HK1151543B - Composition comprising antibody that binds to domain ii of her2 and acidic variants thereof - Google Patents

Description

Compositions comprising antibodies that bind to domain II of HER2 and acidic variants thereof

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application No.61/024825 filed on 30/1/2008, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

Technical Field

The present invention concerns compositions comprising a main species HER2 antibody and acidic variants thereof that bind to domain II of HER 2. The invention also relates to pharmaceutical formulations comprising said composition and to the therapeutic use of said composition.

Background

HER2 antibody

The HER family of receptor tyrosine kinases is an important mediator of cell growth, differentiation and survival. This receptor family includes four distinct members: epidermal growth factor receptor (EGFR, ErbB1 or HER1), HER2(ErbB2 or p 185)^neu) HER3(ErbB3) and HER4(ErbB4 or tyro 2).

EGFR encoded by the erbB1 gene has been considered to be the cause of human malignancies. In particular, increased EGFR expression has been observed in breast, bladder, lung, head, neck and stomach cancers, as well as glioblastoma. Increased expression of the EGFR receptor is often associated with increased production of the EGFR ligand, transforming growth factor alpha (TGF- α), such tumor cells leading to receptor activation through an autocrine stimulatory pathway. BaselgaandMendelsohn, pharmac. ther.64: 127-154(1994). Monoclonal antibodies against EGFR, or its ligand TGF-alpha or EGF, have been evaluated as therapeutic agents for the treatment of such malignancies. See, e.g., BaselgaandMendelsohn, supra; masuiet al, cancer research 44: 1002-1007 (1984); and wuetal, j.clin.invest.95: 1897-1905(1995).

Second member of the HER family, p185^neuThe product of the originally identified transformed gene, neuroblastoma from chemically treated rats. The activated form of the neu proto-oncogene results from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu was observed in breast and ovarian cancers and was associated with poor prognosis (Slamonteal, Science 235: 177-. To date, no point mutations similar to those in the neu protooncogene have been reported for human tumors. Overexpression of HER2 has also been observed in other carcinomas (frequent but heterogeneous due to gene amplification) including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder. See, kingtal, Science 229: 974 (1985); yokoteatal, Lancet 1: 765-; fukushigeet, mol. cellbiol.6: 955- "958 (1986); guerinetal, oncogeneres.3: 21-31 (1988); cohenetal, Oncogene 4: 81-88 (1989); yonemugeatal, cancer res.51: (1034 1991); borstetal, gynecol. oncol.38: 364 (1990); weinereetl, cancer res.50: 421-; kernet, cancer res.50: 5184 (1990); park et al, cancer res.49: 6605 (1989); zhauuetal, mol. carminog.3: 254-; aaslandetal, br.j. cancer 57: 358-363 (1988); williamserial, Pathobiology 59: 46-52 (1991); and mccannetal, Cancer 65: 88-92(1990), etc. HER2 can be overexpressed in prostate cancer (Guetal., cancer Lett.99: 185-9 (1996); RossetalHum. pathol.28: 827-33 (1997); rossetal, Cancer 79: 2162-70 (1997); and sadasivantetanal, j.urol.150: 126-31(1993)).

P185 has been described for rat^neuAnd the human HER2 protein product. Drebin and colleagues prepared a product against rat neu gene, p185^neuThe antibody of (1). See, e.g., drebinet, Cell 41: 695-706 (1985); myersetal, meth.enzyme.198: 277-290 (1991); and WO 94/22478. Drebinet, Oncogene 2: 273-277(1988) reported the correlation with p185^neuThe mixture of antibodies reactive with the two distinct regions of (a) produces a synergistic anti-tumor effect on neu-transformed NIH-3T3 cells implanted in nude mice. See also U.S. patent 5,824,311 issued on 20/10 of 1998.

Hudziakteal, mol.cell.biol.9 (3): 1165-1172(1989) describe the generation of a panel of antibodies to HER2 and identified using the human breast tumor cell line SK-BR-3. The relative cell proliferation of SK-BR-3 cells after exposure to antibody was determined by crystal violet staining of monolayer cells after 72 hours. Using this assay, maximum inhibition was obtained with an antibody designated 4D5, which inhibited cell proliferation by 56%. The other antibodies of the panel reduced cell proliferation to a lesser extent in this assay. Antibody 4D5 was also found to sensitize breast tumor cell lines overexpressing HER2 to the cytotoxic effects of TNF- α. See also U.S. Pat. No.5,677,171 issued 10/14 in 1997. The antibodies discussed in the Hudziak et al article have also been identified in: fendelyetal, cancer research 50: 1550 and 1558 (1990); kottsetal, invtro 26 (3): 59A (1990); sarupetal, growth regulation 1: 72-82 (1991); shepardetal, j.clin.immunol.11 (3): 117-127 (1991); kumaret al, mol.cell.biol.11 (2): 979-; lewis et al, cancer immunol.immunol.37: 255-; pietraset, Oncogene 9: 1829-1838 (1994); vitettaetaet, cancer research 54: 5301-5309 (1994); sliwkowskieet, j.biol.chem.269 (20): 14661-14665 (1994); scottetal, j.biol.chem.266: 14300-5 (1991); d' souzaetal, proc.natl.acad.sci.91: 7202-; lewis et al, cancer research 56: 1457-1465 (1996); and schaeferet al, Oncogene 15: 1385-1394(1997).

Recombinant humanized versions of murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAbHER2, Trastuzumab orU.S. patent 5,821,337) clinically works on patients with metastatic breast cancer that overexpresses HER2 and who have received extensive early anti-cancer therapy (baselgaetal, j.clin.oncol.14: 737-744(1996)). Trastuzumab was marketed by the food and drug administration at 25.9.1998 and can be used to treat metastatic breast cancer patients with tumors that overexpress HER2 protein.

Other HER2 antibodies with various properties are described in the following documents: tagliabuetal, int.j.cancer 47: 933 937 (1991); mckenzieetal, Oncogene 4: 543 and 548 (1989); maieretal, cancer res.51: 5361-5369 (1991); bacusetal, molecular Carcinogenesis 3: 350-362 (1990); stancovskiietal, pnas (usa) 88: 8691 and 8695 (1991); bacusetal, cancer research 52: 2580 + 2589 (1992); xuetal, int.j.cancer 53: 401-408 (1993); WO 94/00136; kasprzykey, cancer research 52: 2771-2776 (1992); hancocketal, cancer res.51: 4575-4580 (1991); shawveret, cancer res.54: 1367-; artemigaetatal, cancer res.54: 3758-3765 (1994); hartrettal, j.biol.chem.267: 15160- > 15167 (1992); U.S. Pat. nos. 5,783,186; and klapperiet, Oncogene 14: 2099-2109(1997).

Homology screening allows two other members of the HER receptor family to be identified: HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968 and Krauseal et al, PNrAS (USA) 86: 9193-. Both receptors show increased expression in at least some breast cancer cell lines.

It is generally found that there are various combinations of HER receptors in cells and that its dimerization is thought to increase the diversity of cellular responses to various HER ligands (Earp et al, Breast cancer research 35: 115-132 (1995)). EGFR is bound by 6 different ligands: epidermal Growth Factor (EGF), transforming growth factor alpha (TGF-. alpha.), amphiregulin, heparin-binding epidermal growth factor (HB-EGF), betacellulin, and epiregulin (Groenenet al, Growth factors 11: 235-257 (1994)). One family of heregulin proteins that results from alternative splicing of a single gene are ligands of HER3 and HER 4. The heregulin family includes the alpha, beta and gamma heregulins (Holmesteal., Science 256: 1205-1210 (1992); U.S. Pat. No.5,641,869; and Schaefertal., Oncogene 15: 1385-1394 (1997)); neu Differentiation Factor (NDF); glial Growth Factor (GGF); acetylcholine Receptor Inducing Activity (ARIA); and sensory and motor neuron derived factor (SMDF). For a review see Groenenet al, growths fans 11: 235-257 (1994); lemke, G, Molec & cell.neurosci.7: 247- "262" (1996); and leeet, pharm.rev.47: 51-85(1995). Three additional HER ligands have recently been identified: neuregulin-2 (NRG-2), which is reported to bind HER3 or HER4 (Changeatal, Nature 387: 509-; neuregulin-3, which binds to HER4(Zhangetal, PNAS (USA)94 (18): 9562-7 (1997)); and neuregulin-4, which binds HER4(Harariet al, Oncogene 18: 2681-89 (1999)). HB-EGF, betacellulin and epiregulin also bind HER 4.

Although EGF and TGF α do not bind HER2, EGF stimulates EGFR and HER2 to form a heterodimer, which activates EGFR and leads to transphosphorylation of HER2 in the heterodimer. Dimerization and/or transphosphorylation appears to activate HER2 tyrosine kinase. See, earpoint, supra. Similarly, an active signaling complex is formed when HER3 is co-expressed with HER2, and antibodies against HER2 disrupt this complex (Sliwkowskieet, J.biol.chem.269 (20): 14661-14665 (1994)). Furthermore, HER3 increased affinity for protein regulation (HRG) to a higher affinity state when co-expressed with HER 2. For HER2-HER3 protein complexes see also leviet, journal of neuroscience 15: 1329-; morrisseyetal, proc.natl.acad.sci.usa 92: 1431-1435 (1995); and lewis et al, cancer res.56: 1457-1465(1996). HER4, like HER3, forms an active signaling complex with HER2 (Carrawayandconttley, Cell 78: 5-8 (1994)).

To target the HER signaling pathway, rhuMAb2C4(Pertuzumab) was developed as a humanized antibody that inhibits dimerization of HER2 with other HER receptors, thereby inhibiting ligand-driven phosphorylation and activation, and downstream activation of the RAS and AKT pathways. In a phase I trial of Pertuzumab as a single agent for treating solid tumors, 3 subjects with advanced ovarian cancer were treated with Pertuzumab. One had a sustained partial response, while the other had stable disease for 15 weeks. Procamsocincclinononcol 22: 192, Abstract771 (2003).

