EGFR targeting drugs given in the preceding paragraph, as well as small molecule HER2 tyrosine kinase inhibitors such as TAK165, available from Takeda, dual HER inhibitors such as EKB-569 (available from Wyeth) which preferably bind EGFR but inhibit both HER2 and EGFR overexpressing cells, GW572016 (available from Glaxo) and the oral HER2 and EGFR tyrosine kinase inhibitor and PKI-166 (available from Novartis); comprehensive HER inhibitors [panHER inhibitors] such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as the ISIS-5132 antisense agent available from ISIS Pharmaceuticals which inhibits Raf-1 signaling; non-HER targeting TK inhibitors such as Imatinib mesylate (Gleevac ™) available from Glaxo; MAPK inhibitor of extracellular regulated kinase I CI-1040 (available from Pharmacia); quinazolines such as PD 153035,4- (3-chloroanilino) quinazolines; pyridopyrimidine; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261, and CGP 62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2,3-d] pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino) phthalimide); tyrphostins containing nitrothiophene groups; PD-0183805 (Wamer-Lamber); antisense molecules (e.g. those that bind to HER-encoding nucleic acid); quinoxalines (US Patent No. 5,804,396); tryphostin (US Patent No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); versatile HER inhibitors such as CI-1033 (Pfizer); Affmitac (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 publications: US Patent No. 5,804,396; WO99 / ​​09016 (American Cyanimid); WO98 / 43960 (American Cyanamid); WO97 / 38983 (Warner Lambert); WO99 / ​​06378 (Warner Lambert); WO99 / ​​06396 (Warner Lambert); WO96 / 30347 (Pfizer, Inc); WO96 / 33978 (Zeneca); WO96 / 3397 (Zeneca); and WO96 / 33980 (Zeneca).

[0135] An anti-angiogenic agent refers to a compound that blocks or interferes to some extent with the development of blood vessels. The anti-angiogenic factors can, for example, be small molecules or antibodies that bind to a growth factor or growth factor receptor involved in promoting angiogenesis. Preferred anti-angiogenic agents are an antibody that binds to Vascular Endothelial Growth Factor (VEGF) such as Bevacizumab (AVASTIN®).

[0136] The term cytokine "is a general term for proteins released by one cell population that interact with other cells as intercellular mediators. Examples of such cytokines are lymphokines, monokines, 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; relaxing; prorelaxin; glycoprotein hormones such as

Follicle Stimulating Hormone (FSH), Thyrotropin (TSH) and Luteinizing Hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor -ai -β; Mullerian inhibitor; mouse gonadotropin peptide; inhibin; activin; vascular endothelial growth factor; an 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 growth factor 1 and 2; Erythropoietin (EPO); osteoinductive factors; interferons such as interferon α, -β, and -γ; colony stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocytomacrophage-CSF (GM-CSF);

and granulocytes-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1?, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12; tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and KIT ligand (KL). The expression cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequences cytokines.

[0137] The formulated antibody is preferably substantially pure and desirably substantially homogeneous (ie free of contaminating proteins etc.). A substantially pure antibody is 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. A substantially homogeneous antibody is a composition that comprises at least about 99% by weight of the antibody based on the total weight of the composition.

[0138] A B-cell surface marker or B-cell surface antigen is an antigen expressed on the surface of a B-cell that may be targeted by an antibody which binds to it. Exemplary B cell surface markers include the leukocyte surface markers CD 10, CD 19, 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, Barclay et al. Academic Press, Harcourt Brace &amp; Co., New York ed.). Other B cell surface markers include RP105, FcRH2, B-cell CR2, CCR6, Ρ2Χ5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRHl, IRTA2, ATWD578, FcRH3, IRTA1, BC FcRHó7, BC FcRH6 B cell surface markers of particular interest are preferably expressed on the surface of B cells as compared to other mammalian non-B cell tissues, and can be expressed on B cell precursors and mature B cells. A preferred B cell surface marker is CD20 or BR3.

[0139] The CD20 or CD20 antigen is an approximately 35-kDa, non-glycosylated phosphoprotein found on the surface of more than 90% of B cells from peripheral blood or lymphoid organs. CD20 is present on both normal B cells and malignant B cells, but is not expressed on stem cells. Other names for CD20 in the literature include B cell restriction antigen

-35 and Bp35. The CD20 antigen is described in Clark et al. Proc. Natl. Acad. Sci. (USA) 82: 1766 (1985), for example.

[0140] For its own purposes only, humanized 2H7 refers to a humanized variant of the 2H7 antibody whose CDR sequences are disclosed in US Patent No. 5,500,362 (Figs. 5 and 6). Examples of humanized 2H7 antibodies include the variants described in WO2004 / 056312 as well as other variants including but not limited to: 2H7vl6, 2H7v31, 2H7v73, 2H7v75, 2H7v96, 2H7vll4, 2H7vll5, 2H7vll6, 2H7v377, etc.

[0141] The humanized 2H7 antibody may contain one, two, three, four, five, or six of the following CDR sequences:

CDR LI RASSSVSYXH sequence where X is M or L (SEQ ID No. 67), for example SEQ ID No. 57 (Figure 18A), L2 CDR sequence of SEQ ID No. 58 (Figure 18A), L3 CDR sequence QQWXFNPPT, where X is S or A (SEQ ID No. 68), for example SEQ ID No. 59 (Fig. 18A), CDR HI sequence of SEQ ID No. 60 (Fig. 18B), CDR H2 sequence AlYPGNGXTSYNQKFKG where X is D or A (SEQ ID No. 69), for example SEQ ID No. 61 (Fig. 18B), and the CDR sequence H3 VVYYSXXYWYFDV where X at position 6 is N, A, Y, W or D and X at position 7 is S or R (SEQ ID No. 70), for example SEQ ID No. 62 (Fig. 18B) .

[0142] The above CDR sequences are generally present within the human light chain variable region and heavy chain variable region framework sequences, as are the substantially human Kappa light chain subgroup I (VlkI) shared FR residues and the substantially human subgroup I heavy chain FR shared III (VhIII). See also WO 2004/056312 (Lowman et al.).

[0143] The heavy chain variable region may be attached to the constant chain region of human IgG, where the region may be, for example, IgG1 or IgG3, including native and variant sequence constant regions. They may include

[0144] Such antibodies may include the heavy variable domain sequences of SEQ ID No. 29 (v16 as shown in Fig. 18B), optionally also including the light domain variable [chain] sequence of SEQ ID No. 26 (v16 as shown in Fig. 18A) which optionally comprises one or more amino acid substitutions at positions 56, 100 and / or 100a, e.g. D56A, N100A or N100Y and / or SOOaR in the heavy [chain] variable domain and one or more amino acid substitutions at positions 32 and / or 92, e.g., M32L and / or S92A in the light [chain] variable domain. Preferably the antibody is an intact antibody comprising the light chain amino acid sequences of SEQ ID No. 63 or 64 and the heavy chain amino acid sequences of SEQ ID No. 65, 66, 71, or 72.

[0145] A preferred humanized 2H7 antibody is ocrelizumab (Genentech).

[0146] The antibody may further comprise at least one amino acid substitution in the Fc region that improves ADCC activity, such as where the amino acid substitutions are at positions 298, 333 and 334, preferably S298A, E333A and K334A, using heavy chain residue numbering Eu. See also U.S. Patent No. 6,737,056B 1, Presta.

[0147] Any of these antibodies may contain at least one substitution in the Fc region that improves FcRn binding or serum half-life, for example a substitution at position 434 of a heavy chain such as N434W. See also U.S. Patent No. 6,737,056B 1, Presta.

[0148] Any of these antibodies may further comprise at least one amino acid substitution in the Fc region that improves CDC activity, for example, comprising at least a substitution at position 326, preferably K326A or K326W. See also U.S. Patent No. 6,528,624B 1 (Idusogie et al.).

[0149] Some preferred humanized 2H7 variants are those that contain a light chain variable domain of SEQ ID No. 26 and a heavy chain variable domain of SEQ ID No. 29, including those with or without Fc region substitutions (if present) and those containing the heavy chain variable domain of SEQ ID No. heavy chain variable with alteration N100A; or D56A and N100A; or D56A, N100Y, and SOOaR; in SEQ ID No. 29 and a light chain variable domain with the M32L alteration; or S92A; or M32L and S92A; in SEQ ID No. 26.

[0150] M34, in the variable heavy chain of 2H7v16, has been identified as a potential source of antibody stability and is another potential candidate for substitution.

[0151] In summary of some of the various preferred embodiments of the invention, the variable region of the 2H7v16 based variants comprises the amino acid sequence of v16, except for the amino acid substitution positions which are set out in the Table below. Unless otherwise noted, 2H7 variants will have the same light chain as v16.

Exemplary Variants of the Humanized 2H7 Antibody [0152]

<td>2H7 version</td><td>Heavy chain changes (V_H.)</td><td>Light chain changes (V_L.)</td><td>Fc changes</td>

<td>16 for reference</td><td></td><td></td><td> -</td>

<td> 31</td><td> -</td><td> -</td><td>S298A, E333A, K334A</td>

<td> 73</td><td>SUN 00 A</td><td>M32L</td><td></td>

<td> 75</td><td>N100A</td><td>M32L</td><td>S298A, E333A, K334A</td>

<td> 96</td><td>D56A, N100A</td><td>S92A</td><td></td>

<td> 114</td><td>D56A, N100A</td><td>M32L, S92A</td><td>S298A, E333A, K334A</td>

<td> 115</td><td>D56A, N100A</td><td>M32L, S92A</td><td>S298A, E333A, K334A, E356D, M358L</td>

<td> 116</td><td>D56A, N100A</td><td>M32L, S92A</td><td>S298A, K334A, K322A</td>

<td> 138</td><td>D56A, N100A</td><td>M32L, S92A</td><td>S298A, E333A, K334A, K326A</td>

<td> 477</td><td>D56A, N100A</td><td>M32L, S92A</td><td>S298A, E333A, K334A, K326A, N434W</td>

<td> 375</td><td> -</td><td> -</td><td>K334L</td>

<td> 588</td><td> -</td><td> -</td><td>S298A, E333A, K334A, K326A</td>

<td> 511</td><td>D56A, N100Y, SlOOaR</td><td>M32L, S92A</td><td>S298A, E333A, K334A, K326A</td>

One preferred humanized 2H7 comprises the 2H7v16 light chain variable [chain] sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPL ^ APSNLASGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID No. 26&gt;;

and the 2H7v16 heavy chain variable domain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGR FTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDWŻGQGTL IDVTV) (SEQGTL No.VTV).

[0154] Where the humanized 2H7v16 antibody is an intact antibody, it may contain a light chain amino acid sequence:

DIQMTQSPSSLSASVGDRVTTTCRASSSVSYMHWYQQKPGKAPKPLIVAPSNLASGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEiCHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID No. 63);

and the heavy chain amino acid sequence of SEQ ID No. 65 or:

-38 EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGR mSVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVVZGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEYKFfNWYYDGYEYHNAKTKPREEOYNSTYRWSYLTYLHODWLNGKEYKCKYSNKALPAPlEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID No. 71).

[0155] Another preferred humanized 2H7 antibody comprises the 2H7v511 variable light [chain] domain sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKR (SEQ ID No. 73) and sequence heavy domain 2H7v511

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGATYPGNGATSYNQKFKGR FTISVDKSKNTLYLQMNSLRAEDTAVYYCARWYYSYRYWYFDVWGQGTLVTV No. 74) (SEQ ID No. 74).

[0156] Where the humanized 2H7v511 antibody is an intact antibody, it may contain a light chain amino acid sequence:

DIQMTQSPSSLSASVGDRVnTCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDF TLIISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKZHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID No. 64) and heavy aminokwasowąłańcucha sequence SEQ ID No. 66 or:

EVQLVESGGGLVQPGGSLRLSCAASGY1FTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGR FTISVDKSKNTLYLQMNSLRAEDTAVYYCARWYYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPŁPKDTLM1SRTPEVTCVWDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRWSVLTVLHQDWLNGKEYKCKVSNAALPAPIAA TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEVESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID No. 72).

[0157] B-cell neoplasm includes non-Hematological lymphoma (NHL), including follicular low-grade NHL, small lymphocyte NHL (SL), follicular medium-grade NHL, diffuse medium-grade NHL, high-grade immunoblastic NHL , high-grade lymphoblastic NHL, high-grade small non-convex NHL, tumor-heavy NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldenstrom's macroglobulinemia; leukemia, including acute leukemia

-39 lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia and chronic myeloblastic leukemia and other hematological malignancies. Such tumors can be treated with antibodies directed against B cell surface markers such as CD20.

[0158] The expression non-Hodgkin's lymphoma or NHL refers to a tumor of the lymphatic system other than Hodgkin's lymphomas. Hodgkin's lymphomas can be generally distinguished from non-Hodgkin's lymphomas by the presence of Reed-Sternberg cells in Hodgkin's lymphomas and their absence in non-Hodgkin's lymphomas. Examples of non-Hodgkin's lymphomas encompassed by this expression include any of those which could be identified by one of skill in the art (e.g. oncologist or pathologist) according to classification schemes known in the art, such as the Revised European-American Lymphoma (REAL) scheme described in Color Atlas of Clinical Hematology, Third Edition; A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Limited 2000) (see especially Figs. 11.57, 11.58 and / or 11.59). More specific examples include, but are not limited to, relapsed or refractory NHL, low-grade first-line NHL, stage III / IV NHL, chemotherapy-resistant NHL, lymphoblastic leukemia and / or B-cell precursor lymphoma, small lymphocyte lymphoma, chronic B-cell lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmic lymphoma, marginal zone B cell lymphoma, splenic marginal zone lymphoma, extra-nodal MALT-type marginal lymphoma, marginal zone nodal lymphoma, hairy cell leukemia, low grade myeloma and / or myeloma, myeloma / follicular myeloma Medium grade / follicular NHL, mantle cell lymphoma, germinal center (follicular) lymphoma, Moderate-grade NHL diffuse, diffuse large B-cell lymphoma, aggressive NHL (including aggressive first-line NHL and aggressive recurrent NHL), NHL recurrent or refractory to autologous stem cell transplantation, primary large B-cell mediastinal lymphoma, primary exudative lymphoma , High-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-convex NHL, High tumor mass NHL, Burkitt's lymphoma, lymphoblastic T-cell precursor (peripheral) leukemia and / or lymphoma, adult cell lymphoma and / or leukemia, chronic T-cell lymphoma and / or prolymphatic leukemia, large granular lymphocytic leukemia, granuloma mycosis and / or Sezary syndrome, extranodal lymphoma of natural cytotoxic cells / T cells (nasal type), enteropathic type T-cell lymphoma, T-cell hepatocellular lymphoma, subcutaneous T-cell lymphoma resembling cellulitis, cutaneous (cutaneous) lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, peripheral T-cell lymphoma (not otherwise specified) ) and angioimmunoblastic T-cell lymphoma.