Antibody variant compositions

Us patent 6,339,142 describes a HER2 antibody composition comprising a mixture of an anti-HER 2 antibody and one or more acidic variants thereof, wherein the amount of the acidic variants is less than about 25%. Trastuzumab is an exemplary HER2 antibody.

The poster "Effect of cell Cultur processing Change human antibody Processes diagnostics" presented at the well-characterized Biotechnology drug conference (WellCharacterizedBiotech pharmaceutical conference) (1/2003) by Reid et al describes an unnamed humanized IgG1 antibody composition that has N-terminal heterogeneity due to the combination of VHS signal peptide, N-terminal glutamine and pyroglutamic acid on its heavy chain.

The speech "the Ideal Chromatographic antibody Characterisation method" made by Harris et al at the IBC antibody Generation conference (IBCAntibody production conference) (month 2 2002) reported a VHS extension on the heavy chain of humanized anti-IgE antibody E25.

The speech made by Rouse et al in WCBP (6-9.1.2004), `TopDOwn ` Glycoprotein CharacteriationbyHighResolutionMassSpectrometrimethyl ItalicationBiopharamcal development ` describes a monoclonal antibody composition due to its light chain on which^-3AHS or^-2The HS signal peptide residues have N-terminal heterogeneity.

The poster "Strategiseof ComperabiltiyStudiesandAssaysforWellCharacterizedBiologicals" presented by JillPorter at IBC conference (9.2000) discusses ZEAX NAPAD^TMHas three additional amino acid residues in its heavy chain.

US2006/0018899 describes a composition comprising the main species Pertuzumab antibody and an amino-terminal leader extension variant of the Pertuzumab antibody, as well as other variant forms.

Summary of The Invention

According to a first aspect, the present invention concerns a composition comprising a main species HER2 antibody that binds to domain II of HER2 and acidic variants thereof, wherein the acidic variants comprise glycated variants (glycatedvariants), disulfide reduced variants (disulfiededvariants), or non-reducible variants (non-reductiblevariants). Preferably, the acidic variants include glycated variants, deamidated variants (deamidatedvariants), disulfide reduced variants, sialylated variants (sialylated variants), and non-reducible variants. Desirably, the amount of the acidic variant is less than about 25%.

In another aspect, the invention provides a composition comprising a main species HER2 antibody and an acidic variant of the main species antibody, wherein the main species HER2 antibody comprises the variable light and variable heavy chains of seq id nos. 3 and 4, and wherein the acidic variant comprises a glycated variant, a deamidated variant, a disulfide reduced variant, a sialylated variant, and a non-reducible variant.

The invention also concerns pharmaceutical formulations comprising said compositions in a pharmaceutically acceptable carrier.

In addition, the invention relates to a method of treating HER2 positive cancer in a patient comprising administering to the patient the pharmaceutical formulation in an amount effective to treat the cancer. For such methods, it is preferred that the main species antibody and acidic variant have essentially the same pharmacokinetics, as demonstrated in the examples herein.

In another aspect, the present invention concerns a method of preparing a pharmaceutical composition comprising: (1) preparing a composition comprising a main species HER2 antibody that binds domain II of HER2 and acidic variants thereof, said acidic variants comprising glycated variants, disulfide reduced variants, or non-reducible variants, and (2) evaluating said acidic variants in said composition and determining the amount thereof to be less than about 25%. In one embodiment, the acidic variant is assessed by a method selected from the group consisting of: ion exchange chromatography (wherein the composition is treated with sialidase); reduced capillary electrophoresis with sodium dodecyl sulfate (CE-SDS); non-reducing CE-SDS; borate chromatography; and peptide mapping.

Brief Description of Drawings

FIG. 1 provides a schematic representation of the structure of HER2 protein, and the amino acid sequences of domains I-IV of its extracellular domain (SEQ ID Nos. 19-22, respectively).

FIGS. 2A and 2B depict the light chain variable region (V) of murine monoclonal antibody 2C4_L) (FIG. 2A) and heavy chain variable region (V)_H) (FIG. 2B) (SEQ ID Nos. 1 and 2, respectively); humanized 2C4 form 574V_LAnd V_HDomains (SEQ ID Nos. 3 and 4, respectively), and human V_LAnd V_HAlignment of the amino acid sequences of the consensus frameworks (hum. kappa.1, light chain. kappa.subgroup I; humIII, heavy chain subgroup III) (SEQ ID Nos. 5 and 6, respectively). Asterisks indicate the difference between humanized 2C4 version 574 and murine monoclonal antibody 2C4 or between humanized 2C4 version 574 and human framework. Complementarity Determining Regions (CDRs) are in parentheses.

FIGS. 3A and 3B show the amino acid sequences of the light chain (SEQ ID No.15) and heavy chain (SEQ ID No.16) of Pertuzumab. CDRs are shown in bold. The carbohydrate module (motif) is attached to Asn299 of the heavy chain.

FIGS. 4A and 4B show the amino acid sequences of the Pertuzumab light chain (SEQ ID No.17) and heavy chain (SEQ ID No.18), each containing the complete amino-terminal signal peptide sequence.

Figure 5 graphically depicts the binding of 2C4 at the heterodimer binding site of HER2, thereby preventing heterodimerization with activated EGFR or HER 3.

Figure 6 depicts HER2/HER3 conjugation to MAPK and Akt pathways.

Figure 7 compares the activity of Trastuzumab and Pertuzumab.

FIGS. 8A and 8B show the amino acid sequences of the Trastuzumab light chain (SEQ ID No.13) and heavy chain (SEQ ID No. 14).

Figures 9A and 9B depict the variant Pertuzumab light chain sequence (seq id No.23) and the variant Pertuzumab heavy chain sequence (seq id No. 24).

Figure 10 shows the experimental design of the isolation, cell culture, recovery, and PK (pharmacokinetics) evaluation and analytical testing of cation-exchanged MP (main peak) and AV (acidic variant). Fresh medium ═ standard medium; spent medium was used as standard medium after 12 days of cell culture, and cells were removed by centrifugation. Dissolved oxygen, pH, and other parameters were not controlled.

Figure 11 shows a typical dionexprop Cation Exchange (CEX) chromatogram from example 1.

Figure 12 shows an analysis of Pertuzumab starting material and CEX fractions. AV ═ acidic variant; MP as the main peak; and BV is a basic variant.

FIG. 13 reveals the CEX of the Major Peak (MP) incorporated into the cell culture and incubated for 12 days.

FIG. 14 depicts the main peak incubation conditions.

Figure 15 outlines the method for characterizing acidic variants.

Figure 16 shows the Pertuzumab concentration versus time in the PK study in example 1.

Figure 17 provides the area under the curve (AUC) and geometric mean ratio from the PK study in example 1.

Detailed description of the preferred embodiments

I. Definition of

The term "main species antibody" refers herein to the amino acid sequence structure of an antibody in a composition that is the predominant number of antibody molecules in the composition. Preferably, the main species antibody is a HER2 antibody, such as an antibody that binds to domain II of HER2, an antibody that inhibits HER dimerization more effectively than Trastuzumab, and/or an antibody that binds to the heterodimeric binding site of HER 2. Preferred embodiments of the main class of antibodies herein are antibodies comprising the light and heavy chain variable region amino acid sequences of seq id nos. 3 and 4, and most preferably the light and heavy chain amino acid sequences of seq id nos. 15 and 16 (Pertuzumab).

An "amino acid sequence variant" antibody is herein an antibody having an amino acid sequence that differs from that of the main species antibody. Typically, amino acid sequence variants will have at least about 70% homology with the main species antibody, and preferably, they will be at least about 80%, and more preferably at least about 90% homologous with the main species antibody. 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 herein include acidic variants (e.g., deamidated antibody variants), basic variants, antibodies having an amino-terminal leader extension (e.g., VHS-) on one or both light chains thereof, antibodies having a C-terminal lysine residue on one or both heavy chains thereof, antibodies having one or more oxidized methionine residues, and the like, and include combinations of heavy and/or light chain amino acid sequence variations.

"acidic variants" refers to variants of the main species of antibody that are more acidic than the main species of antibody. Acidic variants have acquired a negative charge or lost a positive charge relative to the main species antibody. Such acidic variants can be resolved using separation methods that separate proteins according to charge, such as ion exchange chromatography. When separated by cation exchange chromatography, acidic variants of the main species antibody elute before the main peak.

A "disulfide-reducing variant" has one or more disulfide-forming cysteines chemically reduced to the free thiol form. Such variants may be monitored by hydrophobic interaction chromatography or by screening methods such as capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), for example as described in example 1.

Herein, "non-reducible variant" refers to a major class of antibody variants that cannot be chemically reduced to heavy and light chains by treatment with a reducing agent (such as dithiothreitol). Such variants can be assessed by treating the composition with a reducing agent and assessing the resulting composition using methods for assessing protein size, such as capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), for example using the techniques described in example 1 below.

A "glycosylated variant" antibody herein refers to an antibody having one or more carbohydrate moieties attached to it that are different from the one or more carbohydrate moieties attached to the main species of antibody. Examples of glycosylation variants herein include antibodies having a G1 or G2 oligosaccharide structure attached to their Fc region in place of the G0 oligosaccharide structure, antibodies having one or more carbohydrate moieties attached to one or both of their light chains, antibodies having no carbohydrate moieties attached to one or both of their heavy chains, sialylated antibodies, and the like, as well as combinations of these glycosylation changes.

If the antibody has an Fc region, an oligosaccharide structure as shown herein, such as in figure 14, may be attached to one or both heavy chains of the antibody, e.g. at residue 299. For Pertuzumab, G0 is the most predominant oligosaccharide structure, while other oligosaccharide structures such as G0-F, G-1, Man5, Man6, G1-1, G1(1-6), G1(1-3), and G2 are found in lesser amounts in Pertuzumab compositions.

Unless otherwise indicated, "G1 oligosaccharide structures" herein include G1(1-6) and G1(1-3) structures.

For the purposes herein, "sialylated variant" refers to a major class of antibody variants comprising one or more sialylated carbohydrate moieties attached to one or both of its heavy chains. Sialylated variants can be identified by evaluating the composition (e.g., by ion exchange chromatography) with or without sialidase treatment, e.g., as described in the examples.