[0159] An autoimmune disease is a disease or disorder arising from and directed against a subject's own tissues, or co-segregation or manifestation thereof

-40 or a disease state resulting therefrom. Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, atherosclerosis, psoriasis, and ankylosing spondylitis), psoriasis, dermatitis including atopic dermatitis, chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis / dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as progressive systemic sclerosis, inflammatory bowel disease (IBD) (for example, Kron's disease, ulcerative colitis, autoimmune colitis), pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis episcleritis), respiratory distress syndrome including adult respiratory distress syndrome (ARDS), meningitis, IgE-dependent disease, such as anaphylaxis and allergic rhinitis, encephalitis such as Rasmussen's encephalitis, uveitis or autoimmune uveitis, colitis such as microscopic colitis and collagenous colitis, glomerulonephritis (KNN) such as membranous UC (membranous nephropathy), idiopathic membranous UBC, membranous proliferative UBC (BPKZN), including Type I and Type II and rapidly progressive UBC, allergic conditions, allergic reaction, eczema, asthma, conditions including T-cell infiltration and chronic inflammatory reactions, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) such as skin SLE, subacute cutaneous lupus erythematosus, lupus (including nephritis, encephalitis, pediatric, non-renal, discoidal, alopecia), juvenile diabetes (Type I), including pediatric insulin dependent diabetes mellitus (IDDM), adult diabetes mellitus (Type II diabetes), multiple sclerosis (MS) such as spin-optic MS, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T cells, tuberculosis, sarcoidosis, granuloma including lymphomatoid asthma, Wegener's granulomatosis, agranulocytosis, vasculitis (including large vasculitis (including rheumatic polymyalgia and giant cell arteritis (Takayasu)), median vasculitis (including Kawasaki disease and polyarteritis nodosa), CNS vasculitis, systemic necrotizing vasculitis, vasculitis associated with neutrophil cytoplasmic antibodies such as Churg-Strauss systemic vasculitis or syndrome (CSS)), temporary arteritis , aplastic anemia, Coombs anemia, Blackfan-Diamond anemia, hemolytic anemia or autoimmune hemolytic anemia (AIHA) pernicious anemia, red cell aplasia (PRCA), factor VIII deficiency, haemophilia A, autoimmune neutropenia, penctopenia, leukopenia, leukocyte diapedesis diseases, CNS disease, multiple organ failure, antibody-antigen complex disease, disease with the presence of antibodies against the membrane basal glomerulus, antiphospholipid antibody complex, allergic neuritis, Bechet or Behcet disease, Castelman syndrome, Goodpastur's syndrome, Reynaud's syndrome,

-41 Sjogren's syndrome, Stevens-Johnson syndrome, pemphigus such as bullous pemphigoid (including plain and mucosal pemphoid), autoimmune polyglandular syndromes, Reiter's disease, immune nephritis, chronic neuropathy such as IgM polyneuropathy or IgM-dependent nauropathy (thrombocytopenia, developed by patients with problems with the heart muscle, for example), including thrombocytopenic purpura (TTP) and autoimmune or immune dependent thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, autoimmune testicular and ovarian disease including autoimmune orchitis and ovarian inflammation, primary hypothyroidism, hypoparathyroidism autoimmune endocrine diseases including thyroid diseases such as autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Addison's disease, Grave's disease, multi-gland syndromes such as autoimmune polyglandular syndromes (or multi-glandular hormonal disorders syndromes), paraneoplastic syndromes including paraneoplastic syndromes myasthenic Lambert-Eaton syndrome or Lambert-Eaton syndrome, generalized stiffness syndrome, encephalomyelitis such as allergic encephalomyelitis, myasthenia gravis, cerebellar degeneration, inflammation of the limbic system and / or brainstem, neuromyotonia, opsoclonus or myoclonus and opsoclonus syndrome (OMS) and sensory neuropathy, Sheehan's syndrome, autoimmune inflammation liver, chronic hepatitis, lupus hepatitis, chronic active hepatitis and autoimmune chronic active hepatitis, lymphoid interstitial pneumonia, obliterating pneumonia (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), primary biliary cirrhosis, celiac disease (gluten enteropathy), treatment-resistant celiac disease, dermatitis herpetiformis, cryoglobulinemia, lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune inner ear disease (AIED) or autoimmune hearing loss, myoclonus opsoclonus syndrome (OMS), cartilage inflammation such as recurrent cartilage inflammation, alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis non-malignant lymphocytosis, primary lymphocytosis which includes monoclonal B-cell lymphomatosis (e.g., mild monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, canal treatment such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis and CNS channelopathy, autism, focal segmental inflammatory myopathy glomerulonephritis (FSGS), endocrine ophthalmopathy, uveoretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, inflammatory process, gastric atrophy, pre-senile dementia, demyelinating diseases, Dressler's syndrome, alopecia areata, CREST syndrome (calcinosis, Raynaud's symptoms, esophageal dysfunction, sclerosis of the fingers and telangiectasia), autoimmune infertility in men and women, ankylosing spondylitis, mixed connective tissue disease, disease

-42 Chagas, rheumatic disease, recurrent miscarriage, farmer's lung disease, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, avian lung disease, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease , post-transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, myocardial fibrosis, endophthalmitis, erythema haemorrhage and long-term, hemolytic disease of the newborn, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariosis, ciliary body inflammation such as chronic ciliary cellulitis, bi-temporal cellulitis Ciliary or Fuch's ciliary inflammation, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection myocardial disease, Alzheimer's disease, parvovirus infection, rubella virus infection, vaccine syndromes, congenital rubella syndrome, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, follicular interstitial nephritis, thromboembolism thyrotoxicosis, medullary pruritus, and giant cell polymyalgia.

[0160] The tumor necrosis factor receptor superfamily or the TNF receptor superfamily refers to receptor polypeptides bound by the TNF-family of cytokines. Generally, these receptors are Type I transmembrane receptors with one or more cysteine ​​rich repeat sequences in their extracellular domain. The TNF superfamily can be further subdivided into (1) death receptors; (2) decoy receptors; and (3) signal receptors that lack death domains. Death receptors in their cytoplasmic or intracellular region contain a death domain, i.e. a region or sequence that acts to transduce a signal in a cell, which may result in apoptosis or induction of certain genes. Decoy receptors lack a functional death domain and are incapable of transmitting signals that result in apoptosis. Examples of the TNF family of cytokines include Tumor Necrosis Factor alpha (TNFalpha), Tumor Necrosis Factor beta (TNF-beta or lymphotoxin), CD30 ligand, CD27 ligand, CD40 ligand, ΟΧ-40 ligand, ligand

4-1 BB, 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), osteoprotergrin (OPG), APRIL, ligand RANK (also referred to as TRANCE) and TALL1 (also referred to as BlyS, BAFF or THANK) Examples of receptors in the TNF receptor superfamily include: Tumor Necrosis Factor Receptor Type 1 (TNFR1), Tumor Necrosis Factor Receptor Type 2 (TNFR2), P75 Nerve Growth Factor Receptor (NGFR), CD40 B-cell surface antigen, komórek-40 T-cell antigen, Apo-1 receptor (also called Fas or CD95), Apo-3 receptor (also called DR3, swl1, TRAMP, and LARD), receptor called Transmembrane Activator and Interactor CAML or TACI, BCMA, DR4, DR5 protein (alternatively referred to as Apo-2; TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER), DR6, DcRl (also referred to as TRID, LIT or TRAIL-R3), DcR2 (also called TRAIL-R4 or TRUNDD), OPG,

-43 DcR3 (also called TR6 or M68), CAR1, HVEM (also called AT AR or TR2), GITR, ZTNFR-5, NTR-1, TNFL1, CD30, beta lymphotoxin receptor (LTBr), 4-1BB and TR9 receptor (EP988,371A1).

[0161] The expressions Apo-2 ligand, Apo-2L, Apo2L, Apo-2 / TRAIL ligand, and TRAIL are used interchangeably to refer to a polypeptide sequence including amino acid residues 114-281 inclusive, 95-281 inclusive, residues 92-281 inclusive, residues 92-281 91-281 inclusive, residues 41-281 inclusive, residues 39-281 inclusive, residues 15-28 inclusive, or residues 1-281 inclusive, from the amino acid sequence shown in Fig. 24 (SEQ ID No. 46) as well as the biologically active deletion, insertion and / or substituted 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 Figure 24 (SEQ ID No. 46). Apo-2L polypeptides may be encoded by the native nucleotide sequence shown in Figure 24 (SEQ ID No. 45). Optionally, the codon encoding the Proll9 residue (Fig. 24; SEQ ID No. 45) may be CCT or CCG. Optionally, fragments or variants are biologically active and share at least 80% amino acid sequence identity, or at least 90% sequence identity, or at least 95%, 96%, 97%, 98%, or 99% sequence identity to any of the above sequence. The definition includes substituted variants of the Apo-2 ligand, wherein at least one of its native amino acids is substituted with another amino acid, such as an alanine residue. The definition also includes the native sequence of Apo-2 ligand, isolated from a source of Apo-2 ligand or produced by recombinant and / or synthetic methods. The Apo-2 ligand of the invention comprises a polypeptide, referred to as Apo-2 ligand or TRAIL, disclosed in WO97 / 01633 published January 16, 1997, WO97 / 25428 published July 17, 1997, WO99 / ​​36535 published July 22, 1999, WO 01/00832 published 2 January 2001, WO02 / 09755 published February 7, 2002, WO 00/75191 published December 14, 2000, and U.S. Patent No. 6,030,945 issued February 29, 2000. The expressions are used to generally refer to forms of the Apo-2 ligand, which include monomer, dimer, trimer, hexamer, or higher oligomeric forms of the polypeptide. All numbering of the amino acid residues relating to the Apo-2L sequence use the numbering according to Fig. 24 (SEQ ID No. 46), unless specifically stated otherwise.

[0162] The Apo-2 ligand receptor includes the receptors in the art also referred to as DR4 and DR5. Pan et al. described a member of the TNF receptor family, designated DR4 (Pan et al., Science, 276: 111-113 (1997); see also WO98 / 32856 published Jul 30, 1998; WO99 / ​​37684 published Jul 29, 1999; WO 00/73349 published 7 2000 December; US 6,433,147 issued Aug. 13, 2002; US 6,461,823 issued Oct. 8, 2002 and US 6,342,383 issued Jan.29, 2002). Sheridan et al., Science, 277: 818-821 (1997) and Pan et al., Science, 277: 815-818 (1997) have added another receptor for Apo2L / TRAIL (see also WO98 / 51793 published November 19, 1998; WO98 / 41629 published September 24, 1998). This receptor is referred to as DR5 (the receptor is alternatively also referred to as Apo2L; TRAIL-R, TR6, Tango-63, hAPO8, 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); WO98 / 35986

-44 published Aug. 20, 1998; EP870.827 published Oct. 14, 1998; WO98 / 46643 published October 22, 1998; WO99 / ​​02653 published January 21, 1999; WO99 / ​​09165 published February 25, 1999; WO99 / ​​11791 published March 11, 1999; USA 2002/0072091 published Aug 13, 2002; USA 2002/0098550 published December 7, 2001; USA 6,313,269 issued Dec. 6, 2001; USA 2001/0010924 published Aug 2, 2001; USA 2003/01255540 published Jul 3, 2003; USA 2002/0160446 published October 31, 2002, USA 2002/0048785 published April 25, 2002; USA 6,342,369 issued May 27, 2003; US 6,072,047 issued June 6, 2000, US 6,642,358 issued November 4, 2003). As described above, other Apo-2L receptors include DcR1, DcR2, and OPG. The expression Apo-2L receptor includes native sequence receptor and receptor variants. These expressions include the Apo-2L receptor expressed in a variety of mammals, including humans. The Apo-2L receptor can be expressed endogenously as it occurs naturally in various human tissue lines, or it can be expressed by recombinant or synthetic means. "Native Apo-2L receptor sequence" includes a polypeptide having the same amino acid sequence as a naturally derived Apo-2L receptor. Thus, the native sequence of the Apo-2L receptor may take the amino acid sequence of a naturally occurring Apo-2L receptor from any mammal, including humans. Such native Apo2L receptor sequence can be isolated from nature or produced by recombinant or synthetic means. The expression native Apo-2L receptor sequence includes specifically naturally occurring truncated or secreted forms of the receptor (e.g., a soluble form, including, for example, the sequence of an extracellular domain), naturally occurring variant forms (e.g., alternative splicing forms) and naturally occurring allelic variants. Receptor variants may include native sequence Apo-2L receptor fragments or deletion mutants. Figs. 25A-C show the 411 amino acid sequence of the human DR5 receptor together with its nucleotide sequence (SEQ ID Nos. 47 and 48) as published in WO 98/51793 on November 19, 1998. A transcriptional splice variant of the human DR5 receptor is known in the art. This splice variant encodes the 440 amino acid sequence of the human DR5 receptor as shown in Fig. 26A-C, together with its nucleotide sequence (SEQ ID Nos. 49 and 50) and published in WO 98/35986 on August 20, 1998.

[0163] A death receptor antibody refers to an antibody or antibodies targeting a receptor from the tumor necrosis factor receptor superfamily and comprising a death domain capable of signaling apoptosis, and such antibodies include the DR5 antibody and the DR4 antibody.

[0164] A DR5 receptor antibody, DR5 antibody, or anti-DR5 antibody is used in a broader sense to denote antibodies that bind to at least one form of a DR5 receptor or extracellular domain thereof. Optionally, the DR5 antibody is fused or linked to a sequence or heterologous molecule. Preferably, the heterologous sequence allows or helps the antibody form 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 receptor

-45 2L (e.g. DR4, DcRl or DcR2). Optionally, the antibody is an agonist of DR5 signaling activity.

[0165] Optionally, the DR5 antibody of the invention binds to the DR5 receptor over a concentration range of about 0.1 nM to about 20 mM as measured in the BIAcore binding study. Optionally, DR5 antibodies of the invention exhibit an IC50 value from about 0.6 nM to about 18 mM as measured in the BIAcore binding study.

[0166] For its own purposes only, the expression Apomab refers to an agonist antibody that binds to DR5 and comprises the amino acid sequences of [variable] [heavy] and [variable] [chain] light of SEQ ID Nos. 55 and 56. Preferably, Apomab contains the heavy and light chains of SEQ ID Nos. 51 and 52, respectively.

II. Production of Antibodies

[0167] Techniques for producing antibodies that can be formulated according to the invention are followed.

(i) Antigen selection and preparation

[0168] Preferably, the antigen to which the antibody binds is a biologically important glycoprotein and administration of the antibody to a mammal suffering from a disease or disorder may provide a therapeutic benefit to the mammal. However, antibodies directed against non-polypeptide antigens (such as tumor associated glycolipid antigens; see US Patent 5,091,178) are also contemplated.

[0169] Where the antigen is a polypeptide, it may be a transmembrane molecule (e.g., a receptor) or a ligand such as a growth factor. Exemplary antigens include molecules such as renin; growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing hormone; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-l-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor (TF), and von Willebrand factor; anticoagulant factors such as Protein C; atrial natriuretic peptide; pulmonary surfactant; a plasminogen activator such as urokinase or human urine or tissue-specific plasminogen activator (t-PA); bobmesin; thrombia; hematopoietic growth factor; tumor necrosis factor alpha and beta; enkephalinase; RANTES (factor regulated by activation; expression and secretion by normal lymphocytes); human macrophage inflammatory protein (ΜΙΡ-1-alpha); plasma albumin such as human plasma albumin; Mullerian duct inhibitory substance; chain A relaxins; B-chain relaxin; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein such as beta-lactamase; DNase; IgE; a cytotoxic T lymphocyte (CTLA) associated antigen such as

CTLA-4; inhibin; activin; vascular epithelial growth factor (VEGF); hormone or growth factor receptors; protein A or D; rheumatoid factors; a neurotrophic factor, such as brain derived neurotrophic factor (BDNF)

Neurotrophin-3, -4, - 5 or -6 (NT-3, NT-4, NT-5 or NT-6) or a nerve growth factor 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-b1, TGF-b2, TGF-b3, TGF-b4, or TGF-b5; 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; CD proteins such as CD3, CD4, CD8, CD 19, CD20, CD22, and CD40; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic protein (BMP); interferon such as interferon alpha, beta and gamma; colony stimulating factors (CSF), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (IL), e.g. 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; membrane surface proteins; decay accelerating factor; a viral antigen, such as, for example, part of the AIDS envelope [HIV]; transport proteins; homing receptors; addresses; regulatory proteins; integrins such as CD11a, CDI Ib, CDlic, CD18, ICAM, VLA-4 and VCAM; a tumor associated antigen such as the HER2, HER3 or HER4 receptor; and fragments of any of the above-mentioned polypeptides.

[0170] Exemplary molecular targets for the antibodies of the invention include CD proteins such as CD3, CD4, CD8, CD 19, CD20, CD22, CD34, and CD40; members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; B cell surface antigens such as CD20 or BR3; a member of the tumor necrosis receptor superfamily, including DR5; prostate stem cell antigen (PSCA); adhesion molecules such as LFA-1, Maci, p150.95, VLA-4, ICAM-1, VCAM, alpha4 / beta7 integrin and alphav / beta3, including either their alpha or beta subunit (e.g. anti-CD11a, anti-CD18, or anti-CD11b antibodies; growth factors such as VEGF as well as its receptors; tissue factor (TF); tumor necrosis factor (TNF) such as TNF-alpha or TNF-beta, interferon alpha (IFN-alpha); an interleukin such as IL-8; IgE; blood group antigens; flk2 / flt3 receptor; obesity receptor (ESR); mpl receptor; CTLA-4; protein C etc.

[0171] Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for the production of antibodies. For transmembrane molecules such as receptors, fragments thereof (e.g., the extracellular domain of a receptor) may be used as an immunogen. Alternatively, cells expressing the transmembrane molecule can be used as the immunogen. Such cells may be derived from a natural source (e.g. tumor line cells) or may be cells transformed by recombinant techniques to express transmembrane molecules. Other antigens and their forms useful for producing antibodies will be apparent to those skilled in the art.

[0172] For the production of HER2 antibodies, the HER2 antigen to be used in its production can be, eg, a soluble form of the extracellular domain of HER2 or a portion thereof containing the epitope of interest. Alternatively, they can be used to generate antibodies

Cells expressing HER2 on their surface are used (e.g., NIH-3T3 cells transformed to overexpress HER2; or a tumor cell line such as SKBR-3 cells, see Stancovski et al. PNAS (USA) 88: 8691-8695 (1991) ).

(ii) Monoclonal antibodies

[0173] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies making up the population are identical and / or bind to the same epitope, except for possible variants that may arise during the production of the monoclonal antibody. Therefore, a monoclonal dose indicates the nature of the antibody as not being a mixture of discrete antibodies.

[0174] For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or can be produced by recombinant DNA methods (US Patent No. 4,816,567).

[0175] In a 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, lymphocytes can be immunized in vitro. Lymphocytes are then fused to myeloma cells using a suitable cell fusion agent, such as polyethylene glycol, to produce a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0176] The hybridoma cells thus prepared are seeded and cultured in a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of the unfused myeloma parental cells. For example, if the myeloma stem cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then the culture medium used for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent growth of cells lacking HGPRT.

[0177] Preferred myeloma cells are those that fuse efficiently, promote stable, high-level antibody production by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among them, preferred myeloma cell lines are murine myeloma lines, such as those derived from murine MOPC-21 and MPC-11 tumors, available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 or X63-Ag8 cells. -653, available from the American Type Culture Collection, Rockvile, Maryland, USA. Human myeloma and mouse-human hetero-myeloma 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)).

[0178] The culture medium in which the hybridomas are grown is tested for the production of monoclonal antibodies directed against the antigen.

Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay such as a radioimmunoassay (RIA) or enzyme immunoabsorption assay (ELISA).

[0179] The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis from Munson etaL, Anal. Biochem. 107: 220 (1980).

After the identification of hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Additionally, hybridoma cells can be grown in vivo as exudative tumors in an animal.

[0181] Monoclonal antibodies secreted by the subclones are preferably separated from the culture medium, exudate fluid, or serum by conventional antibody purification procedures such as, for example, Protein A Sepharose chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0182] DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be inserted into expression vectors and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that would not otherwise produce the antibody protein, for synthesis. monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria from DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

[0183] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated 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 libraries. Subsequent publications describe the production of high affinity (nM) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)) as well as combinatorial infection and in vivo recombination as a strategy for the construction of very large phage libraries (Waterhouse et al, Nuc. Acids. Res., 21: 2265-2266 (1993)). Thus, these techniques are real

Alternatives to traditional techniques for generating monoclonal antibodies from hybridomas for isolating monoclonal antibodies.

[0184] DNA may also be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains for murine homologous sequences (US Patent No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Sci. USA. , 81: 6851 (1984)), or by covalently linking all or part of a non-immunoglobulin polypeptide coding sequence to an immunoglobulin coding sequence.

[0185] Typically such non-immunoglobulin polypeptides are substitutions for the antibody constant domains or substitutions for the variable domains of one antigen-joining site of an antibody to form a bivalent chimeric antibody having one antigen combining site with antigen specificity and a second antigen combining site with specificity. for a different antigen.

(iii) Humanized antibodies,

[0186] Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, typically taken from an "import" variable domain. Humanization can generally be carried out by 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 for the corresponding human antibody sequences. Accordingly, such "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567) in which substantially less than the entire human variable domain has been replaced with the appropriate 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 have been substituted with residues from analogous sites in rodent antibodies.

[0187] To reduce antigenicity, the choice of human variable domains, both from light and heavy chains, to be used in making the humanized antibodies is very important. According to the so-called "best match" method, the variable domain sequence of a rodent antibody is searched over the entire library of known human variable domain sequences. Then, the human sequence most closely related to the rodent sequence is accepted as the human "framework" (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 framework derived from a sequence that is shared by all human antibodies of a given subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Prestae (a /., J. Immunol., 151: 2623 (1993)). )).

[0188] Furthermore, it is important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are produced by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are widely available and known to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of the sequences of selected candidate immunoglobulins. Reviewing these presentations allows for an analysis of the likely role of the residues in the function of the proposed immunoglobulin sequence, i.e. the analysis of residues affecting the ability of the proposed immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the receiving and import sequence to achieve the desired characteristics of the antibody, such as increased affinity for the target antigen (s). In general, the hypervariable region residues are directly and substantially involved in influencing antigen binding.

[0189] WO01 / 00245 describes the preparation of exemplary humanized HER2 antibodies that bind HER2 and block ligand activation of a HER receptor. The humanized antibody of particular interest blocks EGF, TGF-α and / or HRG dependent MAPK activation substantially as effectively as murine monoclonal antibody 2C4 (or a Fab fragment thereof) and / or binds HER2 essentially as effectively as murine monoclonal antibody 2C4 ( or a Fab fragment thereof). The humanized antibody may, for example, contain non-human hypervariable region residues incorporated into a human heavy variable [chain] domain, and may further include a framework (FR) substitution at a position selected from the group consisting of 69H, 71H, and 73H using the domain numbering system. a variable specified in Kabat et al., Sequences of Proteins of Immunological Interest, Ed. 5th Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one embodiment, the humanized antibody comprises FR substitutions at two or all of positions 69H, 71H, and 73H.

[0190] An exemplary humanized antibody of interest comprises heavy variable domain complementarity determining residues GFTFTDYTMX, where X is preferably D or S (SEQ ID No. 7); DVNPNSGGSIYNQRFKG (SEQ ID No. 8); and / or NLGPSFYFDY (SEQ ID No. 9), optionally including amino acid modifications of these CDR residues, eg, which modifications substantially preserve or improve antibody affinity. For example, the antibody variant of interest has from about one to about seven or about five amino acid substitutions in the above heavy chain variable CDR sequences. Such antibody variants can be produced by affinity maturation, e.g., as described below. A most preferred humanized antibody comprises the amino acid sequence of a heavy chain variable domain in SEQ ID No. 4.

[0191] The humanized antibody may contain complementarity determining residues from the light domain [chain] KASQDVSIGVA (SEQ ID No. 10); SASYXXX, where X at position 5 is preferably R or L, where X at position 6 is preferably Y or E, and X at position 7 is preferably T or S (SEQ ID No. 11); and / or QQYYIYPYT (SEQ ID No. 12), e.g. in addition to the heavy domain variable [chain] CDR residues in the previous paragraph. Such humanized antibodies optionally contain amino acid modifications to the above CDR residues, e.g., which modifications substantially preserve or improve the affinity of the antibody. For example, the antibody variant of interest has from about one to about seven or about five amino acid substitutions in the above light CDR variable sequences. Such antibody variants can be produced by affinity maturation, e.g., as described below. The most preferred humanized antibody comprises the variable light domain amino acid sequence in SEQ ID No. 3.

[0192] The application also contemplates affinity matured antibodies that bind HER2 and block ligand activation of a HER receptor. The parent antibody may be a human antibody or a humanized antibody, e.g., comprising the light and / or heavy variable [chain] variable [domain] sequences of SEQ ID Nos. 3 and 4, respectively (i.e., variant 574). The affinity matured antibody preferably binds to the HER2 receptor with an affinity greater than that of murine 2C4 or variant 574 (e.g. affinity improved from about two or about four to about 100 fold or about 1000 fold, e.g., as assessed by HER2 Extracellular Domain (ECD) ELISA). Exemplary heavy [chain] variable [domain] CDR residues to be substituted include H28, H30, H34, H35, H64, H96, H99, or combinations of two or more (e.g., two, three, four, five, six or seven of these residues) . An exemplary light-chain variable [domain] CDR residue to alter include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97, or combinations of two or more (e.g., two, three, four, five, or up to about ten of these residues).

[0193] Various forms of the humanized antibody or affinity matured antibody are contemplated. For example, the humanized antibody or affinity matured antibody can be an antibody fragment, such as a Fab, that is optionally linked to one or more cytotoxic agent (s) to form an immunoconjugate. Alternatively, the humanized or affinity matured antibody can be a full length antibody, such as a full length IgG antibody.

(iv) Human antibodies

[0194] Human antibodies can be produced as an alternative to humanization. For example, it is now possible to generate transgenic animals (e.g., mice) that are capable, upon 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 heavy chain joining region (Jh) gene in chimeric and germline mutant mice results in complete inhibition of

- 52 endogenous antibodies. Transfer of the human germline immunoglobulin gene system into such mutant mice will result in the production of human antibodies upon antigen exposure. See, e.g., Jakobovits et aL, Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Yearin Immuno., 7:33 (1993); and U.S. Patent Nos. 5,591,669, 5,589,369, and 5,545,807.

[0195] Alternatively, phage display technology (McCafferty et al., Nature 348: 552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from the immunoglobulin variable (V) domain gene repertoire from unimmunized donors. According to this technique, the antibody V domain genes are cloned in frame into a larger or smaller gene for a filamentous bacteriophage coat protein, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particles contain a single copy of the single-stranded DNA of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody exhibiting these features. Thus, the phage mimics some properties of the B cell. Phage display can be performed in a number of ways; for a review thereof see, e.g., Johnson, K. S. and Chiswell, D. J., Cur Opin in Struct Biol 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 various antioxazole antibody systems from a small randomized combinatorial library of V genes derived from the spleens of immunized mice. 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), one can construct a repertoire of V genes from unimmunized human donors and isolate antibodies against a variety of antigen systems (including self antigens). (See also U.S. Patent Nos. 5,565,332 and 5,573,905).

[0196] As discussed above, human antibodies can also be produced using in vitro activated B cells (see US Patents 5,567,610 and 5,229,275).

[0197] Human HER2 antibodies are described in US Patent No. 5,772,997 issued June 30, 1998 and WO 97/00271 published January 3, 1997.

(v) Antibody fragments

[0198] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived from proteolytic digestion of full-length antibodies (see, e.g., 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 from recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F (ab ') 2 fragments (Carter et al.,

[0199] Biotechnology 10: 163-167 (1992)). According to another approach, F (ab ') 2 fragments can be isolated directly from recombinant host cell culture. Other

Techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, 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, eg, as described in US Patent 5,641,870, for example. Such linear antibody fragments can be monospecific or bispecific.

(vi) Bispecific antibodies

[0200] Bispecific antibodies are antibodies that display binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes on the HER2 protein. Other such antibodies may combine the HER2 binding site with the binding site (s) for EGFR, HER3 and / or HER4. Alternatively, the HER2 arm [antibody] may be linked to an arm [antibody] that binds to a leukocyte trigger molecule such as a T cell receptor molecule (e.g. CD2 or CD3), or IgG Fc receptors (FcyR) such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms on cells expressing HER2. Bispecific antibodies can be used to locate cytotoxic agents on HER2-expressing cells. These antibodies have a HER2 binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate, or radioactive hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F (ab ') 2 bispecific antibodies).

[0201] WO 96/16673 describes a HERZ / FcyRIH bispecific antibody and US Patent No. 5,837,234 discloses a HER2 / FcyRI bispecific antibody, IDM1 (Osidem). The HER2 / Fca bispecific antibody is shown in WO98 / 02463. US Patent No. 5,821,337 teaches the knowledge of HER2 / CD3 antibody. MDX-210 is a HER2-FcyRIII bispecific Ab.

[0202] Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain - light chain pairs in which the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). Due to the random selection of immunoglobulin heavy and light chains, these hybridomas (quadromes) potentially produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is relatively cumbersome and the product yield is low. Similar procedures are described in WO 93/08829 and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

[0203] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is to an immunoglobulin heavy chain constant domain, comprising at least part of the hinge region,

- 54 CH2 and CH3. It is preferred that the first heavy chain constant region (CHI) containing the site necessary for light chain binding is present in at least one attachment. DNA encoding heavy chain fusions and, if necessary, immunoglobulin light chain are inserted into separate expression vectors and co-transfected into an appropriate host organism. This allows for great flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where optimal performance is provided by an unequal ratio of the three polypeptide chains used during construction. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are not critical.

[0204] In one preferred embodiment of this approach, the bispecific antibodies consist of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain - light chain pair (providing a second binding specificity) in the other arm. It has been found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations since the presence of an immunoglobulin light chain in only half of the bispecific molecule allows for easy separation. This approach is disclosed in WO 94/04690. For further details on making bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

[0205] According to another approach, described in US Patent No. 5,731,168, the interface between a pair of antibody molecules can be transformed so as to maximize the percentage of heterodimers recovered from recombinant cell culture. A preferred interaction surface comprises at least a portion of the Ch3 domain from the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). On the interface of the second antibody molecule, compensating "depressions" of identical or similar size to the larger side chain (s) are created by replacing large amino acid side chains with smaller ones (eg, alanine or threonine). This provides a mechanism for increasing the yield of heterodimer formation compared to undesirable end products such as homodimers.

[0206] Bispecific antibodies include cross-linked or heteroconjugate antibodies. For example, one of the antibodies in the heteroconjugate can be conjugated to avidin and the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (US Patent No. 4,676,980) and treat HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies can be produced by any convenient means

- 55 cross-linking. Suitable crosslinking agents are well known in the art and are disclosed in US Patent No. 4,676,980 along with a number of crosslinking techniques.

[0207] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be made using chemical binding. Brennan et al., Science, 229: 81 (1985) describe a procedure in which full-length antibodies are proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenate, to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bridges. The Fab 'fragments obtained are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then converted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form a bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0208] Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli cells that can be chemically conjugated to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the preparation of a fully humanized bispecific F (ab ') 2 antibody molecule. Each Fab' fragment was separately secreted from E. coli and subjected to direct in vitro chemical coupling to form the bispecific antibody. The bispecific antibody thus produced was able to bind to cells overexpressing the HER2 receptor and normal human T lymphocytes as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). The leucine zipper peptides of the Fos and Jun proteins were bound to the Fab 'portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The diabody technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993), has provided an alternative mechanism for generating bispecific antibody fragments. The fragments contain a variable domain (V_H.) of the heavy chain, linked to the variable domain (V_L.) of a light chain via a linker that is too short to allow pairing of two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary domains V L and V H of the other fragment, thus creating two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single chain Fv (scFv) dimers has also been demonstrated. See Gruber et al., J. Immunol., 152: 5368 (1994).

[0209] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be produced. Tutte / αΖ. J. Immunol. 147: 60 (1991).

(vii) Other modifications of the amino acid sequence

[0210] Amino acid sequence modification (s) of the described antibodies are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the Antibody are made by introducing appropriate nucleotide changes into the Antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequences of the Antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct ensuring that the final construct possesses the desired characteristics. The amino acid changes can also affect post-translational processes of the Antibody, such as altering the number or position of glycosylation sites.

[0211] A useful method of identifying certain residues or regions of an Antibody that are favorable locations for mutagenesis is called alanine scanning mutagenesis, as described by Cunningham and Wells Science, 244: 1081-1085 (1989). It identifies a residue or group of target residues (e.g. charged residues such as arg, asp, his, lys and glu) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to influence the interaction of the amino acids with the antigen. These amino acid locations, demonstrating functional sensitivity to substitutions, are then refined by introducing additional or different sets of variants on or for the substitution sites. Therefore, while the site for introducing an amino acid sequence change has been determined, the nature of the mutation does not need to be determined. For example, to analyze the effects of a mutation at a given site, ala scanning or random mutagenesis is performed at the target codon or region and the expressed Antibody variants are screened for the desired activity.

[0212] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions as single or multiple amino acid residues. Examples of terminal insertions include Antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the Antibody molecule include fusions to the N or C terminus of an antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0213] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the Antibody molecule replaced by a different residue. The sites of greatest interest for substitution mutagenesis include the hypervariable regions, but changes in the FR or Fc region are also contemplated. Conservative substitutions are shown in Table 1 under the heading preferred substitutions. If such substitutions result in a change in biological activity, then to the products tested

More significant changes, called exemplary substitutions in Table 1, or as further described below in relation to the classes of amino acids may be introduced.