"glycated variant" refers to an antibody to which a sugar (such as glucose) is covalently attached. This addition may occur by reaction of glucose with lysine residues on the protein (e.g., in cell culture media). Glycated variants can be identified by mass spectrometry analysis of reduced antibodies (assessing the increase in mass of the heavy or light chain). Glycated variants can also be quantified by borate chromatography, as explained in example 1 below. Glycated variants differ from glycated variants.

"deamidated" antibody refers to an antibody in which one or more asparagine residues are derived, for example, as aspartic acid, succinimide, or isoaspartic acid. An example of a deamidated antibody is a Pertuzumab variant in which Asn-386 and/or Asn-391 on one or both heavy chains of Pertuzumab is deamidated.

An "amino-terminal leader extension variant" as used herein refers to a main species antibody having an amino-terminal leader sequence of one or more amino acid residues at the amino terminus of any one or more of the heavy or light chains of the main species antibody. Exemplary amino-terminal leader extensions comprise or consist of three amino acid residues, VHS, which are present on one or both light chains of an antibody variant.

"homology" is defined as the percentage of identical residues in amino acid sequence variants after alignment of the sequences and introduction of gaps, if necessary, to achieve maximum percent identity. Methods and computer programs for comparison are well known in the art. One such computer program is "Align 2", authored by Genentech, inc, and submitted to the U.S. copyright office (Washington, DC20559) in 1991, 12, 10 with a user document (userdocumentation).

For purposes herein, "cation exchange analysis" refers to any method of separating a composition comprising two or more compounds according to charge differences using a cation exchanger. Cation exchangers generally comprise covalently bound, negatively charged groups. Preferably, the cation exchanger herein is a weak cation exchanger and/or contains carboxylate/ester functionality, such as PROPACWCX-10 sold by Dionex^TMA cation exchange column.

The "HER receptor" is a receptor protein tyrosine kinase belonging to the HER receptor family, including the EGFR, HER2, HER3, and HER4 receptors, as well as other members of this family to be identified in the future. The HER receptor will typically comprise an extracellular domain which can bind a HER ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxy-terminal signal domain containing several tyrosine residues that can be phosphorylated. Preferably, the HER receptor is a native sequence human HER receptor.

The extracellular domain of HER2 comprises 4 domains: domain I (about amino acid residues 1-195), domain II (about amino acid residues 196-319), domain III (about amino acid residues 320-488) and domain IV (about amino acid residues 489-630) (the residue numbers do not include a signal peptide). See garrettetotal, mol.cell.11: 495-505 (2003); choetal, Nature 421: 756 and 760 (2003); franklinal, cancer cell 5: 317-; or plowmanetal, proc.natl.acad.sci.90: 1746-1750(1993). See also fig. 1 herein.

The terms "ErbB 1", "HER 1", "epidermal growth factor receptor", and "EGFR" are used interchangeably herein to refer to, for example, carpenteal, ann.rev.biochem.56: EGFR disclosed in 881-914(1987), including naturally occurring mutant forms thereof (e.g., deletion mutant EGFR in Humphreyental, PNAS (USA) 87: 4207-4211 (1990)). erbB1 refers to the gene encoding the EGFR protein product.

The expressions "ErbB 2" and "HER 2" are used interchangeably herein and refer to, for example, sembaet al, pnas (usa) 82: 6497-: 230-234(1986) and human HER2 protein (Genebank accession number X03363). The term "erbB 2" refers to the gene encoding human ErbB2, while "neu" refers to the gene encoding rat p185^neuThe gene of (1). Preferred HER2 is native sequence human HER 2.

"ErbB 3" and "HER 3" refer, for example, to U.S. patents 5,183,884 and 5,480,968 and krausset al, pnas (usa) 86: 9193-9197 (1989).

The terms "ErbB 4" and "HER 4" refer herein to, for example, european patent application 599,274; plowmanetal, proc.natl.acad.sci.usa90: 1746 — 1750 (1993); and plowmanetal, Nature 366: 473, 475(1993), including isoforms (isoforms) thereof, for example as disclosed in WO99/19488 published 4/22 in 1999.

"HER ligand" refers to a polypeptide that binds to and/or activates a HER receptor. HER ligands of particular interest herein are natural sequence human HER ligands such as Epidermal Growth Factor (EGF) (Savagesetal, J.biol.chem.247: 7612-7621 (1972)); transforming growth factor alpha (TGF-. alpha.) (Marquardt et al, Science 223: 1079-1082 (1984)); amphiregulin, also known as Schwannoma (schwannoma) or keratinocyte autocrine growth factor (shoyabeta., Science 243: 1074-; betacellulin (Shingel., Science 259: 1604-; heparin-binding epidermal growth factor (HB-EGF) (Higashiyamaet, Science 251: 936-939 (1991)); epiregulin (Toyodaet al, J.biol. chem.270: 7495-7500 (1995); and Komurakakietal. Oncogene 15: 2841-2848 (1997)); alpha-regulin (see below); neuregulin-2 (NRG-2) (Carrawayetal, Nature 387: 512-516 (1997)); neuregulin-3 (NRG-3) (Zhangetal, Proc. Natl. Acad. Sci.94: 9562-9567 (1997)); neuregulin-4 (NRG-4) (Harariet, Oncogene 18: 2681-89 (1999)); or cripto (CR-1) (Kannacetal, J.biol.chem.272 (6): 3330-3335 (1997)). HER ligands that bind EGFR include EGF, TGF- α, amphiregulin, betacellulin, HB-EGF and epiregulin. HER ligands that bind HER3 include heregulin. HER ligands capable of binding to HER4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 and heregulin.

"heregulin" (HRG) as used herein refers to a polypeptide encoded by a heregulin gene, as described in U.S. patent 5,641,869 or marchonnietal, Nature 362: 312, 318 (1993). Examples of heregulins include heregulin-alpha, heregulin-beta 1, heregulin-beta 2, and heregulin-beta 3 (Holmestal, Science 256: 1205-1210 (1992); and U.S. Pat. No.5,641,869); neu Differentiation Factor (NDF) (Pelesetal, Cell 69: 205-216 (1992)); acetylcholine Receptor Inducing Activity (ARIA) (Fallsetal., Cell 72: 801-815 (1993)); glial Growth Factor (GGF) (Marchionnietal, Nature 362: 312-318 (1993)); sensory and motor neuron derived factor (SMDF) (Hoetal., J.biol.chem.270: 14523-; gamma-heregulin (Schaeferet al, Oncogene 15: 1385-1394 (1997)). The term includes biologically active fragments and/or amino acid sequence variants of native sequence HRG polypeptides, such as EGF-like domain fragments thereof (e.g., HRG β 1177-244).

"HER dimer" refers herein to a non-covalently bound dimer comprising at least two different HER receptors. Such complexes may form when cells expressing two or more HER receptors are exposed to a HER ligand, may be isolated by immunoprecipitation and analyzed by SDS-PAGE, as described, for example, by sliwkowskietal, j.biol.chem.269 (20): 14661-. Examples of such HER dimers include EGFR-HER2, HER2-HER3, and HER3-HER4 heterodimers. Furthermore, the HER dimer may comprise two or more HER2 receptors in combination with different HER receptors such as HER3, HER4, or EGFR. Other proteins, such as cytokine receptor subunits (e.g., gp130) can bind to the dimer.

A "heterodimer binding site" on HER2 refers to a region of the extracellular domain of HER2 that, when forming a dimer with EGFR, HER3, or HER4, contacts a region of the extracellular domain of EGFR, HER3, or HER4 or interfaces with a region of the extracellular domain of EGFR, HER3, or HER 4. Said region has been found in domain II of HER 2. Franklinal, cancer cell 5: 317-328(2004).

"HER activation" or "HER 2 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., by phosphorylation of tyrosine residues in a HER receptor or substrate polypeptide, caused by an intracellular kinase domain of the HER receptor). HER activation may be mediated by HER ligands that bind to HER dimers comprising the HER receptor of interest. HER ligands that bind to HER dimers may activate the kinase domain of one or more HER receptors in the dimer, resulting in phosphorylation of tyrosine residues in one or more HER receptors and/or phosphorylation of tyrosine residues in other substrate polypeptides, such as Akt or MAPK intracellular kinases.

The term "antibody" is used herein in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during the course of production of the monoclonal antibody, such as those described herein. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against the same determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a monoclonal antibody originally manufactured by kohleretal, Nature 256: 495(1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. patent No. 4,816,567). "monoclonal antibodies" may also be used, for example, as described in clacksonet, Nature 352: 624-: the technique described in 581-597(1991) was isolated from phage antibody libraries.

Monoclonal antibodies specifically include "chimeric" antibodies wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrisonetal, Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable region antigen binding sequences derived from a non-human primate (e.g., old world monkey, ape, etc.) and human constant region sequences.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

"intact antibody" refers to a polypeptide comprising an antigen-binding variable region and a light chain constant region (C)_L) And heavy chain constant region C_H1、C_H2 and C_H3. The constant region may be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof. Preferably, the whole antibody has one or more effector functions and comprises an oligosaccharide structure attached to one or both of its heavy chains.

Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis: downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as the segment from the amino acid residue at position Cys226 or Pro230 to the carboxyl terminus thereof. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region may be eliminated, for example, during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Thus, a complete antibody composition may include a population of antibodies in which all K447 residues have been eliminated, a population of antibodies in which no K447 residues have been eliminated, or a population of antibodies in which antibodies having K447 residues and antibodies having no K447 residues are mixed.

Unless otherwise indicated herein, the numbering of the residues of the immunoglobulin heavy chain is that of the EU index as in kabat et al, sequences of proteins of immunologica interest, 5th ed. "EU index as in Kabat" refers to the residue numbering of the human IgG1EU antibody.

The heavy chain constant regions of intact antibodies can be classified into different "classes" (class) according to their amino acid sequence. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant regions corresponding to the different antibody classes are referred to as α, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell, followed by causing lysis of the target cell. The main cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravechandkinet, annu.rev.immunol.9: 457-92(1991) page table 3 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. patent 5,500,362 or 5,821,337. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models, such as clynesetal, pnas (usa) 95: 652-.