Table 1

<td>The rest is original</td><td>Sample substitutions</td><td>Favorable substitutions</td>

<td>Ala (A)</td><td>Val; Leu; How much</td><td>Val</td>

<td>Arg (R)</td><td>Lys; Gin; Asn</td><td>Lys</td>

<td>Asn (N)</td><td>Gin; His; Asp, Lys; Arg</td><td>Gin</td>

<td>Asp (D)</td><td>Glu; Asn</td><td>Glu</td>

<td>Cys (C)</td><td>Cheese; Ala</td><td>Cheese</td>

<td>Gln (Q)</td><td>Asn; Glu</td><td>Asn</td>

<td>Glu (E)</td><td>Asp; Gin</td><td>Asp</td>

<td>Gly (G)</td><td>Ala</td><td>Ala</td>

<td>His (H)</td><td>Asn; Gin; Lys; Arg</td><td>Arg</td>

<td>He (I)</td><td>Leu; Val; Underworld; Ala; Phe; Norleucine</td><td>Leu</td>

<td>Leu (L)</td><td>Norleucine; How much; Val; Underworld; Ala; Phe</td><td>How much</td>

<td>Lys (K)</td><td>Arg; Gin; Asn</td><td>Arg</td>

<td>Met (M)</td><td>Leu; Phe; How much</td><td>Leu</td>

<td>Phe (F)</td><td>Trp; Leu; Val; How much; Ala; Tyr</td><td>Tyr</td>

<td>Pro (P)</td><td>Ala</td><td>Ala</td>

<td>Cheese (S)</td><td>Thr</td><td>Thr</td>

<td>Thr (T)</td><td>Val; Cheese</td><td>Cheese</td>

<td>Trp (W)</td><td>Tire; Phe</td><td>Tyr</td>

<td>Tyr (Y)</td><td>Trp; Phe; Thr; Cheese</td><td>Phe</td>

<td>Val (V)</td><td>How much; Leu; Underworld; Phe; Ala; Norleucine</td><td>Leu</td>

[0214] Substantial modifications to the biological activity of the antibody are made by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone around the substitution, for example, in the form of a sheet or helix conformation (b) of the charge or hydrophobicity of the molecule in place

- 58 target or (c) side chain size. Amino acids can be grouped according to the similarities in the properties of their side chains (in AL Lehninger, Biochemistry, 2nd ed., Pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polame: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q) (3) acidic: Asp (D), Glu (E) (4) basic: Lys (K), Arg (R), His (H)

[0215] Alternatively, naturally occurring residues can be grouped based on common side chain properties:

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

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

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues influencing chain positioning: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

[0216] Non-conservative substitutions will result in the exchange of a member of one of these classes for another class.

[0217] Any cysteine ​​residue not involved in maintaining the proper conformation of the Antibodies may be substituted, generally with serine, to improve the oxidation stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bond (s) can be added to the antibody to improve its stability (especially where the antibody is an antibody fragment such as an Fv fragment).

[0218] A particularly preferred type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Overall, the resulting variant (s) selected for further development will have improved biological properties as compared to the parent antibody from which they were generated. A convenient method of producing such substitution variants involves affinity maturation using phage display. Briefly, several sites of the hypervariable region (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody variants thus produced are displayed in monovalent form on filamentous phage particles as fused to the gene III product of M13 packaged within each particle. Subsequently, the phage-displayed variants are screened for their biological activity (e.g. binding affinity). Scanning mutagenesis can be performed to identify the hypervariable region sites proposed for modification

- 59 alanine to identify hypervariable region residues contributing significantly to antigen binding. Alternatively or additionally, it may be advantageous to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and its antigen. Such contact and adjacent residues are candidates for substitution in accordance with the techniques developed herein. Once such variants are produced, a panel of variants is screened as described, and an antibody with outstanding properties in one or more relevant studies may be selected for further development.

[0219] Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. Changing means removing one or more carbohydrate molecules found on the antibody and / or adding one or more glycosylation sites that are not present on the antibody.

[0220] Antibody glycosylation is typically either N-glycosylation or O-glycosylation. Nglycosylation refers to the attachment of a carbohydrate molecule to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of a carbohydrate molecule to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-glycosylation refers to the attachment of one or more N-acetylgalactosamine, galactose or xylose sugars to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0221] Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Variation can also be made by adding or substituting one or more serine or threonine residues in the original antibody sequence (for O-glycosylation sites).

[0222] Where the antibody contains an Fc region, the attached carbohydrate can be altered. For example, antibodies with a mature carbohydrate structure lacking fucose attached to the Fc region of an antibody are described in US Patent Application No. 2003/0157108 A1, Presta, L. See also US 2004/0093621 A1 (Kyowa Hakko Kogyo Co., Ltd). References of antibodies with an N-acetylglucosamine (GIcNAc) split in a carbohydrate attached to the Fc region of an antibody are found in WO03 / 011878, JeanMairet et al. and U.S. Patent No. 6,602,684, Umana et al. Antibodies with at least one galactose residue in an oligosaccharide attached to the Fc region of an antibody have been reported in WO97 / 30087, Patel et al. See also WO98 / 58964 (Raju, S.) and WO99 / ​​22764 (Raju, S.) for antibodies with altered carbohydrates attached to their Fc regions. Antibody compositions comprising a main species antibody with such carbohydrate structures attached to the Fc region are contemplated herein.

[0223] Nucleic acid molecules encoding amino acid variant sequences of an 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 naturally occurring amino acid sequence variants) or preparation by oligonucleotide-directed (or site-directed) mutagenesis, PCR-based mutagenesis, and cassette mutagenesis of a pre-prepared variant or non-variant antibody version.

(viii) Searching for antibodies with desired properties

[0224] Techniques for producing antibodies have been described above. Moreover, antibodies with certain biological characteristics can be selected as desired.

[0225] To identify antibodies that block ligand activation of the HER receptor, the ability of the antibody to block binding of the HER receptor ligand to cells expressing the HER receptor (e.g., in combination with another HER receptor with which the HER receptor of interest forms HER heterooligomers) may be determined. . For example, cells naturally expressing or transfected to express HER receptors from HER heterooligomers can be incubated with the antibody and then exposed to labeled HER ligand. Then, the ability of the HER2 antibody to block ligand binding to the HER receptor in the HER herero-oligomer can be assessed.

[0226] For example, inhibition of HRG binding to MCF7 breast cancer cell lines by HER2 antibodies can be performed on ice using MCF7 monolayers in 24-well plate format substantially as described in WO01 / 00245. HER2 monoclonal antibodies can be added to each well and incubated for 30 minutes. Then labeled rHRGpi 177-224 (25 pm) can be added¹²⁵And, incubation can be continued for 4 to 16 hours. Dose response curves can be prepared for the antibody of interest and an IC50 value calculated. In one embodiment, an antibody that blocks HER receptor activation with a ligand for inhibiting HRG binding to MCF7 cells will have an IC50 of about 50nM or less in this assay, more preferably 10nM or less. Where the antibody is an antibody fragment such as a Fab fragment, the IC50 for inhibiting HRG binding to MCF7 cells in this assay may be, for example, about 100nM or less, more preferably 50nM or less.

[0227] Alternatively or additionally, the ability of the HER2 antibody to block HER ligand stimulated tyrosine phosphorylation of the HER receptor present in a HER hetero-oligomer may be assessed. For example, cells endogenously expressing HER receptors or transfected to express them can be incubated with the antibody and then tested for HER ligand dependent tyrosine phosphorylation activity using an anti-phosphotyrosine monoclonal [antibody] (optionally conjugated to a detectable label). The receptor kinase activity assay, described in US Patent No. 5,766,863, is also available for determining HER receptor activation and the blocking of this activity by the antibody.

[0228] One can screen for an antibody that inhibits HRG stimulation of p 180 tyrosine phosphorylation in MCF7 cells essentially as described in WO01 / 00245. For example, MCF7 cells can be plated in 24-well plates and HER2 monoclonal antibodies added to each well and incubated for 30 minutes at room temperature; then rHRGp1 177-244 can be added to each well to a final concentration of 0.2 nM and incubation continued for 8 minutes. Media can be aspirated from each well and the reaction stopped by adding 100 µl of SDS sample loading buffer (5% SDS, 25 mM DTT and 25 mM Tris-HCl, pH 6.8). Each sample (25 μΐ) can be separated by electrophoresis on a 4-12% gradient gel (Novex) and then electrophoretically transferred to a polyvinylidene difluoride membrane. Anti-phosphotyrosine [antibody] immunoblots (at 1 pg / ml) can be induced and the intensity of the dominant reactive band at Mr ~ 180,000 can be quantified with a reflection densitometer. The selected antibody preferably will significantly inhibit the stimulation of p18O tyrosine phosphorylation by HRG to about 0-35% of the control of this assay. Dose-response curves can be prepared to inhibit the stimulation of p18O tyrosine phosphorylation by HRG as determined by reflection densitometry and calculate an IC50 for the antibody of interest. In one embodiment, an antibody that blocks ligand activation of the HER receptor to inhibit HRG stimulation of p18O tyrosine phosphorylation will have an IC50 of about 50nM or less, more preferably 10nM or less in this assay. Where the antibody is an antibody fragment such as a Fab fragment, the IC50 for inhibiting HRG stimulation of p18O tyrosine phosphorylation in this assay may be, for example, about 100nM or less, more preferably 50nM or less.

[0229] The effect of the antibody on inhibiting MDAMB-175 cell growth can also be assessed, eg, essentially as described in Schaefer et al. Oncogene 15: 1385-1394 (1997). According to this study, MDA-MB-175 cells can be treated with HER2 monoclonal antibody (10pg / mL) for 4 days and stained with crystal violet. Incubation with the HER2 antibody may have a growth inhibitory effect on this cell line similar to that exhibited by monoclonal antibody 2C4. In a further embodiment, 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 extent than monoclonal antibody 4D5 (and optionally to a greater extent than monoclonal antibody 7F3), both in the presence and absence of exogenous HRG.

[0230] The HER2 antibody of interest can block the heregulin-dependent association of HER2 with HER3 in MCF7 and SK-BR-3 cells, as determined by a co-immunoprecipitation experiment such as that described in WO011 / 00245, substantially more effectively than the antibody. monoclonal 4D5, and preferably substantially more effective than monoclonal antibody 7F3.

[0231] To identify growth inhibitory HER2 antibodies, one can screen for antibodies that inhibit the growth of tumor cells that overexpress HER2. IN

In one embodiment, the selected growth inhibitory antibody is capable of inhibiting the growth of SK-BR-3 cells in cell culture by about 20-100%, and preferably by about 50-100% at an antibody concentration of about 0.5 to 30 pg. / ml. The SK-BR-3 assay described in US Patent No. 5,677,171 can be performed to identify such antibodies. According to this study, SK-BR-3 cells are grown in a 1: 1 mixture of F12 and DMEM media, supplemented with 10% fetal bovine serum, glutamine and streptomycin penicillin. SK-BR-3 cells are seeded at 20,000 cells in a 35mm cell culture dish (2ml / 35mm dish). 0.5 to 30 pg / ml of HER2 antibody is added per plate. After six days, the number of cells is counted as compared to the untreated cells using a COULTER ™ electronic cell counter. Antibodies that inhibit the growth of SK-BR-3 cells by about 20-100% or about 50-100% can be selected as growth inhibitory antibodies. See US Patent No. 5,677,171 for studies to screen for growth inhibitory antibodies such as 4D5 and 3E8.

[0232] To select for apoptosis-inducing HER2 antibodies, an annexin binding study using BT474 cells is available. BT474 cells are grown and plated as discussed in the previous paragraph. The medium is then removed and replaced with fresh medium alone or medium containing 10pg / ml of the monoclonal antibody. After a three-day incubation period, monolayers are washed with PBS and then trypsinized a [plate]. The cells are then centrifuged and suspended in Ca-binding buffer²⁺ and in test tubes, prepare aliquots as discussed above for the cell death study. The tubes are then given labeled annexin (e.g. annexin V-FTIC) (1 µg / ml). Samples can be analyzed using a FACSCAN ™ flow cytometer and FACSCONVERT ™ CellQuest software (Becton Dickinson). Those antibodies that induce annexin binding at statistically significant levels compared to controls are selected as apoptosis-inducing antibodies. In addition to the annexin binding assay, a DNA staining assay using BT474 cells is available. To perform this assay, BT474 cells treated with the antibody of interest as described in the previous two sections were incubated for 2 h. at 37 ° C with 9pg / ml HOECHST 33342 □ and then analyzed on an EPICS ELITE ™ flow cytometer (Coulter Corporation) using MODFIT LT ™ software (Verity Software House). Antibodies that induce changes in the percentage of apoptotic cells that are 2 times or greater (and preferably 3 times or greater) than the untreated cell (up to 100% apoptotic cells) may be selected as pro-apoptotic antibodies in this study. See WO98 / 17797 for studies to screen for apoptosis-inducing HER2 antibodies such as 7C2 and 7F3.

[0233] To screen for antibodies binding an epitope on HER2 bound by an antibody of interest, routine cross-blocking studies can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). to assess whether the antibody cross-blocks the binding of an antibody, such as 2C4 or Pertuzumab, to HER2.

Alternatively or additionally, epitope mapping may be performed by methods known in the art and / or the antibody-HER2 structure may be examined (Franklin et al. Cancer Cell 5: 317-328 (2004)) to see if the HER2 domain (s) is / are bound to the antibody.

(ix) Immunoconjugates

[0234] Disclosed are immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g. i.e. radioconjugate).

[0235] Chemotherapeutic agents useful in generating such immunoconjugates have been described above. Conjugates of the antibody and one or more small molecule toxins such as calicheamicin, maytansine (US Patent No. 5,208,020), trichotene, and CC 1065 are also contemplated.

[0236] The antibody may be conjugated to one or more maytansine molecules (eg, from about 1 to about 10 maytansine molecules per antibody molecule). For example, maytansine can be converted to Mai-SS-Me, which can be reduced to Mai-SH3 and reacted with a modified antibody (Chari et al. Cancer Research 52: 127-131 (1992)) to form an antibody immunoconjugate. .

[0237] Another immunoconjugate of interest comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics at sub-picomolar concentrations is capable of producing double-stranded DNA breaks. Structural analogs of calicheamicin that may be used include, but are not limited to, γι \ α₂ \ a₃ \ Nacetyl- γι \ PSAG i Θ¹1 (Hinman et al. Cancer Research 53: 3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928 (1998)). See also US Patent No. 5,714,586; 5,712,374; 5,264,586; and 5,773,001.

[0238] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding diphtheria toxin active fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), cucumber balm inhibitor, curcine, crotin, soapwort inhibitor, gelonin, mitogelin, restryctocin, phenomycin, enomycin and tricotecans. See, for example, WO 93/21232 published October 28, 1993.

[0239] Disclosed is an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

[0240] A variety of radioactive isotopes are available for the production of radioactively conjugated HER2 antibodies. Examples include At²¹¹,1¹³¹,1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes Lu.

[0241] The antibody and cytotoxic agent conjugates can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyl dithiol) propionate (SPDP), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolate (IT), difunctional imide ester derivatives (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), Bisazide compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniobenzoyl) ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds ( such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugating a radionucleotide to an antibody. See WO94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, an acid labile linker, a peptidase sensitive linker, a dimethyl linker, or a disulfide-containing linker may be used (Chari et al. Cancer Research 52: 127-131 (1992)).

[0242] Alternatively, a fusion protein comprising an anti-HER2 antibody and a cytotoxic agent may be created, e.g., by recombinant techniques or peptide synthesis.

[0243] The antibody can be conjugated to a "receptor" (such as streptavidin) for use in tumor pre-targeting, where the antibody-receptor conjugate is administered to a patient followed by removal of unbound conjugate from circulation with a cleansing agent, followed by administration " ligand "(eg avidin), conjugated to a cytotoxic agent (eg radionucleotide).

(x) Other antibody modifications

[0244] Other modifications to the antibody are contemplated. For example, the antibody can be linked to one or more non-protein polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody may also be encapsulated in prepared microcapsules, for example, by coacervation techniques or by interfacial polymerization (for example, in hydroxymethylcellulose or gelatin microcapsules, respectively, and poly (methylmetacylate) microcapsules in a colloidal drug delivery system (for example, liposomes, microspheres). microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed. (1980).

[0245] It may be desirable to modify the antibody of the invention with respect to effector function, e.g., so as to enhance the antibody dependent cellular cytotoxicity (ADCC) and / or complement dependent cellular cytotoxicity (CDC). This can be achieved by making one or more amino acid substitutions in the Fc region of an antibody. Alternatively or additionally, cysteine ​​residues (s) may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in that region. The thus generated homodimeric antibody may exhibit improved internalization capacity and / or enhanced complement dependent cell killing and antibody dependent cellular cytotoxicity (ADCC). See Caron et aL, J. Exp Med. 176: 1191-1195 (1992) and Shopes, BJ Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be made using heterobifunctional cross-linkers, as described in Wolff et al. Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody can be produced having dual Fc regions and thereby enhanced complement lysis and ADCC capacity. See Stevenson et al. Anti-Cancer Drug Design 3: 219-230 (1989).

[0246] WO00 / 42072 (Presta, L.) describes antibodies with improved ADCC function in the presence of human effector cells, wherein the antibodies contain amino acid substitutions in their Fc region. Preferably, an antibody with improved ADCC comprises substitutions at positions 298, 333 and / or 334 in the Fc region. Preferably, the altered Fc region is a human IgG1 Fc region including or including substitutions at one, two or three of these positions.

[0247] Antibodies with altered C1q binding and / or complement dependent cell cytotoxicity (CDC) are described in WO99 / ​​51642, US Patent No. 6,194,551B1, US Patent No. 6,242,195B1, US Patent No. 6,528,624B1 and US Patent No. 6,538 .124 (Idusogie et aL). These antibodies contain an amino acid substitution at one or more of positions 270, 322, 326, 327, 329, 313, 333, and / or 334 of their Fc region.

[0248] To increase the serum half life of the antibody, a salvage receptor binding epitope may be introduced into the antibody (especially an antibody fragment) as described in, for example, US Patent No. 5,739,277. The expression salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (e.g. IgGi, IgG₂, IgG₃ or IgG₄), which is responsible for increasing the serum half-life of IgG molecules, in vivo. Antibodies with substitutions in their Fc region and increased serum half-life are also described in WO00 / 42072 (Presta, L.).

[0249] Engineered antibodies with three or more (preferably four) functional antigen binding sites are also contemplated (US Application No. US2002 / 0004587 A1, Miller et al.).

[0250] The disclosed HER2 antibodies can also be formulated as immunoliposomes. Antibody-containing liposomes are prepared by methods known in

The state of the art, such as that described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545; and WO97 / 38731 published October 23, 1997. Liposomes with enhanced circulation time are disclosed in US Patent No. 5,013,556.

[0251] Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with a defined hole size to obtain liposomes of the desired diameter. Fab 'fragments of the antibody of the invention can be conjugated to the liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982). The chemotherapeutic agent is optionally included in the liposome. See Gruber et al., J. National Cancer Inst. 81 (19) 1484 (1989).

(ix) Exemplary Antibodies

[0252] Exemplary antibodies that can be formulated according to the 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 B cell surface markers such as CD 19, CD20 (for example Rituximab (RITUXAN®) and humanized 2H7), CD22, CD40, or BR3;

antibodies that bind to IgE, including Omalizumab (XOLAIR®) commercially available from Genentech, E26 (Figs 17A-B), HAE1 (Figs 17A-B), an IgE antibody with an amino acid substitution at position 265 of its Fc region (USA 2004/0191244 A1), Hu-901 (Figs. 17A-B), an IgE antibody as in WO2004 / 070011 or an antibody (including antibody fragments and full-length antibodies) containing the variable domains of any of these 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 2, 1998; U.S. Patent No. 5,091,313 issued Feb. 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 epithelial growth factor (VEGF) or its receptor, including Bevacizumab (AVASTIN ™), commercially available from Genentech, and Ranibizumab (LUCEIMS ™);

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 humanized variants thereof (WO00175348);

Anti-CD11a antibodies, including efalizumab (RAPTIVA®) (US 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 (US Patent No. 5,622,700 issued April 22, 1997 or as in WO 97/26912 published July 31, 1997); anti-Apo-2 receptor antibodies (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 Sep. 30, 1997, Lorenz et al. J. Immunol. 156 (4): 1646-1653 ( 1996), and Dhainaut et al. Crit Care Med.

23(9):1461-1469 (1995));

anti-Tissue Factor (TF) (European Patent No. 0 420 937 BI granted November 9, 1994);

anti-human a4p integrin₇ (WO 98/06248 published February 19, 1998);

anti-EGFR antibodies, including chimeric or humanized antibody 225, as in WO 96/40210 published December 19, 1996;

anti-CD3 antibodies such as OKT3 (US Patent No. 4,515,893 issued May 7, 1985);

anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT®) and (ΖΕΝΑΡΑΧ®) (See US Patent No. 5,693,762 issued December 2, 1997);

anti-CD4 antibodies such as the cM-7412 antibody (Choy et al. Arthritis Rheum 39 (1): 52-56 (1996));

anti-CD52 antibodies such as CAMPATH-1H (Riechmann et al. Nature 332: 323337 (1988);

anti-Fc receptor antibodies such as the anti-FcyRI M22 antibody as in Graziano et al. J. Immunol. 155 (10): 4996-5002 (1995);

carcinogenic anti-antigen (CEA) antibodies such as hMN-14 (Sharkey et al. Cancer Res. 55 (23Suppl): 5935s- 5945s (1995);

antibodies directed against breast epithelial cells, including huBrE3, 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 cancer cells such as C242 (Litton et al. Eur J. Immunol. 26 (1): 1-9 (1996)); anti-CD38 antibodies, e.g. AT 13/5 (Ellis et al. J. Immunol. 155 (2): 925-937 (1995));

anti-CD33 antibodies such as Hu M195 (Jurcie 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. Cancer Res 55 (23 Suppl): 5899s-5907s (1995);

Anti-EpCAM antibodies such as 17-1A (PANOREX®);

anti-GpIIb / IIa antibodies such as abciximab or c7E3 Fab (REOPRO®);

anti-RSV antibodies such as MEDI-493 (SYNAGIS®);

anti-CMV antibodies such as PROTOVIR®;

anti-HIV antibodies such as PRO542;

anti-hepatitis antibodies such as the OSTAVIR® anti-Hep B antibody;

OvaRex anti-CA 125 antibody; anti-idiotypic BEC2 antibody against GD3 epitope;

anti-av33 antibody, VITAMIN®;

anti-human renal tumor cell antibody such as ch-G250; ING-1;

anti-human 17-1A antibody (3622W94);

anti-human colorectal tumor (A33);

anti-human melanoma antibody R24, directed against GD3 ganglioside;

anti-human squamous cell carcinoma (SF-25); and anti-human leukocyte antigen (HLA) antibodies, such as Smart ID 10 and anti-HLA antibody DR Oncolym (Lym-1).

(xi) Antibody variant compositions

[0253] Formulations containing compositions that comprise a mixture of a main species antibody and one or more variants thereof are disclosed. Where the main species binds HER2, preferably the HER2 antibody (one or both of the major types of HER2 antibody and antibody variants thereof) is one that binds to Domain II of HER2, inhibits HER dimerization more effectively than Trastuzumab and / or binds to HER2. a heterodimeric binding site on HER2. The major type antibody may be one that comprises the amino acid sequences of a light chain variable domain and a heavy chain variable domain of SEQ ID Nos. 3 and 4, and most preferably it comprises a light chain amino acid sequence selected from SEQ ID Nos. 15 and 23 and a heavy chain amino acid sequence selected from SEQ ID Nos. 16 and 24.

[0254] The formulated HER2 antibody composition may comprise a mixture of a major type HER2 antibody and an amino acid sequence variant thereof including an amino terminal extension in the form of a leader sequence. Preferably, the amino-terminus extension leader sequence is on the light chain of the variant antibody (e.g., on one or two light chains of the variant antibody). The main type HER2 antibody or antibody variant can be a full-length antibody or antibody fragment (e.g., Fab fragments of F (ab ')₂), but preferably both are full-length antibodies. An antibody variant may include an amino terminal extension in the form of a leader sequence on any one or more

-69 of your heavy or light chains. Preferably, the amino-terminal extension leader sequence is on one or two light chains of the antibody. The amino terminus extension leader sequence preferably comprises or consists of VHS-. The presence of an amino-terminal leader in the form of a leader in the composition can be detected by a variety of analytical techniques including, but not limited to, sequence analyzes.

N-terminal, charge heterogeneity study (e.g., cation exchange chromatography or zone capillary electrophoresis), mass spectrometry, etc. The amount of antibody variant in a composition generally ranges from an amount that is the limit of detection in any test (preferably N-terminal sequence analyzes). ) used to detect a variant to an amount less than that of the main species antibody. Generally, about 20% or less (e.g. from about 1% to about 15%, for example, from 5% to about 15%) of the antibody molecules in the composition contain an amino terminal extension in the form of a leader sequence. Such percentages are preferably determined using quantitative N-terminal sequence analyzes or cation exchange analyzes (preferably using a high resolution weak cation exchange column such as a PROPAC WCX-10 ™ cation exchange column). In addition to the amino-terminal leader sequence variants, further changes to the amino acid sequence of the main species and / or variant antibody are contemplated, including, but not limited to, an antibody having a C-terminal lysine residue on one or both of its heavy chains, a deamidated variant of the antibody. e.t.c.

[0255] Furthermore, the main species or variant antibody may further comprise changes in glycosylation, non-limiting examples of which include a HER2 antibody having a G1 or G2 oligosaccharide structure fused to its Fc region, a HER2 antibody having a carbohydrate molecule fused to its light chain (e.g. one or two carbohydrate molecules attached to one or two light chains of the antibody), the HER2 antibody comprising a non-glycosylated heavy chain.

III. Preparation of Formulations

[0256] The invention provides, in a first aspect, a stable pharmaceutical formulation as defined in claim 1. However, the antibody in the formulation may be an antibody fragment comprising an antigen binding region, such as a Fab fragment or an F (ab ') 2.

[0257] A pharmaceutical formulation is disclosed comprising or consisting essentially of a full length deamidation or aggregation susceptible IgG1 antibody 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 from about 60mM to about 250mM; and polysorbate 20 in an amount from about 0.01% to about 0.1%.

[0258] A pharmaceutical formulation is disclosed comprising an antibody that binds to HER2 domain II in a histidine buffer at a pH of about 5.5 to about 6.5, a saccharide, and a surfactant. For example, the formulation may include Pertuzumab w

Amounts from about 20mg / mL to about 40mg / mL, histidine acetate buffer, sucrose, and polysorbate 20, wherein the formulation pH is from about 5.5 to about 6.5.

[0259] The formulation is especially useful for antibodies prone to deamidation and / aggregation and / or fragmentation such that the buffer delays deamidation and / or aggregation and / or fragmentation of the formulated antibody. Additionally, unlike other histidine buffers prepared with HCl, the histidine-acetic buffer lacks chloride ion, which was found to be beneficial in that this buffer, when combined with a saccharide, had the same protective effect on the antibody as polysorbate 20 and it was stable and compatible with storage in stainless steel tanks. Thus, in addition to a composition itself containing an antibody prone to deamidation, aggregation and / or fragmentation, the invention provides a method of reducing deamidation, aggregation and / or fragmentation of a therapeutic monoclonal antibody (e.g., relative to a composition at a different pH or in a different buffer) comprising formulating the antibody in a histidine-acetate buffer, pH 5.5 to 6.5. In this embodiment, deamidation, aggregation, and / or fragmentation may be determined or measured before and after the antibody formulation, with the formulated antibody exhibiting acceptable deamidation, aggregation, and / or fragmentation in the formulation during storage.

[0260] Examples of CD20 antibodies that may be formulated include: C2B8, now referred to as Rituxim abem (RITUXAN®), commercially available from Genentech (see also US Patent No. 5,736,137,); murine antibody 2B8, labeled with yttrium- [90], designated Y2B8 or Ibritumomab Tiuxetan ZEVALIN®, commercially available from Biogen-Idee (see also US Patent No. 5,736,137); mouse IgG2a BI, also referred to as Tositumomab, optionally labeled 1311 to form the 13H-B1 antibody (Iodine Tositumomab 1131, BEXXAR ™) (US Patent No. 5,595,721); murine monoclonal antibody 1F5 (Press et al. Blood 69 (2): 584-591 (1987) and variants thereof, including framework-stuck or humanized 1F5 (WO03 / 002607, Leung, S.); (ATCC HB- 96450); murine antibody 2H7 and chimeric 2H7 (Clark et al. PNAS 82: 1766-1770 (1985); U.S. Patent No. 5,500,362); humanized 2H7; huMax-CD20 (WO04 / 035607, Genmab, Denmark); AME-133 (Applied Molecular Evolution); The A20 antibody or variants thereof, such as the A20 antibody chimeric or humanized (cA20, hA20, respectively) (USA 2003/0219433, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, ΒΕΙ, or NU-B2 available from International Leukocyte Typing Workshop (Valentine et al., In: Leukocyte Typing III (Mc Michael, Ed., pp. 440, Oxford University Press (1987)).

[0261] The CD20 antibody may be a humanized 2H7 antibody. For example, 2H7vl6 and 2H7v511. Humanized 2H7vl6 can be an intact antibody or antibody fragment comprising the light chain variable domain and heavy chain variable domain sequences in Figures 18A-B (SEQ ID Nos. 26 and 29). Where the humanized 2H7v16 antibody is a full length antibody, it may comprise the light and heavy chain amino acid sequences of SEQ ID Nos. 63 and 65.

[0262] Where the antibody binds to VEGF, it preferably comprises variable domain sequences as shown in Fig. 19. Most preferred anti-VEGF antibody is a full length humanized IgG1 antibody, Bevacizumab (AVASTIN ™), commercially available from Genentech.

[0263] Where the formulated antibody binds IgE, it may be selected from the group consisting of: E25, Omalizumab (XOLAIR®) commercially available from Genentech (see also Figs 17A-B), HAE1 (Figs 17A-B), IgE antibodies with amino acid substitution at position 265 of your Fc region (USA 2004/0191244 A1) , Hu-901 (Figs. 17A-B), IgE antibodies as in WO2004 / 070011, or antibodies (including antibody fragments and full-length antibodies) containing the variable domains of any of these IgE antibodies.

[0264] Where the antibody binds to a receptor from the tumor necrosis factor (TNF) superfamily or to the death receptor, it preferably binds to DR5 and is preferably an agonist antibody. Publications in this area include Sheridan et al., Science, 277: 818-821 (1997), Pane (a), Science, 277: 815-818 (1997), WO98 / 51793 published November 19, 1998; WO98 / 41629 published September 24, 1998; Screaton et al., Curr. Biol., 7: 693-696 (1997); Walczak et al., EMBO I, 16: 5386-5387 (1997); Wu et al., Nature Genetics, 17: 141-143 (1997); WO98 / 35986 published August 20, 1998; EP870.827 published Oct. 14, 1998; WO98 / 46643 published October 22, 1998; WO99 / ​​02653 published January 21, 1999; WO99 / ​​09165 published February 25, 1999; WO99 / ​​1179 published March 11, 1999; US 2002/0072091 published August 13, 2002; 2002/0098550 published December 7, 2001; 6,313,269 issued December 6, 2001; 2001/0010924 published August 2, 2001; US 2003/01255540 published Jul 3, 2003; US 2002/0160446 published October 31, 2002, US 2002/0048785 published April 25, 2002; US 6,342,369 issued Feb. 2002; 6,569,642 issued May 27, 2003, 6,072,047 issued June 6, 2000, 6,642,358 issued November 4, 2003; IS 6.743.625 issued June 1, 2004). The most preferred DR5 antibody is Apomab.

[0265] Each of the formulations noted above contains a buffer, preferably a histidine buffer, and most preferably a histidine-acetate buffer having a pH of from 5.5 to 6.5, preferably from 5.8 to 6.2, such as about 6.0. The buffer concentration is dictated, at least in part, by the desired pH. Exemplary buffer concentrations are in the range of from about 10mM to about 200mM, preferably from about 10mM to about 40mM, most preferably about 20mM.

[0266] The concentration of the antibody in the formulation is preferably from about 10mg / mL to about 250mg / mL. The concentration of the antibody can be determined based on the intended use and mode of administration of the formulation. For example, where the formulation is for IV administration (e.g., HER2 antibody), the concentration of the antibody in the formulation is preferably from about 20mg / mL to about 40mg / mL. In an exemplary formulation of Pertuzumab, intended for intravenous (IV) administration, the antibody concentration was from about 20 mg / mL to about 40 mg / mL, most preferably about 30 mg / mL.

[0267] Where an antibody is for SQ or IM administration (e.g. for an anti-IgE antibody) higher concentrations of the antibody may be desirable. Such significantly higher antibody concentrations can 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.

[0268] Where the formulation comprises a DR5 antibody such as Apomab, exemplary antibody concentrations are from about 10mg / mL to about 30mg / mL, for example about 20mg / mL of DR5 antibody; such formulations are useful for intravenous administration.