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

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 a native sequence human FcR. In addition, a preferred FcR is one that binds an IgG antibody (gamma receptor), including receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB contains in its cytoplasmic domain an immunoreceptor tyrosine-based inhibitory motif (ITIM) (see for review)Annu, rev, immunol.15: 203-234(1997)). For review of FcR seeSee ravechandkinet, annu.rev.immunol.9: 457-492 (1991); capeletal, immunology 4: 25-34 (1994); and dehaasetal, j.lab.clin.med.126: 330-341(1995)). The term "FcR" encompasses other fcrs herein, including those that will be identified in the future. The term also includes the neonatal receptor, FcRn, which is responsible for transfer of maternal IgG to the fetus (guyeret al, j.immunol.117: 587(1976) and kimetal, j.immunol.24: 249 (1994)).

"complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to solubilize a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) that complexes with a cognate antigen. To assess complement activation, CDC assays may be performed, such as Gazzano-santoroet, j.immunol.methods 202: 163 (1996).

"native antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (V) at one end_H) Followed by a plurality of constant regions. Each light chain has a variable region (V) at one end_L) And the other end is a constant region. The constant region of the light chain is aligned with the first constant region of the heavy chain, and the variable region of the light chain is aligned with the variable region of the heavy chain. It is believed that particular amino acid residues form an interface between the light and heavy chain variable regions.

The term "variable" refers to the fact that certain portions of the variable regions differ widely in antibody sequence and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called hypervariable regions in the light and heavy chain variable regions. The more highly conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FRs, which largely adopt a β -sheet conformation, connected by three hypervariable regions that form loops and, in some cases, form part of the β -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and together with the hypervariable regions of the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, sequences of proteins of immunological interest, 5th edition, public health service, national institutes of health, Bethesda, Md, 1991). The constant regions are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as participation of antibodies in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable regions typically comprise amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34(L1), 50-56(L2) and 89-97(L3) in the light chain variable region and 31-35(H1), 50-65(H2) and 95-102(H3) in the heavy chain variable region; Kabat et al, sequence of proteins of immunologicalcales interest, 5th edition, public health service, national institutes of health, Bethesda, MD 1991) and/or those from the "high-variable loop" (e.g., residues 26-32(L1), 50-52(L2) and 91-96(L3) in the light chain variable region and 26-32(H1), 53-55(H2) and 96-101(H3, Biomusic J917: 1987: 901)). "framework region" or "FR" residues refer to variable region residues other than the hypervariable region residues defined herein.

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

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain variable region and one light chain variable region in tight, non-covalent association. It is in this construction thatIn this case, the three hypervariable regions of each variable region interact with each other at V_H-V_LThe surface of the dimer defines an antigen binding site. The six hypervariable regions together confer antigen-binding specificity to the antibody. However, even a single variable region (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

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

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

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein the domains are present on a single polypeptide chain. Preferably, the Fv polypeptide is at V_HAnd V_LFor a review of scFv see Pl ü ckthun, in the "Thermatologyof monoclonal antibodies", Vol 113, Rosenburg and Moore eds, Springer-Verlag, New York, 269, 315, 1994, HER2 antibody scFv fragments are described in WO93/16185, U.S. Pat. No.5,571,894, and U.S. Pat. No.5,587,458.

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments are in the same polypeptide chain (V)_H-V_L) In (b) comprises a linked heavy chain variable region (V)_H) And light chain variableZone (V)_L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and two antigen binding sites are created. Diabodies are described more fully in, for example, EP404,097; WO 93/11161; and hollingerial, proc.natl.acad.sci.usa90: 6444-6448(1993).

"humanized" forms of non-human (e.g., rodent) antibodies refer to chimeric antibodies that contain, at a minimum, sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are those in which residues from a hypervariable region of a human immunoglobulin (recipient antibody) are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. Typically, the humanized antibody will comprise substantially no less than at least one, and typically two, variable regions in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. Optionally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See joneset al, Nature 321: 522-525 (1986); riechmannetal, Nature 332: 323-329 (1988); and Presta, curr, op, struct, biol.2: 593-596(1992).

Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or TrastuzumabAs described in table 3 of U.S. patent 5,821,337, expressly incorporated herein by reference; humanized 520C9(WO93/21319) and humanized 2C4 antibodies, as described herein.

For purposes herein, "Trastuzumab"),And "huMAb 4D 5-8" refers to an antibody comprising the light and heavy chain amino acid sequences in seq id nos. 13 and 14, respectively.

Herein, "Pertuzumab" and "rhuMAb 2C 4" refer to antibodies comprising the amino acid sequences of the light and heavy chain variable regions in seq id nos. 3 and 4, respectively. If Pertuzumab is a whole antibody, it preferably comprises the light and heavy chain amino acid sequences in seq id nos. 15 and 16, respectively.

"naked antibody" refers to an antibody (as defined herein) unconjugated to a heterologous molecule, such as a cytotoxic moiety or a radioactive label.

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

A HER2 antibody that "inhibits HER dimerization more effectively than Trastuzumab" refers to an antibody that reduces or eliminates HER dimers more effectively (e.g., at least about 2-fold more effectively) than Trastuzumab. Preferably, such an antibody inhibits HER dimerization at least about as effectively as an antibody selected from the group consisting of: murine monoclonal antibody 2C4, a Fab fragment of murine monoclonal antibody 2C4, intact Pertuzumab, and a Fab fragment of Pertuzumab. Inhibition of HER dimerization can be assessed directly by studying HER dimers, or by assessing HER activation or downstream signaling caused by HER dimerization, and/or by assessing antibody-HER 2 binding sites, and the like. Assays used to screen for antibodies capable of inhibiting HER dimerization more effectively than Trastuzumab are described in agusetal, cancer cell 2: 127-. By way of example only, inhibition of HER dimerization can be analyzed by evaluating, for example, the following: inhibition of HER dimer formation (see, e.g., FIGS. 1A-B and WO01/00245 of Agusetal, cancer cell 2: 127-137 (2002)); reduction of HER ligand activation in cells expressing HER dimers (e.g., FIGS. 2A-B of WO01/00245 and Agusetal, cancer cell 2: 127-137 (2002)); blockade of HER ligand binding to HER dimer expressing cells (e.g., WO01/00245 and Agusetal, cancer cell 2: 127-137(2002) FIG. 2E); inhibition of cell growth of cancer cells expressing HER dimers (e.g., MCF7, MDA-MD-134, ZR-75-1, MD-MB-175, T-47D cells) in the presence (or absence) of a HER ligand (e.g., WO01/00245 and Agusetal, FIGS. 3A-D of cancer cell 2: 127-137 (2002)); inhibition of downstream signaling (e.g., inhibition of HRG-dependent AKT phosphorylation or inhibition of HRG-or TGF-dependent MAPK phosphorylation) (see, e.g., WO01/00245 and Agusetal, cancer cell 2: 127-137(2002) FIGS. 2C-D). Whether an antibody inhibits HER dimerization can also be assessed by studying the antibody-HER 2 binding site, for example by assessing the structure or model, such as crystal structure, of an antibody that binds to HER2 (see, e.g., franklinal, cancer cell 5: 317-.

The HER2 antibody may be more effective (e.g., at least 2-fold more effective) than Trastuzumab in "inhibiting HRG-dependent AKT phosphorylation" and/or inhibiting "HRG-or TGF α -dependent MAPK phosphorylation" (see, e.g., Agusetal, cancer cell 2: 127-137(2002) and WO 01/00245).

The HER2 antibody may be an antibody that "does not inhibit cleavage of the extracellular domain of HER 2" (Molinaetal., cancer Res.61: 4744-4749 (2001)).

A HER2 antibody that "binds to the heterodimeric binding site of HER2 binds to residues in domain II (and optionally also binds to other domains other than the extracellular domain of HER2, such as residues in domains I and III) and is at least to some extent able to sterically hinder the formation of HER2-EGFR, HER2-HER3, or HER2-HER4 heterodimers. Franklinal, cancer cell 5: 317-.

An antibody that "binds to domain II of HER 2" binds to residues in domain II and optionally other domains of HER2, such as residues in domains I and III.

"growth inhibitory agent" as used herein refers to a compound or composition that inhibits the growth of a cell, particularly a HER-expressing cancer cell, in vitro or in vivo. Thus, the growth inhibitory agent may be an agent that significantly reduces the percentage of HER expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a position outside the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M-phase blockers include the vinca alkaloids (vincristine and vinblastine), paclitaxel, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that block G1 also spill over into S phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in the molecular Basisis cancer, edited by Mendelsohn and Israel, Chapter 1, entitled "Cellcycerrection, oncogenes, and Andanitioplastic vehicles", Murakani et al, WBSaunders, Philadelphia, 1995, especially page 13.

Examples of "growth inhibitory" antibodies are those that bind to HER2 and inhibit the growth of cancer cells that overexpress HER 2. Preferred growth inhibitory HER2 antibodies inhibit the growth of SK-BR-3 breast tumor cells in cell culture by greater than 20%, preferably greater than 50% (e.g., from about 50% to about 100%) at an antibody concentration of about 0.5-30 μ g/ml, wherein growth inhibition is determined 6 days after exposure of the SK-BR-3 cells to the antibody (see U.S. Pat. No.5,677,171 issued 10/14/1997). 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 murine monoclonal antibody 4D5, such as Trastuzumab.

Antibodies that "induce apoptosis" are those that induce programmed cell death as measured by annexin V binding, DNA fragmentation, cell contraction, endoplasmic reticulum expansion, cell rupture and/or membrane vesicle formation (referred to as apoptotic bodies). The cell is typically a cell that overexpresses the HER2 receptor. Preferably, the cell is a tumor cell, such as a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. In vitro, the cell may be an SK-BR-3, BT474, Calu3 cell, MDA-MB-453, MDA-MB-361 or SKOV3 cell. There are a variety of methods available for assessing cellular events associated with apoptosis. For example, Phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by DNA laddering (laddering); whereas nuclear/chromatin condensation accompanying DNA fragmentation can be assessed by any increase in hypodiploid cells. Preferably, the antibody that induces apoptosis is one that results in an about 2 to 50 fold, preferably about 5 to 50 fold, most preferably about 10 to 50 fold, increase in induction of annexin binding relative to untreated cells in an annexin binding assay using BT474 cells (see below). Examples of HER2 antibodies that induce apoptosis are 7C2 and 7F 3.