[0269] The formulation to be administered is preferably an aqueous (non-lyophilized) formulation and has not been previously lyophilized. Although the formulation may be lyophilized, it preferably is not. However, freezing the aqueous formulation without the simultaneous drying that takes place during freeze-drying is specifically contemplated to facilitate longer storage, for example, in a stainless steel tank.

[0270] Preferably, the formulation further comprises a saccharide, most preferably a disaccharide such as trehalose or sucrose. Generally, the saccharide is included in an amount to reduce the formation of soluble aggregates such as those appearing during freezing / thawing. Exemplary saccharide concentrations range from about 10mM to about IM, for example from about 60mM to about 250mM, and most preferably about 120mM for a HER2 antibody formulation and about 240mM for a DR5 antibody formulation.

[0271] Although the formulation containing the histidine-acetate buffer and the saccharide was found to be stable, the formulation optionally further comprises a surfactant such as polysorbate, most preferably polysorbate 20. Generally, the surfactant is included in an amount that reduces the formation of insoluble aggregates (such as those appearing). while shaking or transferring). Preferably, the surfactant concentration is from about 0.0001% to about 1.0%, most preferably from about 0.01% to about 0.1%, such as about 0.02%.

[0272] Optionally, the formulation does not contain a toning amount of a salt such as sodium chloride.

[0273] The formulation is generally sterile, and can be accomplished according to procedures known to those skilled in the art for the preparation of sterile pharmaceutical formulations suitable for administration to human subjects, including filtration through sterile filter membranes before or after preparation of the formulation.

[0274] Furthermore, a formulation is desirably one that has been shown to be storage-stable. Various stability studies are available to the skilled person to confirm the stability of the formulation. For example, a formulation may be one that has been shown to be storage stable: 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 1 year; and / or about -20 ° C for at least 3 months. Stability can be tested by assessing the physical stability, chemical stability, and / or biological activity of the antibody in the formulation near the time of formulation as well as after storage at the temperatures noted. Physical and / or stability can be assessed qualitatively and / or quantitatively in a variety of ways, including assessment of aggregate formation (for example, using size exclusion chromatography, turbidity measurements, and / or by visual inspection); estimating the heterogeneity of the charge, z

Using cation exchange chromatography or zone capillary electrophoresis; amino terminal or carboxyl terminal sequence analysis; mass spectrometric analyzes; SDS-PAGE analysis to compare whole and reduced antibodies; peptide map analysis (e.g., tryptic or LYS-C); evaluating the biological activity or antigen-binding function of the antibody; etc. Instability may result in aggregation, deamidation (e.g. deamidation of Asn), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomerization), em cleavage / hydrolysis / fragmentation (e.g. fragmentation of the linker region), formation of succinate, unpaired cysteine ​​(cysteines), N-terminal extension, C-terminal processing, differences in glycosylation, etc. Activity the biological or antigen-binding function can be assessed using various techniques available to those skilled in the art.

[0275] As noted above, freezing the formulation is specifically contemplated. Therefore, the formulation can be tested for freeze-thaw stability.

[0276] Methods are provided for making pharmaceutical formulations, comprising making a formulation as described, and assessing the physical stability, chemical stability, or biological activity of the monoclonal antibody in the formulation.

[0277] The formulation may be provided inside a vial with a syringe-pierceable stopper, preferably in an aqueous form. Desirably, the vial is kept at about 28 ° C until administered to the individual in need of it. The vial may be, for example, a 20cc vial (for example for a 420mg dose) or a 50cc vial (for example for a 1050mg dose). For DR5 antibody such as Apomab, the formulation can be provided in a 5cc glass vial (e.g. 5.5ml fill).

[0278] Formulation may be provided inside a stainless steel reservoir. The formulation in a stainless steel tank is optionally frozen and non-freeze dried.

[0279] 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) provided that they do not adversely affect the desired characteristics of the formulation. The acceptable carriers, excipients, or stabilizers at the dosages and concentrations employed are non-toxic to the recipient and include; additional buffering agents; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradable polymers such as polyesters; preservatives; and / or salt-forming counterions such as sodium.

IV. Treatment with an Antibody Formulation

[0280] The invention enables a method of treating a disease or disorder in a subject, comprising administering to the subject a formulation described herein in an amount effective to treat the disease or disorder.

[0281] Where the antibody in the formulation binds to HER2, it is preferably used for the treatment of cancer. The cancer will generally contain HER2-expressing cells such that the HER2 antibody will be able to bind to the cancer cells. Thus, it relates to a method of treating HER2-expressing cancer in a subject comprising administering a HER2 antibody pharmaceutical formulation to the subject in an amount effective to treat the cancer. The various cancers that can be treated with the composition are listed above in the definition section.

[0282] It is also contemplated that the HER2 antibody formulation can be used to treat a variety of non-malignant diseases or disorders that include autoimmune diseases (e.g. psoriasis), ednometriosis, scleroderma, restenosis, polyps such as colon polyps, nasal or digestive polyps; fibroadenoma, respiratory disease (see definition above); cholecystitis, neurofibromatosis; polycystic kidney disease; inflammatory diseases, skin diseases including psoriasis and dermatitis; vascular diseases (see definition below); conditions involving inappropriate proliferation of epithelial cells; stomach ulcers; Menetrier's disease, secreting adenomas or protein-loss syndrome; renal dysfunction; angiogenic disorders; eye diseases such as age related macular degeneration; putative ocular histoplasmosis syndrome, retinal neovascularization resulting from hypertrophic diabetic retinopathy, retinal vascularization, diabetic retinopathy, or age-related macular degeneration; pathologies associated with bone diseases such as osteoarthritis, rickets, and osteoporosis; damage following cerebral ischemia; fibrotic or edema diseases such as liver cirrhosis, pulmonary fibrosis, sarcoidosis, thyroiditis, hyperglycemia, Rendu-Osler-Weber disease, chronic obliterating pulmonary disease, or edema following burns, trauma, radiation, strokes, hypoxia or ischemia; skin hypersensitivity, diabetic retinopathy and diabetic nephropathy, Gullain-Barre syndrome, graft versus host disease, or graft rejection; Paget's disease; osteoarthritis; photoaging (e.g., caused by irradiation of human skin with UV); benign prostatic hyperplasia; some bacterial infections, including pathogenic bacteria selected from adenoviruses, hantaviruses, Borrelia burgdorferi, Yersinia spp. and Bordetella pertussis; thrombosis due to platelet aggregation, reproductive conditions such as endometriosis, uterine endometriosis, ovarian hyperstimulation syndrome, pre-eclampsia, functional uterine bleeding, or menstrual disorders; synovitis; atheroma; acute and chronic nephropathies (including hypertrophic glomerulonephritis and diabetes-induced renal failure); eczema; hypertrophic scarring; endotoxic shock and mycosis; familial adenomatous polyposis; neurodegenerative diseases (e.g., 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 lungs, kidneys or liver; T cell-dependent hypersensitivity; infantile hypertrophic pyloric stenosis; urinary syndrome

-75 obstructive; psoriatic arthritis; and Hashimoto's disease. Preferred non-malignant indications for treatment include psoriasis, endometriosis, scleroderma, vascular diseases (e.g., restenosis, atherosclerosis, coronary disease or hypertension), colon polyps, fibroadenoma, or respiratory diseases (e.g., asthma, chronic bronchitis or cystic fibrosis). ).

[0283] Where the antibody in the formulation binds to a B cell surface marker such as CD20 or B R3, the formulation can be used to treat B cell neoplasms such as NHL or CLL, autoimmune disease, transplant rejection, or to block an immune response. to a foreign antigen such as an antibody, toxin, gene therapy viral vector, transplant, infectious agent, or alloantigen (see WO 01/03734, Grillo-Lopez et al.).

[0284] Where the antibody in the formulation is an IgE antibody, it can be used to treat an IgE-mediated disorder (USSN 2004/0197324 A1, 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, Wiskott-Aldrich syndrome, thymic alymphoplasia, IgE myeloma and the graft versus host reaction.

[0285] Antibodies that bind to a receptor of the TNF superfamily (for example, which bind to DR5) or bind to VEGF (or its receptor) can be used to treat cancer, various forms of which are described in the definition section above. Preferably, the cancer treated with the DR5 antibody formulation is a solid tumor or NHL.

[0286] Where the indication is cancer, the patient may be treated with a combination of the antibody formulation and a chemotherapeutic agent. Combined administration comprises co-administration or simultaneous administration, using separate formulations or a single pharmaceutical formulation, and sequential administration in either order, preferably there is a time when both (or all) active ingredients exhibit their biological activity simultaneously. Thus, the chemotherapeutic agent may be administered before or after administration of the composition. The distribution of time between the at least one administration of the chemotherapeutic agent and the at least one administration of the composition is preferably about 1 month or less, and more preferably about 2 weeks or less. Alternatively, the chemotherapeutic agent and the composition are administered to a patient simultaneously in a single formulation or in separate formulations.

[0287] Treatment with the formulation will result in an improvement in the signs or symptoms of cancer or disease. For example, where the disease being treated is cancer, such therapy may result in improved survival time (overall survival and / or progression free survival) and / or may result in an objective clinical response (partial or complete). Moreover, treatment with the combination of a chemotherapeutic agent and an antibody formulation can result in a synergistic or more than additive benefit to the patient.

[0288] Preferably, the antibody in the formulation is a naked antibody. However, the administered antibody can be conjugated to a cytotoxic agent. Preferably,

The immunoconjugate and / or the antigen to which it is bound is / are intemalized by the cell, resulting in enhanced therapeutic efficacy of the immunoconjugate in killing the cancer cells to which it binds. In a preferred embodiment, the cytotoxic agent in the cancer cell targets or interacts with the nucleic acid. Examples of such cytotoxic agents include maytansinoids, calicheamicins, ribonuclease, and DNA endonuclease.

[0289] The formulation is administered to a human patient by known means such as intravenous administration, e.g., by bolus injection, or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebral, subcutaneous, intraarticular, intrasynovial, intrathecal administration. orally, topically or inhaled. It is preferred to administer the antibody composition intravenously, intramuscularly, or subcutaneously, with intravenous administration being most preferred.

[0290] For subcutaneous delivery, the formulation can be administered via a syringe; an injection device (e.g., INJECTEASE ™ and GENJECT ™ devices); an injection pen (such as GENPEN ™); a needleless device (e.g., MEDIJECTOR ™ and BIOJECTOR ™); or a subcutaneous patch delivery system.

[0291] To prevent or treat a disease, the appropriate dose of the antibody will depend on the type of disease being treated, as defined above, the severity and course of the disease, whether the antibody is being administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history, and response to the antibody and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or during serial therapy. Depending on the type and severity of the disease, the initial proposed dose for administration to a patient is about 1 gg / kg to about 50 mg / kg (e.g. 0.1-20 mg / kg) of a HER2 or DR5 antibody, or during one or more treatments, for example. the number of separate applications, or by continuous infusion. The dosage of the antibody will generally range from about 0.05mg / kg to about 10mg / kg. When a chemotherapeutic agent is administered, it is usually administered in doses known to the chemotherapeutic agent or, optionally, lowered due to the combined action of the drugs or the negative side effects attributed to the administration of the therapeutic agent. Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by one of skill in the art. Preparation and dosing schedules for such chemotherapeutic agents are also described in Chemotherapy Service Ed., MC Perry, Williams &amp; Wilkins, Baltimore, MD (1992).

[0292] Other therapeutic dosage regimens may be combined with the antibody, including, but not limited to: a second (third, fourth, etc.) chemotherapeutic agent (s) (ie, cocktails of various therapeutic agents); other monoclonal antibody; growth inhibitory agent; a cytotoxic agent; a chemotherapeutic agent; a drug that targets EGFR; a tyrosine kinase inhibitor; an anti-angiogenic agent; and / or a cytokine; e.t.c.

[0293] In addition to the above-mentioned therapeutic administration regimens, the patient may undergo surgical removal of cancer cells and / or radiation therapy.

V. Crafted Items

[0294] A manufactured item is disclosed that contains the pharmaceutical formulation of the invention and provides instructions for its use. The article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (such as dual chamber syringes), and test tubes. The container can be made of various materials such as glass or plastic. The container holds the formulation, and a label on or associated with the container may indicate methods of use. The container containing the formulation may be a reusable vial which allows for repeated administration (e.g., 2-6 administrations) of the prepared formulation. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package insert with instructions for use, as noted in the previous section.

[0295] The invention will be better understood with reference to the following examples. However, they should not be interpreted as limiting the scope of the invention.

EXAMPLES

Stable, Fluid Formulation of Pertuzumab

[0296] These examples describe the development and stability testing of stable liquid formulations containing Pertuzumab at a protein concentration ranging from about 10 mg / mL -180 mg / mL. The selected formulations had low turbidity and were physically and chemically stable. The chloride ion was removed from the formulation to reduce the risk of corrosion. The formulation was isotonic and suitable for subcutaneous or intramuscular delivery. The formation of insoluble aggregates under agitation stress was prevented using a histidine-acetate formulation with sucrose without the need for polysorbate 20.

Analytical methods

Color, Appearance and Clarity (CAC)

[0297] The color, appearance and clarity of the samples were determined by visually inspecting the vials for white and black backgrounds under fluorescent light at room temperature.

UV concentration measurements

[0298] First, aliquots of the liquid product were diluted in the formulation buffer such that A_max at around 278 nm it was in the range of 0.5-1.0 absorbance units. The absorbance of the diluted samples was measured under UV in a quartz cuvette with an optical path length of 1 cm on an HP 8453 spectrophotometer. The absorbance was measured at 278 nm and 320 nm. The absorbance at 320 nm was used to correct light scattering by the ze background

-78 due to larger aggregates, bubbles and particles. A blank for the measurements was performed in the formulation buffer. Protein concentration was assessed using an absorption of 1.50 (mg / mL) '¹ cm '¹.

pH measurements

[0299] The pH was measured at room temperature using a RADIOMETER COPENHAGEN PHM82 ™ pH meter. The probe used was a combined glass / reference electrode with a radiometer adapter (Sigma, Cat # E-5759). Standard solutions of pH 4.01 and pH 7.00 (EM Science) were used for the calibration of the pH meter.

Ion exchange chromatography (ΙΕΧ)

[0300] Cation exchange chromatography was used to measure the charge changes of the variants. This study uses a DIONEX PROPAC WCX-10 ™ column on an HP 1100D HPLC system. Samples were diluted to 1 mg / mL in Mobile Phase A containing 20 mM MES at pH 6.0. Then 50 mL of the diluted sample was applied to the column, stored at ambient temperature. The peaks were eluted with a slight NaCl gradient using a mobile [phase] B containing 20 mM MES, 250 mM NaCl, pH 6.0. The eluent was monitored at 280 nm. Data were analyzed using HP CHEMSTATION ™ software (Rev A08.03).

Zone Capillary Electrophoresis (CZE)

[0301] The purity of the Fab and F (ab ') 2 fragments was determined by CZE. This study was performed on a BIORAD BIOFOCUS ™ 3000 ™ capillary electrophoresis system with a BIOCAP XL ™ 50 pm ID capillary, 44.6 cm overall length and 40 cm to the detector.

Steric Exclusion Chromatography (SEC)

[0302] Steric exclusion chromatography was used for aggregate and fragment counting. This test uses a TSK G3000 SWXL ™ column, 7.8 x 300 mm and runs on an HP 1100 ™ HPLC system. The samples were diluted to 10 mg / mL in the mobile phase and the injection volume was 20 pL. The mobile phase was 100 mM K2HPO4 at pH 6.8 and the protein was eluted isocratic in a gradient at 0.5 mL / min for 45 minutes. The absorption of the eluate was monitored at 280 nm. Integrations were done using HP CHEMSTATION ™ software (Rev A08.03).

Biological Activity

[0303] 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

[0304] Pertuzumab Fab and F (ab ') 2 antibody fragments were formulated at a protein concentration of 1.0 mg / mL under the following buffer conditions:

mM citrate, 140 mM NaCl, pH 4.0;

mM succinate, 140 mM NaCl, pH 5.0;

- 7,910 mM succinate, 140 mM NaCl, pH 6.0;

mM histidine, 140 mM NaCl, pH 7.0; and mM glycylglycine, 140 mM NaCl, pH 8.0.

[0305] Each formulation was filtered and then aliquoted into 3 cc WHEATON ™ USP Type I glass vials sealed with TEFLON ™ coated gray butyl stoppers. Samples were stored at 40 ± 2 ° C. Drug product stability analyzes showed that Fab and F (ab ')₂ they were most stable at pH between 5.0 and 6.0.

Table 2. Effect of pH on the degradation of Fab or F (ab ') and stored at 40 ° C

<td>Formulation pH</td><td colspan="2">Fab</td><td colspan="2">F (ab ')₂</td>

<td></td><td>% Of CZE Main Peak</td><td>% Major Peak SEC</td><td>% Of CZE Main Peak</td><td>% Major Peak SEC</td>

<td> 4,0</td><td> 74,1</td><td> 96,7</td><td> 43,6</td><td> 89,4</td>

<td> 5,0</td><td> 83,2</td><td> 96,4</td><td> 65,4</td><td> 94,0</td>

<td> 6,0</td><td> 82,9</td><td> 96,2</td><td> 69,0</td><td> 92,3</td>

<td> 7,0</td><td> 83,9</td><td> 96,4</td><td> 62,3</td><td> 91,3</td>

<td> 8,0</td><td> 72,7</td><td> 96,4</td><td> 49,2</td><td> 89,8</td>

EXAMPLE 2

[0306] Pertuzumab was formulated into 20 mM histidine acetate buffer with 120 mM sucrose and 0.02% polysorbate 20. The formulation pH was adjusted with acetic acid to a final pH between 5.0 and 7.0. The protein concentration was 30 mg / mL. Each formulation was filled into 3 cc USP Type I glass vials and stored at 40 ° C for stability studies. The results showed that Pertuzumab was the most stable around pH 6.0.

Table 3. Effect of pH on the degradation of Pertuzumab stored at 40 ° C

<td>Formulation pH</td><td>Temperature (° C)</td><td>Storage time (weeks)</td><td>% SEC Monomer</td><td>% Major Peak ΙΕΧ</td>

<td> 5,0</td><td> 40</td><td> 2</td><td> 99,4</td><td> 57,4</td>

<td> 5,5</td><td> 40</td><td> 2</td><td> 99,4</td><td> 59,2</td>

<td> 6,0</td><td> 40</td><td> 2</td><td> 99,4</td><td> 60,6</td>

<td> 6,5</td><td> 40</td><td> 2</td><td> 99,3</td><td> 60,5</td>

<td> 7,0</td><td> 40</td><td> 2</td><td> 99,1</td><td> 54,0</td>

<td> 5,0</td><td> 40</td><td> 4</td><td> 97,3</td><td> 48,1</td>

<td> 5,5</td><td> 40</td><td> 4</td><td> 99,1</td><td> 50,5</td>

<td> 6,0</td><td> 40</td><td> 4</td><td> 99,1</td><td> 53,3</td>

<td> 6,5</td><td> 40</td><td> 4</td><td> 99,0</td><td> 52,3</td>

<td> 7,0</td><td> 40</td><td> 4</td><td> 98,6</td><td> 42,3</td>

EXAMPLE 3

[0307] Pertuzumab formulations with a protein concentration of 100 mg / mL were prepared with the following excipients:

(1) 10 mM histidine-HCl, 240 mM 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;

[0308] Each formulation was filled into a 3 cc FORMA VITRUM ™ USP Type I glass vial sealed with FLUROTEC ™ coated butyl rubber stoppers. The samples were stored at 30 ° C and 40 ° C and the stability was assessed for quality (CAC) and purity (SEC, IEC). The stability results showed that Pertuzumab in the histidine-phosphate buffer degraded much faster than in other histidine buffers when stored at 40 ° C (Fig. 8 and Fig. 9).

EXAMPLE 4

[0309] Pertuzumab was concentrated by ultrafiltration / diafiltration to 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.

[0310] The haze of each formulation was measured prior to filtration. The results, as shown in Fig. 10, showed that Pertuzumab samples formulated in the histidine-acetate and histidine-HCl buffers had lower amounts of insoluble aggregates than those in the histidine-sulfate buffer.

EXAMPLE 5

[0311] Pertuzumab was formulated at 30 mg / mL in [buffer] 20 mM histidine-acetate, 120 mM sucrose, 0.02% polysorbate 20, pH 6.0. Pertuzumab was filled into miniature 316L stainless steel and HASTELLOY ™ reservoirs. All samples were stored at -20 ° C and 5 ° C and evaluated for quality (CAC), purity (SEC, IEC) and strength (UV-Vis). Stability analyzes showed that Pertuzumab was stable in this formulation when stored at -20 ° C and 5 ° C for at least 3 months. Formulation

- 81 chloride free is compatible with 316L stainless steel and HASTELLOY ™ reservoir.

Table 4. Pertuzumab Stability in Stainless Steel Reservoirs

<td>Reservoirs</td><td>Temp (° C)</td><td>Time (Months)</td><td>CAC</td><td>Spec. UV (mg / mL)</td><td>SEC (% monomer)</td><td>IEC (% of main peak)</td>

<td></td><td></td><td> 0</td><td>Passed^and</td><td> 29,0</td><td> 99,8</td><td> 67,9</td>

<td rowspan="2">316L</td><td> -20</td><td> 3</td><td>Passed</td><td> 28,9</td><td> 99,7</td><td> 66,8</td>

<td> 5</td><td> 3</td><td>Passed</td><td> 28,7</td><td> 99,7</td><td> 66,8</td>

<td rowspan="2">HASTELLOY ™</td><td> -20</td><td> 3</td><td>Passed</td><td> 29,1</td><td> 99,7</td><td> 66,8</td>

<td> 5</td><td> 3</td><td>Passed</td><td> 28,8</td><td> 99,7</td><td> 67,7</td>

^and Rated for Color, Appearance and Clarity: Clear to slightly opalescent, colorless to pale yellow solution.

EXAMPLE 6

[0312] Pertuzumab was formulated using tangential flow filtration (TFF). The final formulation contains 20 mM histidine acetate, 120 mM sucrose, 0.02% polysorbate 20, pH 6.0 at a protein concentration of 30 mg / mL. The samples were filled in 20 ml FORMA VITRUM ™ USP Type I glass vials, closed with a butyl rubber stopper coated with 20 mm FLUROTEC ™ and sealed with aluminum flip-top caps. All samples were stored at -70 ° C, 5 ° C, 15 ° C, and the stability was assessed for quality (CAC), purity (SEC, IEC), strength (UV-Vis) and potency (Bioassay). The results showed that Pertuzumab is stable in this formulation when stored at 5 ° C and 15 ° C for at least 3 months.

Table 5. Stability of Pertuzumab in glass vials

<td>Temp. (° C)</td><td>Time (Months)</td><td>CAC</td><td>Spec. UV (mg / mL)</td><td>SEC (% monomer)</td><td>IEC (% of main peak)</td><td>Biological test (% specific activity)</td>

<td></td><td> 0</td><td>Passed</td><td> 29,2</td><td> 99,8</td><td> 64,1</td><td> 83</td>

<td> -70</td><td> 1</td><td>Passed</td><td> 29,7</td><td> 99,8</td><td> 65,2</td><td> 92</td>

<td></td><td> 3</td><td>Passed</td><td> 30,7</td><td> 99,8</td><td> 67,0</td><td> 93</td>

<td> 5</td><td> 3</td><td>Passed</td><td> 30,4</td><td> 99,7</td><td> 67,2</td><td> 90</td>

<td> 15</td><td> 1</td><td>Passed</td><td> 29,7</td><td> 99,7</td><td> 64,4</td><td> 78</td>

<td></td><td> 3</td><td>Passed</td><td> 30,4</td><td> 99,7</td><td> 65,5</td><td> 93</td>

EXAMPLE 7

[0313] 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, pH 6.0;

(3) 20 mM succinate at pH 6.0; and (4) 20 mM succinate, 240 mM sucrose at pH 6.0.

[0314] Each formulation was added with a different concentration of polysorbate 20. All samples were filled into 3 cc USP Type I glass vials and mixed horizontally at 70 rpm at room temperature for up to 7 days. The turbidity stability of each sample was assessed at the 7 day time point. The results showed that the use of polysorbate 20 in the final formulation was effective in preventing the formation of insoluble aggregates. See Fig. 11.

EXAMPLE 8

[0315] Pertuzumab was prepared in the following formulations:

(1) 25 mg / mL Pertuzumab, 10 mM Histidine-HCl, 240 mM sucrose, pH 6.0;

(2) 50 mg / mL Pertuzumab, 10 mM Histidine-HCl, 240 mM sucrose, pH 6.0;

(3) 60 mg / mL Pertuzumab, 20 mM histidine-acetate, 120 mM sucrose, pH 6.0.

[0316] Various amounts of polysorbate 20 were added to each formulation. All samples were filled into 3 cc USP Type I glass vials and mixed horizontally at 70 rpm at room temperature for up to 7 days. The physical stability of each sample for turbidity was assessed at the 7 day time point. The results showed that the use of polysorbate 20 in the histidine-HCl and sucrose formulation was effective in preventing the formation of insoluble particles. The histidine-acetate and sucrose formulation appeared to have the same protein protective effect as polysorbate 20. See Fig. 12.

EXAMPLE 9

[0317] Pertuzumab was formulated as follows:

(1) 100 mg / mL protein, 10 mM Histidine-HCl, pH 6.0;

(2) 100 mg / mL protein, 20 mM succinate, pH 6.0;

(3) 60 mg / mL protein, 20 mM histidine-acetate, pH 6.0.

[0318] Each formulation was mixed with different amounts of sucrose. All samples were sterile filled in 3 cc USP Type I glass vials. They were then frozen at -70 ° C and thawed at 5 ° C, three times. The physical stability of each sample was determined after three freeze-thaw cycles. The results showed that sucrose prevents the formation of soluble aggregates in the freeze-thaw process. See Fig. 13.

- 83 EXAMPLE 10

[0319] A preferred Pertuzumab formulation for therapeutic use consists essentially of 30mg / mL Pertuzumab in [buffer] 20mM histidine-acetate, 120mM sucrose, 0.02% polysorbate at pH 6.0.

<td>Relationship</td><td>Concentration</td><td>Quantity / L</td>

<td>Pertuzumab</td><td>30 mg / mL</td><td>30 g</td>

<td>L-Histidine MW = 155.16 g / mol</td><td>20 mm</td><td>3.10g</td>

<td>Ice Octic Acid MW = 60.05 g / mol Density = 1.05 g / cm 3</td><td>ll.6mM</td><td>0.66 mL</td>

<td>Saccharose MW = 342.3 g / mol</td><td>120 mm</td><td>41.1 g</td>

<td>Polysorbate 20 Density = 1.012 g / cm³</td><td>0.02% (w / v)</td><td>0.2 mL</td>

<td colspan="3">MW: Molar mass</td>

Vial configuration for 420mg dose:

Vial: 20 cc Formal Vitrum Type I glass

Cork: 20mm DAIKYO GRAY ™, laminated with fluoro-resin

Cap: 20mm aluminum, flip-top

Filling volume: 14.50 mL

Delivery: 14.0 mL of Pertuzumab in an IV bag with saline.

Vial configuration for a dose of 1050mg:

Vial: 50 cc. Formal Vitrum Type I glass

Cork: 20mm DAIKYO GRAY ™, laminated with fluoro-resin

Cap: 20mm aluminum, flip-top

Filling volume: 36.0 mL

Delivery: 35.0 mL of Pertuzumab in an IV bag with saline.

- 84 EXAMPLE 11

[0320] This example is for another formulation of Pertuzumab used in Phase I and Phase II clinical trials. The composition consists of 25 mg / ml Pertuzumab, 10 mM Histidine-HCl buffer, 240 mM sucrose, 0.02% Polysorbate 20, pH 6.0.

<td>Ingredient</td><td>Concentration</td>

<td>Pertuzumab</td><td>25 mg / ml</td>

<td>L-His HCl.H₂O (MW 209.6)</td><td>1.12 mg / ml (0.0125 M)</td>

<td>L-His (MW 155.2)</td><td>0.72 mg / ml (0.0099 M)</td>

<td>Sucrose (MW342.3)</td><td>82.15 mg / ml (0.240 M)</td>

<td>Polysorbate 20</td><td>0.2 mg / ml (0.02%)</td>

EXAMPLE 12

[0321] Cellular apoptosis depends on internal and external pathways. Chemotherapy can cause cell damage and can trigger apoptosis through internal pathways in response to cell damage. 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 TumourNecrosis Factor Superfamily. Nature Reviews 2: 420-430 (2002)). Death receptors, such as DR4 and DR5, located on the surface of cells, trigger apoptosis through an external pathway that does not involve p53. Agonists such as Apo2L bind to DR4 and DR5 receptors and activate caspases 8 and 10 via the Fas-associated death domain. Then, to induce apoptosis, casapases 8 and 10 activate caspases 3, 6 and 7. Molecular signaling of death receptors on tumor cells has the therapeutic potential to eliminate cancer cells that are resistant to conventional therapies, and molecules such as Apo2L are currently undergoing clinical evaluation.

[0322] Apomab is a full length humanized IgG1 of CHO origin constructed with a lambda light chain. It is an agonist anti-DR5 antibody that has shown the induction of apoptosis in various tumor cell lines. Preclinical studies using a tumor-implanted mouse model showed that Apomab exhibited similar or improved tumor reduction compared to Apo2L. Apomab is evaluated as an anti-cancer agent for use in advanced solid tumors and Non-Hodgkin's Lymphoma (NHL). The amino acid sequences of the Apomab heavy and light chains used in these experiments are shown in Figures 27 and 28.

- 85 Preparation of Antibody Formulations

[0323] The recombinantly produced Apomab showed a very dilute protein concentration at high pH. The material was concentrated to approximately 20 mg / mL and exchanged into 20 mM sodium acetate buffer, pH 5.0 using a Millipore Labscale tangential flow filtration (TFF) system with a MILLIPORE PELLICON ™ XL membrane, PLCGC10, 50 cm. Apomab samples were formulated in various 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 membrane dialysis with a molecular weight threshold of 10,000 Da (Pierce, Inc. ). In the final dialysis, 240 mM trehalose was added. After dialysis, 0.02% TWEEN 20 □ was added to the formulation and samples were filtered through 0.22 µm filters (Millipore, Inc.). Sterile 3 cc glass vials (Forma Vitrum, Inc.) were filled with Apomab in a volume of 0.5 mL and sealed with 13 mm stoppers (Daikyo, Inc). Protein stability was assessed at 70 ° C, 5 ° C, 30 ° C and 40 ° C with storage up to 3 months.

Stability of Apomab Formulations

[0324] For drug product stability testing, a formulated batch of Apomab was filled into 5 cc FORMA VITRUM® glass vials. Vials were filled with 5.5 mL of formulated antibody, fitted with 20 mm DAIKYO® stoppers and stored at 70 ° C, 5 ° C, 30 ° C and 40 ° C in an upright position.

[0325] For drug substance stability testing, an aliquot of formulated Apomab was sterile filtered through a 0.22 µm filter and 10 mL was filled into 20cc 316L autoclaved stainless steel mini tanks. The reservoirs were placed vertically at -20 ° C and 5 ° C. A 1 mL aliquot was aseptically removed from the mini-reservoirs at predetermined time intervals to assess protein quality. The control vials were 1 mL aliquots in 3 cc glass vials stored at -20 ° C.

Color, Appearance and Clarity

[0326] The clarity, appearance and color of the samples were visually inspected under fluorescent light using a light control station with a black and white background. For drug substance analyzes, samples in mini-reservoirs were transferred to 3 cc glass vials for visual inspection.

pH

[0327] The pH was measured at room temperature using the THERMO ORION SURE-FLOW ROSS ™ semi-micro pH electrode for measuring buffers or the THERMO ORION GLS ™ combined micro pH electrode for measuring the pH of screened protein samples, the Beckman microelectrode probe for toxicological stability of the samples. The pH / ion meter METERLAB ™ pHM240 (Radiometer Analytical) was calibrated daily with buffer standards (EM Science) at pH 7 and pH 4.

Concentration

[0328] Protein concentration was determined by ultraviolet absorption spectroscopy using an AGILENT 8453 spectrophotometer. Samples were diluted as appropriate for

- formulated with blank buffer so as to give an absorption of 0.5 to 1.0. The device was zeroed with a dilution 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 shift and light scattering. Protein concentrations were calculated using the following equation:

Conc. (mg / mL) = (A279 - A320) X dilution factor adsorption factor in cm ^ mg / mL)¹

[0329] The sequence based adsorption coefficient was initially determined to be 1.32 cnT ^ mg / mL) '¹ and this value was used for the pH screening. Later value 1.7 cnf ^ mg / mL) '¹ was determined on the basis of amino acid analyzes and proteolytic methods, and this value was used for stability analyzes of Apomab used in the collective toxin studies.