"epitope 2C 4" refers to the region in the extracellular domain of HER2 to which antibody 2C4 binds. To screen for Antibodies that bind the epitope of 2C4, a conventional cross-blocking assay can be performed, such as described in Antibodies, Arabidopsis Manual, ColdSpringHarbor laboratory, EdHarlow and DavidLane, 1988. Alternatively, epitope mapping can be performed using methods known in the art to assess whether an antibody binds the 2C4 epitope of HER2, and/or the antibody-HER 2 structure (franklinal, cancer cell 5: 317-328(2004)) can be studied to understand which domain/domains of HER2 an antibody binds. Epitope 2C4 comprises residues from domain II in the extracellular domain of HER 2. 2C4 and Pertuzumab bind the extracellular domain of HER2 at the junction of domains I, II and III. Franklinal, cancer cell 5: 317-328(2004).

"epitope 4D 5" refers to the region of the HER2 ectodomain to which antibodies 4D5 (ATCCRL 10463) and Trastuzumab bind. This epitope is close to the transmembrane domain of HER2 and within domain IV of HER 2. To screen for Antibodies that bind the epitope of 4D5, a conventional cross-blocking assay can be performed, such as described in Antibodies, Arabidopsis Manual, ColdSpringHarbor laboratory, EdHarlow and DavidLane, 1988. Alternatively, epitope mapping can be performed to assess whether an antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues within the region from about residue 529 to about residue 625; see FIG. 1).

"epitope 7C2/7F 3" refers to the region 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. To screen for Antibodies that bind the epitope 7C2/7F3, a conventional cross-blocking assay can be performed, such as described in Antibodies, Arabidopsis, laboratory, ColdSpringHarbor laboratory, EdHarlow and DavidLane, 1988. Alternatively, epitope mapping can be performed to determine whether an antibody binds to the 7C2/7F3 epitope on HER2 (e.g., any one or more residues within the region of about residue 22 to about residue 53 of HER2 in figure 1).

"treatment" refers to both therapeutic treatment and prophylactic measures. Subjects in need of treatment include subjects already suffering from the disease as well as subjects in whom the disease is to be prevented. Thus, a patient to be treated herein may have been diagnosed as having a disease or may have a predisposition to or susceptibility to developing a disease.

The terms "cancer" and "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, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumor (including carcinoid tumor, gastrinoma and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, biliary tract tumor, and head and neck cancer.

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

A "HER 2 positive cancer" is a cancer comprising cells that have HER2 protein present on their cell surface.

A cancer that "overexpresses" a HER receptor is one that has significantly higher levels of HER receptor, such as HER2, at its cell surface as compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or increased transcription or translation. Overexpression of a HER receptor can be determined in a diagnostic or prognostic assay by assessing an increase in the level of HER protein present on the surface of a cell (e.g., by immunohistochemistry assay, IHC). Alternatively, or in addition, the level of HER-encoding nucleic acid in the cell may be measured, for example, by fluorescence in situ hybridization (FISH; see WO98/45479 published 10.1998), Southern blotting or Polymerase Chain Reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR). HER receptor overexpression may also be studied by measuring shed antigens (e.g., HER extracellular domain) in biological fluids such as serum (see, e.g., U.S. Pat. No. 4,933,294 issued at 6/12 th 1990; WO91/05264 issued at 4/18 th 1991; U.S. Pat. No.5,401,638 issued at 28 rd 3 th 1995; and Siasetal, J.Immunol.Methods 132: 73-80 (1990)). In addition to the above assays, the skilled artisan can also utilize a variety of in vivo assays. For example, cells in the patient may be exposed to an antibody optionally labeled with a detectable label, such as a radioisotope, and binding of the antibody to cells in the patient may be assessed, for example, by externally scanning for radioactivity or by analyzing a biopsy taken from a patient that has been previously exposed to the antibody.

In contrast, a cancer that "does not overexpress the HER2 receptor" is one that does not express higher than normal levels of HER2 receptor as compared to a non-cancerous cell of the same tissue type.

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

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. The term is intended to include radioisotopes (e.g., At)²¹¹、I¹³¹、I¹²⁵、y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、p³²And radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and/or variants thereof.

"chemotherapeutic agent" refers to a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamideAlkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzotepa (benzodopa), carboquone (carboquone), metotepipa (meturedpa), and uredepa (uredpa); ethyleneimine and methylmelamine including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethlamelamine; annonaceous acetogenins (especially bullatacin and bullatacin); -9-tetrahydrocannabinol (dronabinol),) β -lapachone (lapachone), lapachol (lapachol), colchicines (colchicine), betulinic acid (betulinic acid), camptothecin (camptothecin) (including the synthetic analogue topotecan)CPT-11 (irinotecan),) Acetyl camptothecin, scopolectin (scopolectin) and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); podophyllotoxin (podophylotoxin); podophyllinic acid (podophylinaci)d) (ii) a Teniposide (teniposide); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin (dolastatin); duocarmycins (including the synthetic analogs KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin (spongistatin); nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chloramphahazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine hydrochloride), melphalan (melphalan), neomustard (novembichin), benzene mustard (phenyleneterenine), prednimustine, trofosfamide (trofosfamide), uracil mustard (uracilmustard); nitroureas such as carmustine (carmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ranimustine (ranimustine); antibiotics, such as enediynes (enediyne) antibiotics (e.g., calicheamicin, especially calicheamicin γ 1I and calicheamicin ω I1 (see, e.g., Agnew, chem. Intl. Ed. Engl. 33: 183-) (1994)); anthracycline (kinemicin) antibiotics, including kinemicin a; epothilones (esperamicins); and neocarcinostatin (neomycin) chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin (acarinomycin), actinomycin (actinomycin), anthranilic (authramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), carbacetin, carminomycin (carminomycin), carcinomycin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditorexin (detortuicin), 6-diaza-5-oxo-L-norleucine, doxorubicin (doxorubicin) (including.Morpholino doxorubicin, cyano morpholino doxorubicin, 2-pyrrol doxorubicin and doxorubicin hydrochloride liposome injectionLiposomal doxorubicin TLCD-99PEGylated liposomal doxorubicinAnd doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcellomycin), mitomycin (mitomycin) such as mitomycin C, mycophenolic acid (mycophenolic acid), nogomycin (nogalamycin), olivomycin (olivomycin), pelomycin (peplomycin), potfiromycin, puromycin (puromycin), griseofulvin (quelamycin), rodobicin (rodorubicin), streptonigrin (streptanigrin), streptozotocin (streptazocin), tubercidin (tubicin), ubenimex (ubenimex), purified stastin (zinostat), zorubicin (zorubicin); antimetabolites, such as methotrexate, gemcitabine (gemcitabine)Tegafur (tegafur)Capecitabine (capecitabine)epothilone and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate (trimetrexate); purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mantotan (mitotane), trostan (trilostane); folic acid supplements, such asFolic acid; acegulonone (acegultone); an aldophosphamide glycoside (aldophosphamideglycoside); aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); defofamine; dimecorsine (demecolcine); diazaquinone (diaziqutone); edenisol (elfornitine); hydroxypyrazole acetate (ellitiniumacetate); etoglut (etoglucid); gallium nitrate; a hydroxyurea; lentinan (lentinan); lonidamine (lonidamine); maytansinoids (maytansinoids), such as maytansine (maytansinoid) and maytansinol (maytansinol); ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide; procarbazine (procarbazine);polysaccharide complexes (jhsnaral products, Eugene, OR); razoxane (rizoxane); rhizomycin (rhizoxin); sisofilan (sizofiran); germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2, 2', 2 "-trichlorotriethylamine; trichothecenes (trichothecene), especially the T-2 toxin, veracurin a, bacillosporin a (roridina) and serpentinin (anguidine)); urethane (urethan); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); thiotepa; taxols (taxoid), e.g. paclitaxel (paclitaxel)Albumin-engineered nanoparticle dosage form (ABRAXANE) for paclitaxel^TM) And docetaxel (doxetaxel)Chlorambucil (chlorenbucil); 6-sulfurGuanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (oxaliplatin), and carboplatin; vinca alkaloids (vincas) which prevent tubulin polymerization to form microtubules include vinblastine (vinblastine)Vincristine (vincristine)Vindesine (vindesine)And vinorelbine (vinorelbine)Etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); leucovorin (leucovovin); nuantro (novantrone); edatrexate (edatrexate); daunorubicin (daunomycin); aminopterin; ibandronate (ibandronate); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoid acids, such as retinoic acid, including bexarotene (bexarotene)Diphosphonates (bisphosphates), such as clodronate (e.g. clodronate)Or)、etidronateNE-58095, zoledronic acid/zoledronic acid salt/esterAlendronate (alendronate)pamidronateTiludronate (tirudronate)Or risedronate (risedronate)Troxacitabine (1, 3-dioxolane nucleoside cytosine analogues), antisense oligonucleotides, in particular those which inhibit the expression of genes in signaling pathways involving adhesive cell proliferation, such as, for example, PKC- α, Ralf, H-Ras and epidermal growth factor receptor (EGF-R), vaccines, such asVaccines and gene therapy vaccines, e.g.A vaccine,A vaccine anda vaccine; topoisomerase 1 inhibitors (e.g. topoisomerase 1 inhibitors))；rmRHBAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine (perifosine), COX-2 inhibitors (such as celecoxib (celecoxib) or etoricoxib (etoricoxib)), proteosome inhibitors (such as PS 341); bortezomibCCI-779; tipifammi (R11577); orafenaib, ABT 510; bcl-2 inhibitors, such as oblimersensodiumpixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy) and FOLFOX (oxaliplatin)^TM) Abbreviations for treatment regimens combining 5-FU and leucovorin (leucovorin).