Ion exchange chromatography

[0330] Ion-exchange chromatography was performed on a 1100 series HPLC (Agilent Technologies, Inc.), equipped with a diode detector. Chromatography was performed on a PROPAC WCX-I0 ™ column (Dionex) (4 x 250 mm) with a flow rate of 0.5 mL / min and a column temperature of 40 ° C. Mobile phase A was 25 mM sodium phosphate, pH 6.5. Mobile phase B was 100 mM sodium chloride in the same buffer as mobile phase A. The column was equilibrated with 100% mobile phase A. For the screening of the pH samples, Apomab 20 mg was applied to the column and the absorption was monitored at 214 nm. Protein was eluted from the column with the following gradient:

<td>Time (min)</td><td>% A</td><td>% B</td>

<td> 0</td><td> 100</td><td> 0</td>

<td> 50</td><td> 0</td><td> 100</td>

<td> 51</td><td> 100</td><td> 0</td>

<td> 70</td><td> 100</td><td> 0</td>

[0331] For the stability analyzes of the material used in the toxicology studies, 30 mg of Apomab was applied to the column and the absorption was monitored at 280 nm. Protein was eluted from the column with the following gradient:

Gradient: Time (min)% A% B

1000

40,0 4060

41,0 0100

- 87 45,0 0100

45,1 1000

60,0 1000

Spherical Exclusion Chromatography

[0332] Spherical exclusion chromatography was performed on a 1100 series HPLC (Agilent Technologies, Inc.), equipped with a diode detector. Apomab at 50 pg was applied to a TSK Gel 3000SWXL ™ column (7.8 x 300 mm) and passed at a flow rate of 0.9 mL / min for 20 minutes for pH sample testing and 0.5 mL / min for 30 minutes for samples of toxicological stability with the mobile phase in the form of 0.20 M potassium phosphate, 0.25 M potassium chloride, pH 6.2. Absorption was monitored at 280 nm.

Power

[0333] The purpose of the potency bioassay was to measure the ability of Apomab to kill Colo205 cells using ALAMARBLUE ™. Colo205 is a colon cancer cell line that expresses both DR5 and DR4 death receptors. This test introduces a fluorimetric / colorimetric growth index based on the detection of metabolic activity. ALAMARBLUE ™ is a redox dye which is blue and non-fluorescent in its oxidized state. The intracellular metabolic reduction turns it red, which is also fluorescent. Changes in color and fluorescence are proportional to metabolic activity and the number of viable cells. The signal decreases as the cell dies. Apomab was diluted with anti-Fc medium, then Colo 205 cells were added to the Apomab samples and incubated at 37 ° C for 48 hours. ALAMARBLUE ™ was added for the last 2-3 hours. Plates were read at excitation at 530 nm and emission at 590 nm to obtain relative fluorescence units (RFU). Data were analyzed using KALEIDAGRAPH ™. A kill dilution curve was constructed.

RESULTS

Formulation pH Screening

[0334] The effect of pH on antibody stability was tested using Apomab prepared from an unamplified, stable cell line. For these analyzes, Apomab was formulated at 20 mg / mL of antibody in 20 mM sodium acetate buffer at pH 4.0, 4.5, 5.0, 5.5; 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 -70 ° C, 5 ° C, 30 ° C and 40 ° C, and the protein stability was determined by various analytical tests, including CAC, pH, concentration, SEC and IEC. No significant changes in CAC, pH, or protein concentration were observed during storage of the samples.

[0335] Analysis of the samples by SEC showed that no significant changes occurred during storage at 5 ° C and -70 ° C. However, degradation occurred during storage at 30 ° C and 40 ° C (Fig. 20), observed as the formation of antibody fragments and soluble aggregates. To compare the formulations during storage, the kinetics of the antibody monomers were monitored and first order rate constants were calculated. The resulting antibody monomer loss rate profile at pH is shown in Fig. 21. The optimal conditions for antibody monomer stability were obtained by formulating histidine acetate in a buffer at pH 6.0.

[0336] Apomab charge heterogeneity was monitored by IEC. No significant changes in the IEC profile occurred during storage at 5 ° C and -70 ° C. However, the degradation observed as formation of acidic or basic variants appeared depending on the formulation (Fig. 22). In general, more basic variants were made in the lower pH formulation, and more acidic variants were made in the higher pH formulation. To compare the formulations during storage, the kinetics of the major IEC peak was monitored and first order rate constants calculated. The resulting rate profile of the loss of the IEC major peak at pH is shown in Fig. 23. The rate constants observed with IEC were about 10-fold higher than those of SEC (Fig. 21). Therefore, the loss of the major IEC peak was the major degradation of the antibody that will ultimately limit the shelf life of the product. In addition, as observed by SEC, the optimal antibody stability to stabilize the major IEC peak was obtained by formulation in a histidine acetate buffer at pH 6.0.

[0337] Following analysis of the pH screening data described above, an Apomab formulation was selected that contained 20 mg / mL of antibody in 20 mM histidine acetate, 240 mM trehalose, 0.02% polysorbate 20, pH 6.0. For the drug product, the vial configuration consisted of 5.5 mL filling a 5 cc FORMA VITRUM ™ vial with a 20 mM DAIKYO ™ West stopper. Apomab was stored in stainless steel containers.

[0338] The stability of Apomab Medicinal Product was assessed in the 5cc glass vial configuration described above. The vials were stored at -70 ° C (controls), 5 ° C, 30 ° C and 40 ° C. Samples were taken at specified time intervals and analyzed by the following tests: color, appearance, clarity (CAC), pH, protein concentration, SEC, IEC and strength. The results of these tests are shown in Table 6 for samples stored at -70 ° C and 5 ° C and Table 7 for samples stored at 30 ° C and 40 ° C.

Table 6. Stability Data for Apomab Stored at -70 ° C and 5 ° C

<td>Temp KI ares (° C) Temporal Color</td><td>pH</td><td>Concentration (mg / mL)</td><td>SEC (% monomer u)</td><td>IEC (% of main peak)</td><td>Power (% of Specific Activity)</td>

<td>Criteria Report Report Acceptance:</td><td> 6,0 ±0,3</td><td> 20±2</td><td> &gt; 95%</td><td>Report</td><td> 60-140%</td>

<td>ON</td><td>T = 0</td><td>Transparent</td><td>Colorless</td><td> 5,9</td><td> 20,2</td><td> 99,8</td><td> 63</td><td> 94</td>

<td> -70</td><td>One month</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,5</td><td> 99,8</td><td> 63</td><td> 86</td>

<td> -70</td><td>2 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,4</td><td> 99,7</td><td> 64</td><td> 91</td>

<td> -70</td><td>3 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,5</td><td> 99,7</td><td> 63</td><td> 83</td>

<td> -70</td><td>6 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,4</td><td> 99,7</td><td> 64</td><td> 85</td>

<td> -70</td><td>9 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,4</td><td> 99,8</td><td> 65</td><td> 89</td>

<td> -70</td><td>12 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,8</td><td> 99,7</td><td> 63</td><td> 107</td>

<td> 5</td><td>One month</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,5</td><td> 99,7</td><td> 63</td><td> 89</td>

<td> 5</td><td>2 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,4</td><td> 99,7</td><td> 64</td><td> 99</td>

<td> 5</td><td>3 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,6</td><td> 99,7</td><td> 63</td><td> 84</td>

<td> 5</td><td>6 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,5</td><td> 99,7</td><td> 64</td><td> 93</td>

<td> 5</td><td>9 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,6</td><td> 99,7</td><td> 64</td><td> 88</td>

<td> 5</td><td>12 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,7</td><td> 99,6</td><td> 64</td><td> 106</td>

Table 7. Stability Data for Apomab Stored at 30 ° C and 40 ° C

Power

IEC (% (%

<td>Temp</td><td>Point</td><td>Concentration</td><td>SEC (%</td><td>main 0</td><td>A specific</td>

<td>(° C)</td><td>time Klarow Color pH</td><td>(mg / mL)</td><td>monomer</td><td>peak)</td><td>Activity</td>

<td>nity</td><td> )</td><td>you)</td>

<td>Criteria Report Report 6.0 0.3</td><td> 20 ± 2 &gt; 95%</td><td>60-140% report</td>

<td>Acceptance:</td><td></td><td></td>

<td>30 1 month Review Colorless 6.0</td><td> 20,6 98,2</td><td> 59 91</td>

<td>ysty ny</td><td></td><td></td>

<td>30 2 Review Colorless 6.0</td><td> 20,3 97,4</td><td> 54 80</td>

<td>ysty ny months</td><td></td><td></td>

<td>30 3 Review Colorless 6.0</td><td> 20,6 97,2</td><td> 49 74</td>

<td>ysty ny months</td><td></td><td></td>

<td>30 6 Review Colorless 6.0</td><td> 20,2 94,1</td><td> 37 51</td>

<td>months of ysty ny</td><td></td><td></td>

<td>30 9 Review Slightly 6.0</td><td> 20,4 93,2</td><td> 31 55</td>

<td>ysty months</td><td></td><td></td>

<td>yellow</td><td></td><td></td>

<td>30 12 Review Slightly 6.0</td><td> 20,6 91,6</td><td> 25 59</td>

<td>vivacious</td><td></td><td></td>

<td>yellow months</td><td></td><td></td>

<td>40 1 month Review Colorless 6.0</td><td> 20,4 96,6</td><td> 44 79</td>

<td>ysty ny</td><td></td><td></td>

<td>40 2 Review Colorless 6.0</td><td> 20,0 93,7</td><td> 31 64</td>

<td>ysty ny months</td><td></td><td></td>

<td>40 3 Review Slightly 5.9</td><td> 20,3 91,5</td><td> 22 53</td>

<td>ysty months</td><td></td><td></td>

<td>yellow</td><td></td><td></td>

<td>40 6 Review Slightly 6.0</td><td> 20,2 83,9</td><td>NT 26</td>

<td>ysty months</td><td></td><td></td>

<td>yellow</td><td></td><td></td>

<td>40 9 Review Yellow 5.9</td><td> 20,3 78,8</td><td>NT 25</td>

<td>ysty months</td><td></td><td></td>

<td>40 12 Review Yellow 5.9</td><td> 20,5 71,4</td><td>NT 31</td>

<td>vivacious</td><td></td><td></td>

<td>months</td><td></td><td></td>

<td colspan="3">NT = not counted</td>

[0339] After 13 months of storage at -70 ° C and 5 ° C, no change in protein quality was observed. For example, the pH remained at 6.0 0.3, Apomab appeared a clear and colorless liquid, the protein concentration remained at 20.0 2.0 mg / mL, and the monomer% was unchanged. In addition, there was no significant change in the% IEC major peak, and the% specific activity, as determined by the cell killing power test, was within the assay precision range of 60% to 140% of specific activity. The results showed that Apomab stored in 5 cc glass vials was stable for at least 12 months at 5 ° C.

[0340] Table 7 shows that changes in protein quality occurred at 30 ° C and 40 ° C. The SEC shows a decrease in the% monomer with an increase in mostly fragments. The aggregates also grew at higher temperatures, but at a much slower rate. However, aggregates grew significantly after 6 months at 40 ° C. The% of IEC main peak decreased with corresponding increase in acid variants. The basic peaks decreased slightly after 2 months at 40 ° C and 9 months at 30 ° C. After six months of storage at 40 ° C, degradation occurred to such an extent that the major IEC peak could no longer be merged. Cell killing bioassay showed% loss of specific activity at higher temperature with longer storage time. Protein concentration and pH were unchanged. The solution turned slightly yellow after 3 months at 40 ° C and 9 months at 30 ° C and turned yellow after 9 months at 40 ° C.

Stability of the Drug Substance

[0341] The freeze-thaw stability data of the drug substance are shown in Table 8.

Table 8. Freeze-Thaw Stability Data for Miniature Stainless Steel Tanks filled with Apomab

<td>Temp (° C) (Freeze / Defrost)</td><td>Frozen e Defrost Cycle No.</td><td>Clarity</td><td>Colour</td><td>pH</td><td>Concentration (mg / m L)</td><td>SEC (% Monomer)</td>

<td>Acceptance Criteria:</td><td></td><td>Report</td><td>Report</td><td> 6,0 ±0,3</td><td> 20,0 ± 2,0</td><td> &gt;95%</td>

<td>Control</td><td> 0</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,9</td><td> 99,6</td>

<td>(not frozen)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> -20/25</td><td> 1</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,8</td><td> 99,6</td>

<td> -20/25</td><td> 2</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,8</td><td> 99,6</td>

<td> -20/25</td><td> 3</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,9</td><td> 99,6</td>

Table 9. Stability Data for Miniature Stainless Steel Tanks filled with Apomab

<td>Temp (° C)</td><td>Point Time</td><td>Ki rity</td><td>Colour</td><td>pH</td><td>Concentration (mg / mL)</td><td>SEC (% monomer)</td><td>IEC (% of major peak)</td><td>Power (% of Specific) Activity)</td>

<td colspan="2">Criteria Acceptance:</td><td>Report</td><td>Report</td><td> 6,0 ±0,3</td><td> 20±2</td><td> &gt; 95%</td><td>Report</td><td> 60- 140%</td>

<td>ON</td><td>T = 0</td><td>Transparent</td><td>Colorless</td><td> 5,9</td><td> 20,0</td><td> 99,7</td><td> 63</td><td> 88</td>

<td> -20</td><td>One month</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,6</td><td> 99,7</td><td> 63</td><td> 107</td>

<td> -20</td><td>3 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,6</td><td> 99,7</td><td> 63</td><td> 82</td>

<td> -20</td><td>6 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,3</td><td> 99,7</td><td> 64</td><td> 92</td>

<td> -20</td><td>9 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,6</td><td> 99,7</td><td> 64</td><td> 92</td>

<td> -20</td><td>12 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 21,2</td><td> 99,7</td><td> 65</td><td> 94</td>

<td> 5</td><td>One month</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,5</td><td> 99,7</td><td> 62</td><td> 95</td>

<td> 5</td><td>3 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,7</td><td> 99,6</td><td> 62</td><td> 71</td>

<td> 5</td><td>6 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,4</td><td> 99,5</td><td> 62</td><td> 84</td>

<td> 5</td><td>9 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 20,8</td><td> 99,4</td><td> 61</td><td> 84</td>

<td> 5</td><td>12 months</td><td>Transparent</td><td>Colorless</td><td> 6,0</td><td> 21,3</td><td> 99,2</td><td> 59</td><td> 82</td>

No significant changes in the chemical characteristics of the protein were observed after freezing it at -20 ° C for at least 15 hours and thawing at ambient temperature three times.

For example, Apomab appeared to be a clear and colorless liquid, the pH remained at 6.0 + - 0.3, and the percentage of the monomer peak in SEC was unchanged.

[0342] The stability of Apomab in stainless steel tanks was assessed at -20 ° C and 5 ° C (Table 9).

[0343] Samples were aseptically removed from mini-reservoirs at specified intervals and analyzed.

[0344] Apomab showed no change in protein quality at 5 ° C as judged by pH, CAC, protein concentration and% major peak in IEC, but lost 0.1% monomer in SEC every 3 months. Reduced potency was observed when stored at 5 ° C for 3 months. However, sample strength increased again at the 6 and 9 month time points. Thus, the observed power differences at the 3 month time points were attributed to the study variability. Apomab showed no change in protein quality at -20 ° C as judged by pH, CAC, protein concentration,% monomer in SEC,% major peak in IEC and no significant change in potency. Stability data showed Apomab to be stable for at least 1 year at - 20 ° C and three months at 5 ° C.

[0345] A formulation screen was performed to select a formulation for Apomab. A review of the pH, covering the pH range from 4.0 to 7.0, using sodium acetate, histidine acetate, and sodium phosphate as buffers with 240 mM trehalose dihydrate and 0.02% polysorbate 20 showed Apomab to be the most stable in a solution at pH 6 , 0. Thus, a formulation consisting of 20 mM histidine acetate, 240 mM trehalose, 0.02% polysorbate 20, pH 6.0 was developed and experimentally shown to be stable. Using this formulation, Apomab was shown to be stable for at least 12 months at 5 ° C. In addition, Apomab has been shown to be stable for at least 12 months at - 20 ° C and three months at 5 ° C when stored in 316L stainless steel containers. Apomab has also been shown to be stable when subjected to up to 3 freeze / thaw cycles.

Piotr Godlewski

Patent Attorney