The definition also includes anti-hormonal agents that act to modulate or inhibit the effects of hormones on tumors, such as anti-estrogens with mixed agonist/antagonist properties (profiles), including tamoxifen (tamoxifen)4-hydroxy tamoxifen, toremifene (toremifene)Idoxifene (idoxifene), droloxifene (droloxifene), raloxifene (raloxifene)Trioxifene (trioxifene), keoxifene, and Selective Estrogen Receptor Modulators (SERMs), such as SERM 3; pure antiestrogens without agonist properties, such as fulvestrantAnd EM800 (such agents may block Estrogen Receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroid aromatase inhibitors, such as formestane (formestane) and exemestane (exemestane)And non-steroidal aromatase inhibitors such as anastrozole (anastrozole)Letrozole (letrozole)And aminoglutethimide (aminoglutethimide), and other aromatase inhibitors, including vorozole (vorozole)Megestrol acetate (megestrolacete)Fadrozole (fadrozole), imidazole (imidazole); lutenizinghormon releasing hormone agonists, including leuprolideGoserelin (goserelin), buserelin (buserelin) and triptorelin (triptorelin); sex steroids including progestogens such as megestrol acetate (megestrolacetate) and medroxyprogesterone acetate (medroxyprogesterone acetate), estrogens such as diethylstilbestrol (diethylstilbestrol) and bemese's force (premarin), and androgens/retinoid acids such as fluoxymesterone (fluoroxymesterone), all trans-retinoic acid (transretinic acid), and fenretinide (fenretinide); onapristone (onapristone); anti-pregnenones; an estrogen receptor down-regulation modulator (ERD); anti-androgens such as flutamide (flutamide), nilutamide (nilutamide), and bicalutamide (bicalutamide); testolactone (testolactone); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing.

As used herein, the term "EGFR-targeting agent" refers to a therapeutic agent that binds to EGFR and optionally inhibits EGFR activation. Examples of such agents include antibodies and small molecules that bind EGFR. Examples of antibodies that bind EGFR include MAb579 (ATCCRLHB 8506), MAb455 (ATCCRLHB 8507), MAb225 (ATCCRL 8508), MAb528 (ATCCRL 8506)ATCCCRL8509) (see Mendelsohn et al, U.S. patent 4,943,533, and variants thereof, such as chimerization 225(C225 or Cetuximab;) And reconstituted human 225(H225) (see WO96/40210 to ImcloneSestemsInc.; IMC-11F8, fully human EGFR-targeting antibodies (Imclone); antibodies that bind to type II mutant EGFR (see U.S. Pat. No.5,212,290), humanized and chimeric antibodies that bind to EGFR, as described in U.S. Pat. No.5,891,996, and human antibodies that bind to EGFR, such as ABX-EGF or Panitumumab (see WO98/50433 to Abgenix/Amgen), EMD55900 (Stragliototatal. Eur.J.Cancer 32A: 636-640(1996)), EMD7200(matuzumab), humanized EGFR antibodies (EMD/Merkek) that are directed against EGFR and compete for binding to GmTGF- α (see, E35469, WO 35597, WO 354665, α, and chimeric antibodies that bind to EGFR 5527, such as described in PCT WO 43927, WO 35927, WO 35598, WO 435445, WO 4665, WO 25, WO 35599, WO 3546, WO 25, and WO 35599, such as EP, WO 35598, WO 4665, WO 25, WO 3, WO 3,4665, WO 3, WO 25, WO 3, WO 3,4665, WO 3, WO 48, WO 3, WO 3,598,4665, WO 3, WO 3,4665, WO 3, WO 3,598, WO 3, WO 3,598,598, WO 3, WO,genentech/osipharaceuticals); PD183805(CI1033, 2-propenamide, N- [4- [ (3-chloro-4-fluorophenyl) amino group]-7- [3- (4-morpholinyl) propoxy]-6-quinazolinyl]Dihydrochloride, PfizerInc.); ZD1839, gefitinib (IRESSA)^TM4- (3 '-chloro-4' -fluoroanilino) -7-methoxy-6- (3-morpholinopropoxy) quinazoline, AstraZeneca); ZM105180 ((6-amino-4- (3))-methylphenyl-amino) -quinazoline, Zeneca); BIBX-1382(N8- (3-chloro-4-fluoro-phenyl) -N2- (1-methyl-piperidin-4-yl) -pyrimido [5, 4-d]Pyrimidine-2, 8-diamine, boehringer ingelheim); PKI-166((R) -4- [4- [ (1-phenylethyl) amino)]-1H-pyrrolo [2, 3-d]Pyrimidin-6-yl]-phenol); (R) -6- (4-hydroxyphenyl) -4- [ (1-phenylethyl) amino group]-7H-pyrrolo [2, 3-d]Pyrimidines); CL-387785(N- [4- [ (3-bromophenyl) amino)]-6-quinazolinyl]-2-butyneamide (butyrnamide)); EKB-569(N- [4- [ (3-chloro-4-fluorophenyl) amino group]-3-cyano-7-ethoxy-6-quinolinyl]-4- (dimethylamino) -2-butenamide (butenamide), Wyeth); AG1478 (Sugen); AG1571(SU 5271; Sugen); dual EGFR/HER2 tyrosine kinase inhibitors, such as lapatinib (GW572016 or N- [ 3-chloro-4- [ (3-fluorophenyl) methoxy)]Phenyl radical]6[5[ [ 2-methylsulfonyl group ]]Ethyl radical]Amino group]Methyl radical]-2-furyl radical]-4-quinazolinamine (quinazolamine); Glaxo-SmithKline) or cyanoguanidinoquinazoline (cyanoguanidinequizoline) and cyanoamidinoquinazolinamine (cyanoamidinoquinazolinamine) derivatives.

"tyrosine kinase inhibitor" refers to a molecule that inhibits the tyrosine kinase activity of a tyrosine kinase, such as a HER receptor. Examples of such inhibitors include the EGFR-targeting drugs mentioned in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724,714, an oral ErbB2 receptor tyrosine kinase selective inhibitor (PfizeandOSI); dual HER inhibitors that preferentially bind EGFR but inhibit HER2 and EGFR-overexpressing cells, such as EKB-569 (available from Wyeth); lapatinib (GW 572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors, such as canertinib (CI-1033; Pharmacia); raf-1 inhibitors, such as the antisense agent ISIS-5132, available from ISISISISP pharmaceuticals, which inhibits Raf-1 signaling; non-HER targeted TK inhibitors, such as Imatinibmesylate (GLEEVAC), available from Glaxo^TM) (ii) a MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD153035, 4- (3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP59326, CGP60261 and CGP62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2, 3-d]A pyrimidine; curcumin (diferuloylmethane, 4, 5-bis (4-fluoroanilino) -phthalimide); tyrphostins containing nitrothiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. patent 5,804,396); trypostins (U.S. Pat. No.5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis/ScheringAG); pan HER inhibitors such as CI-1033 (Pfizer); affinitac (ISIS 3521; ISIS/Lilly); imatinibmesylate (Gleevec; Novartis); PKI166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/ScheringAG); INC-1C11 (Imclone); cyanoguanidine quinazoline and cyanoamidine quinazolinamine derivatives; or any of the following patent publications: us patent 5,804,396; WO99/09016(American cyanimid); WO98/43960(American cyanamid); WO97/38983 (WarnerLambert); WO99/06378 (WarnerLambert); WO99/06396 (WarnerLambert); WO96/30347(Pfizer, Inc.); WO96/33978 (Zeneca); WO96/3397 (Zeneca); WO96/33980 (Zeneca); and US 2005/0101617.

"anti-angiogenic agent" refers to a compound that blocks or interferes to some extent with vascular development. The anti-angiogenic factor can be, for example, a small molecule or an antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. Preferred anti-angiogenic factors in this context are antibodies that bind to Vascular Endothelial Growth Factor (VEGF), such as Bevacizumab

The term "cytokine" refers to the generic term for a protein released by one cell population that acts on another cell as an intercellular mediator. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; (ii) a relaxin; a prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); a liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian (Mullerian) inhibitory substances; mouse gonadotropin-related peptides; a statin; activin (activin); vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-derived growth factor; transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); osteoinductive factor (osteoinductive factor); interferons such as interferon- α, - β, and- γ; colony Stimulating Factors (CSFs), such as macrophage CSFs (M-CSF), granulocyte-macrophage CSFs (GM-CSF), and granulocyte CSFs (G-CSF); interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

HER2 antibody variant compositions

The present invention is concerned, at least in part, with certain HER2 antibody compositions. Preferably, the HER2 antibody (either or both of the main species HER2 antibody and antibody variants thereof) is an antibody that binds to domain II of HER2, inhibits HER dimerization more effectively than Trastuzumab, and/or binds to the heterodimeric binding site of HER 2. Preferred embodiments of the main class of antibodies herein are antibodies comprising the light and heavy chain variable region amino acid sequences of seq id nos. 3 and 4, and most preferably the light and heavy chain amino acid sequences of seq id nos. 15 and 16 (Pertuzumab).

The compositions herein comprise a main species HER2 antibody that binds to domain II of HER2 and acidic variants thereof, wherein the acidic variants include one, two, or three of glycated variants, disulfide reduced variants, and non-reducible variants. The acidic variants in the composition can include one, two, three, four, or five of a saccharification variant, a deamidation variant, a disulfide reduction variant, a sialylation variant, and a non-reducible variant. Preferably, the total amount of all acidic variants in the composition is less than about 25%. In one embodiment, the glycated variant, the deamidated variant, the disulfide reduced variant, the sialylated variant, and the non-reducible variant comprise at least about 75-80% of the acidic variant in the composition.

The present invention also concerns compositions comprising a main species HER2 antibody and an acidic variant of the main species antibody, wherein the main species HER2 antibody comprises the variable light and variable heavy chain sequences in seq id nos. 3 and 4, and wherein the acidic variant comprises one, two, three, four, or five of a glycated variant, a deamidated variant, a disulfide reduced variant, a sialylated variant, and a non-reducible variant.

The present invention provides a method of preparing a pharmaceutical composition comprising: (1) preparing a composition comprising a main species HER2 antibody that binds domain II of HER2 and acidic variants thereof, including glycated variants, disulfide reduced variants, or non-reducible variants, and (2) evaluating the acidic variants in the composition and determining the amount thereof to be less than about 25%. The method encompasses combining the composition before, during, or after step (2) with a pharmaceutically acceptable carrier. In one embodiment, the composition evaluated in step (2) is in a pharmaceutically acceptable carrier.

In one embodiment, at least about 75-80% of the acidic variants (constituting less than about 25% of the composition) are selected from the group consisting of: glycated variants, deamidated variants, disulfide reduced variants, sialylated variants, and non-reducible variants.

Acidic variants can be assessed by a variety of methods, but preferably the methods include one, two, three, four, or five of the following: ion Exchange Chromatography (IEC), wherein the composition is treated with a sialidase before, after, and/or during IEC (e.g., for assessing sialylated variants); reducing CE-SDS (e.g., for assessing disulfide reducing variants); non-reducing CE-SDS (e.g., for evaluating non-reducible variants); borate chromatography (e.g., for assessing glycated variants); and peptide mapping (e.g., for assessing deamidation variants).

In one embodiment, the total acidic variants are analyzed by ion exchange chromatography, for example using weak cation exchangers and/or cation exchangers with carboxylate functionality (e.g., using a dionex prop wcx-10 chromatography column). In one embodiment of such chromatography, the chromatography conditions involve: the buffer solution A is 20mM Tris, pH6.0; the buffer solution B is 20mM Tris, 200mM NaCl, pH6.0; and a gradient of 1.0mL/min of 0.5% buffer B.

The compositions optionally comprise an amino-terminal leader extension variant. Preferably, the amino-terminal leader extension is located on a light chain of the antibody variant (e.g., on one or both light chains of the antibody variant). The main species HER2 antibody or antibody variant can be an intact antibody or an antibody fragment (e.g. Fab or F (ab')₂Fragments), but preferably both are intact antibodies. The antibody variants herein may comprise an amino-terminal leader extension on any one or more of its heavy or light chains. Preferably, the amino-terminal leader extension is located on one or both light chains of the antibody. The amino-terminal leader extension preferably comprises or consists of VHS-. The presence of the amino-terminal leader extension in the composition can be detected by a variety of analytical techniques, including, but not limited to, N-terminal sequence analysis, assays for charge heterogeneity (e.g., cation exchange chromatography or capillary zone electrophoresis), mass spectrometry, and the like. The amount of antibody variant in the composition will generally range from an amount that constitutes the lower limit of detection of any assay (preferably cation exchange analysis) used to detect the variant to an amount that is less than the amount of the major species of antibody. Typically, about 20% or less (e.g., from about 1% to about 15%, such as from 5% to about 15%, and preferably from about 8% to about 12%) of the antibody molecules in the composition comprise an amino-terminal leader extension. Such percentage amounts are preferably determined by cation exchange analysis.

Other amino acid sequence alterations are contemplated for the main species of antibodies and/or variants, including, but not limited to, antibodies comprising a C-terminal lysine residue on one or both of its heavy chains (such antibody variants may be present in amounts of about 1% to about 20%), antibodies having one or more oxidized methionine residues (e.g., Pertuzumab comprising oxidized met-254), and the like.

Furthermore, in addition to the sialylation variants discussed above, the main species antibody or variant may comprise additional glycosylation variations, non-limiting examples of which include antibodies comprising a G1 or G2 oligosaccharide structure attached to its Fc region, antibodies comprising a carbohydrate moiety attached to its light chain (e.g., one or more carbohydrate moieties such as glucose or galactose attached to one or both light chains of the antibody, e.g., attached to one or more lysine residues), antibodies comprising one or two non-glycosylated heavy chains, and the like.

Optionally, an antibody comprising one or two light chains, wherein either or both of the two light chains comprise the amino acid sequence in seq id No.23 (including variants thereof, such as those disclosed herein). The antibody further comprises one or two heavy chains, wherein either or both of the two heavy chains comprise the amino acid sequence of seq id No.16 or seq id No.24 (including variants thereof, such as those disclosed herein).

The compositions may be recovered from genetically engineered cell lines expressing the HER2 antibody, from, for example, Chinese Hamster Ovary (CHO) cell lines, or may be prepared by peptide synthesis.

Production of HER2 antibodies

Exemplary techniques for generating antibodies for use in accordance with the present invention are described below. The HER2 antigen used to generate the antibody may be, for example, the soluble form of the extracellular domain of HER2 or a portion thereof containing the desired epitope. Alternatively, cells expressing HER2 on their cell surface (e.g., NIH-3T3 cells transformed to overexpress HER 2; or cancer cell lines such as SK-BR-3 cells, see Stancovskiet al, PNAS (USA) 88: 8691-8695(1991)) may be used to generate antibodies. Other forms of HER2 that can be used to generate antibodies will be apparent to those skilled in the art.

(i) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible mutations that may occur during the production of the monoclonal antibody, such as those variants described herein. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

For example, monoclonal antibodies can be produced by a method originally described by kohlereteal, Nature 256: 495(1975), or can be prepared by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, monoclonal antibodies: PriniplsasanPractice, 59-103, academic Press, 1986).

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

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these cells, preferred myeloma cell lines are murine derived myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstituteCellDistributionCenter (SanDiege, California, USA), and SP-2 or X63-Ag8-653 cells available from American TypeCultureCollection (Rockville, Maryland, USA). Human myeloma and mouse-human heteromyeloma cell lines for generating human monoclonal antibodies have also been described (Kozbor, J.Immunol.133: 3001 (1984); and Brodeur et al, monoclonal antibody production techniques and applications, 51-63, Marcel Dekker, Inc., NewYork, 1987).

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

The binding affinity of a monoclonal antibody can be determined, for example, by the methods of munson et al, anal. biochem.107: 220(1980) by Scatchard analysis.

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

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

DNA encoding the monoclonal antibody is readily isolated and sequenced by conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells are preferred sources of the DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell, such as an e.coli cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell that does not otherwise produce antibody proteins, to obtain synthesis of monoclonal antibodies in the recombinant host cell. A review article on recombinant expression of DNA encoding antibodies in bacteria includes skerraet, curr. 256-charge 262(1993) and Pl ü ckthun, Immunol. Revs.130: 151-188(1992).

In another embodiment, the method may be selected from the group consisting of using mccaffeterteytal, Nature 348: 552 (1990) and isolating monoclonal antibodies or antibody fragments from phage antibody libraries. Clacksonetal, Nature 352: 624-: 581-597(1991) describes the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the generation of high affinity (nM range) human antibodies by chain shuffling (Marksetal, Bio/Technology 10: 779-. These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

The DNA may also be modified, for example, by replacing the homologous murine sequences with the coding sequences for the human heavy and light chain constant regions (U.S. Pat. No. 4,816,567; and Morrisonetal, Proc. Natl. Acad. Sci. USA 81: 6851(1984)), or by covalently joining the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide.

Typically, such non-immunoglobulin polypeptides are substituted for the constant regions of an antibody, or they are substituted for the variable regions of one antigen-binding site of an antibody, to produce a chimeric bivalent antibody comprising one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for a different antigen.

(ii) Humanized antibodies

Methods for humanizing non-human antibodies have been described in the art. Preferably, the humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable region. Humanization can be carried out essentially according to the method of Winter and co-workers (Joneset al, Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323-327 (1988); Verhoeyenet et al, Science 239: 1534-1536(1988)), by replacing the corresponding human antibody sequences with hypervariable region sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which essentially less than the entire human variable region is replaced with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

The choice of human variable regions, including light and heavy chains, used to make humanized antibodies is important for reducing antigenicity. The entire library of known human variable region sequences was screened for rodent antibody variable region sequences according to the so-called "best-fit" method. The closest human sequence to rodents is then selected as the human Framework Region (FR) of the humanized antibody (Simsetal., J.Immunol.151: 2296 (1993); Chothiacetal., J.mol.biol.196: 901 (1987)). Another approach uses specific framework regions derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carteretal, Proc. Natl. Acad. Sci. USA 89: 4285 (1992); Prestaetal, J. Immunol.151: 2623 (1993)).

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

U.S. patent No.6,949,245 describes the generation of exemplary humanized HER2 antibodies that bind to HER2 and block ligand activation of the HER receptor. Humanized antibodies of particular interest herein block EGF, TGF- α and/or HRG mediated MAPK activation substantially as effectively as the intact murine monoclonal antibody 2C4 (or Fab fragment thereof) and/or bind HER2 substantially as effectively as the intact murine monoclonal antibody 2C4 (or Fab fragment thereof). The humanized antibodies herein may, for example, comprise non-human hypervariable region residues which are incorporated into the human heavy chain variable region, and may further comprise a Framework Region (FR) substitution at a position selected from the group consisting of 69H, 71H and 73H, using the variable region numbering system set forth in Kabat et al, sequence of proteins of immunological interest, 5th edition, public health service, national institutes of health, Bethesda, MD, 1991. In one embodiment, the humanized antibody comprises FR substitutions at two or all positions 69H, 71H and 73H.

The exemplary humanized antibody of interest herein comprises the heavy chain variable region complementarity determining residue GFTFTDYTMX, wherein X is preferably D or S (SEQ ID NO: 7); DVNPNSGGSIYNQRFKG (SEQ ID NO: 8); and/or NLGPSFYFDY (seq id no: 9), optionally comprising amino acid modifications of those CDR residues, e.g., wherein the modifications substantially maintain or improve the affinity of the antibody. For example, an antibody variant of interest may have from about 1 to about 7 or about 5 amino acid substitutions in the heavy chain variable region CDR sequences described above. Such antibody variants can be prepared by affinity maturation, e.g., as described below. Most preferred humanized antibodies comprise seq id no: 4, and (b) the heavy chain variable region amino acid sequence of seq id No. 4.

For example, in addition to those heavy chain variable region CDR residues in the preceding paragraph, the humanized antibody may comprise light chain variable region complementarity determining residue KASQDVSIGVA (SEQ ID NO: 10); SASYX¹X²X³Wherein X is¹Preferably R or L, X²Preferably Y or E, and X³Preferably T or S (SEQ ID NO: 11); and/or QQYYIYPYT (SEQ ID NO: 12). Such humanized antibodies optionally comprise amino acid modifications of the CDR residues described above, e.g., wherein the modifications substantially maintain or improve the affinity of the antibody. For example, an antibody variant of interest may have from about 1 to about 7 or about 5 amino acid substitutions in the light chain variable region CDR sequences described above. Such antibody variants can be prepared by affinity maturation, e.g., as described below. Most preferred humanized antibodies comprise seq id no: 3, and a light chain variable region amino acid sequence.

Affinity matured antibodies that bind to HER2 and block ligand activation of HER receptors are also contemplated by the present application. The parent antibody may be a human or humanized antibody, e.g. comprising light and/or heavy chain variable region sequences of seq id no: 3 and 4 (i.e., variant 574). The affinity matured antibody preferably binds HER2 receptor with a higher affinity than the intact murine 2C4 or intact variant 574 (e.g., an increase in affinity of about 2-fold or about 4-fold to about 100-fold or about 1000-fold as assessed by HER2 extracellular domain (ECD) ELISA). Exemplary heavy chain variable region CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or a combination of two or more (e.g., 2,3, 4,5, 6, or 7 of these residues). Examples of CDR residues of the light chain variable region for alteration include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97, or a combination of two or more (e.g., 2 to 3,4, 5, or up to about 10 of these residues).

Various forms of humanized antibodies or affinity matured antibodies are also contemplated. For example, the humanized or affinity matured antibody may be an antibody fragment, such as a Fab, which is optionally conjugated to one or more cytotoxic agents to produce an immunoconjugate. Alternatively, the humanized antibody or affinity matured antibody may be an intact antibody, such as an intact IgG1 antibody.

(iii) Human antibodies

As an alternative to humanization, human antibodies can be generated. For example, it is now possible to generate transgenic animals (e.g., mice) that are capable of generating a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of large numbers of human germline immunoglobulin genes in such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., jakobovitset, proc.natl.acad.sci.usa90: 2551 (1993); jakobovitset, Nature 362: 255-258 (1993); bruggermannetal, yearin immune.7: 33 (1993); and U.S. Pat. Nos. 5,591,669, 5,89,369 and 5,545,807.

Alternatively, phage display technology (McCaffertytal, Nature 348: 552-553(1990)) can be used to generate human antibodies and antibody fragments in vitro from a repertoire of immunoglobulin variable (V) region genes from an unimmunized donor. According to this technique, antibody V region genes are cloned in-frame to the major or minor coat protein genes of filamentous phage such as M13 or fd and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle comprises a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody displaying those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for reviews see, e.g., Johnson, kevin s.andchiswell, davidj, currentopinionstructurals biology 3: 564-571(1993). Several sources of V gene segments are available for phage display. Clacksonetal, Nature 352: 624-628(1991) a large number of different anti-oxazolone antibodies were isolated from a random combinatorial library of small V genes derived from the spleen of immunized mice. May be substantially in accordance with marksetal, j.mol.biol.222: 581-597(1991) or Griffithal, EMBOJ.12: 725-734(1993) construct a V gene repertoire from non-immunized human donors and isolate antibodies against a number of different antigens, including self-antigens. See also U.S. Pat. nos. 5,565,332 and 5,573,905.

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

Human HER2 antibodies are described in U.S. Pat. No.5,772,997 issued at 30.6.1998 and in WO97/00271 issued at 3.1.1997.

(iv) Antibody fragments

Various techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoetoet, journal of Biochemical and physical methods 24: 107-117(1992) and Brennaet, Science 229: 81 (1985)). However, these fragments can now be produced directly from recombinant host cells. For example, antibody fragments can be isolated from phage antibody libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and coupled by chemical means to form F (ab')₂Fragments (Carteretal, Bio/Technology 10: 163-. According to another method, F (ab') can be isolated directly from recombinant host cell cultures₂And (3) fragment. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; us patent 5,571,894; and U.S. patent 5,587,458. The antibody fragment may also be a "linear antibody," e.g., as described in U.S. Pat. No.5,641,870. The linear antibody fragment may be monospecific or bispecific.

(v) Bispecific antibodies

Bispecific antibodies refer to antibodies having binding specificity for at least two different epitopes. Exemplary bispecific antibodies can bind to two different species of HER2 proteinOther such antibodies may combine a HER2 binding site with the binding site of EGFR, HER3, and/or HER4 alternatively, the HER2 arm may be combined with an arm that binds a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2 or CD3) or an Fc receptor of IgG (fcyr), such as fcyri (CD64), fcyrii (CD32), and fcyriii (CD16), such that the cellular defense mechanism is focused on HER2 expressing cells₂Bispecific antibodies).

WO96/16673 describes a bispecific HER 2/FcyRIII antibody, while U.S. Pat. No.5,837,234 discloses a bispecific HER 2/FcyRI antibody IDM1 (Osidem). WO98/02463 shows a bispecific HER2/Fc α antibody. U.S. Pat. No.5,821,337 teaches a bispecific HER2/CD3 antibody. MDX-210 is a bispecific HER2-Fc γ RIII antibody.

Methods for making bispecific antibodies are known in the art. The traditional generation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millsteineteal, Nature 305: 537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is rather cumbersome and the product yield is low. WO93/08829 and Traveckereral, EMBOJ.10: 3655-3659(1991) disclose a similar procedure.

According to a different approach, an antibody variable region with the desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant region sequence. Preferably, the fusion uses an immunoglobulin heavy chain constant region comprising at least part of the hinge, CH2, and CH3 regions. Preferably, there is a first heavy chain constant region (CH1) in at least one of the fusions that includes the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields, this provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, where expression of at least two polypeptide chains in the same ratio results in high yields or where the ratio is of no particular significance, it is possible to insert the coding sequences for two or all three polypeptide chains into the same vector.

In a preferred embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. Since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient way of isolation, it was found that this asymmetric structure facilitates the separation of the desired bispecific complex from the undesired immunoglobulin chain combinations. This method is disclosed in WO 94/04690. For additional details on the generation of bispecific antibodies see, e.g., surehetal, methods enzymology 121: 210(1986).

According to another approach described in U.S. Pat. No.5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise at least part of C_H3 antibody constant region. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size for large side chains are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody of the heterologous conjugate may be conjugated to avidin and the other antibody to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treating HIV infection (WO91/00360, WO92/200373, and EP 03089). Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of crosslinking techniques.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennanetal, Science 229: 81(1985) describes the proteolytic cleavage of intact antibodies to F (ab')₂A method for fragmenting. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reverted back to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immobilization reagents for enzymes.

Recent advances have made it easier to recover Fab' -SH fragments directly from E.coli, which can be chemically coupled to form bispecific antibodies. Shalabyetal, j.exp.med.175: 217-225(1992) describes the generation of fully humanized bispecific antibodies F (ab')₂A molecule. Each Fab' fragment was separately secreted by E.coli and subjected to directed chemical coupling in vitro to form bispecific antibodies. The bispecific antibody so formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Also described is the direct preparation and isolation of the bis from recombinant cell culturesVarious techniques for specific antibody fragments. For example, bispecific antibodies have been generated using leucine zippers. Kostelnyetal, j.immunol.148 (5): 1547-1553(1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. Prepared from hollingerial, proc.natl.acad.sci.usa 90: 6444-. The fragment comprises light chain variable regions (V) linked by linkers_L) And heavy chain variable region (V)_H) The linker is too short to allow pairing between the two domains on the same strand. Thus, V on a segment is forced_HAnd V_LDomain and complementary V on another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single chain fv (sFv) dimers has also been reported. See grubbertal, j.immunol.152: 5368(1994).

Antibodies with more than two titers are contemplated. For example, trispecific antibodies can be prepared. Tuttetal, j.immunol.147: 60(1991).

(vi) Other amino acid sequence modifications

Amino acid sequence modifications of the HER2 antibody described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of the HER2 antibody are prepared by introducing appropriate nucleotide changes into the HER2 antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues in the amino acid sequence of the HER2 antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired properties. Amino acid changes may also alter post-translational processing of the HER2 antibody, such as changing the number or position of glycosylation sites.

One method that can be used to identify certain residues or regions of HER2 antibody that are preferred mutagenesis positions is referred to as "alanine scanning mutagenesis", e.g. CunninghamandWells, Science 244: 1081-1085 (1989). Here, one or a group of target residues (e.g. charged residues such as arginine, aspartic acid, histidine, lysine and glutamic acid) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the HER2 antigen. Those amino acid positions that show functional sensitivity to substitution are then refined by introducing more or other variants at or for the substitution site. Thus, although the site of introduction of an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the consequences of a mutation at a specified site, alanine scanning mutagenesis or random mutagenesis is performed at the target codon or region and the expressed HER2 antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the HER2 antibody with an N-terminal methionyl residue or an antibody fused to a cytotoxic polypeptide. Other insertional variants of the HER2 antibody molecule include fusion enzymes at the N-or C-terminus of the HER2 antibody (e.g. for ADEPT) or polypeptides that increase the serum half-life of the antibody.

Another class of variants are amino acid substitution variants. These variants have at least one amino acid residue in the HER2 antibody molecule replaced with a different residue. Sites of most interest for substitutional mutagenesis include the hypervariable regions or CDRs, but FR or Fc region alterations 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, more substantial changes, referred to as "exemplary substitutions" in Table 1, or as described further below with respect to amino acid species, can be introduced and the products screened.

TABLE 1

Original residues	Example alternatives	Preferred alternatives
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp；Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine	Leu
			Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; leu; met; phe; ala; norleucine	Leu

Substantial modification of antibody biological properties is accomplished by selecting substitutions that differ significantly in their effectiveness in maintaining: (a) the structure of the polypeptide backbone of the surrogate region, e.g., as a folded sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Amino acids can be grouped according to their similarity in side chain properties as follows (A.L. Lehninger, in Biochemistry, 2 nd edition, 73-75, Worthpublishers, New York, 1975): (1) non-polar: 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), Gln (Q)

(3) Acidic: asp (D), Glu (E)

(4) Basic: lys (K), Arg (R), His (H)

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

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

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

(3) acidic: asp and Glu;

(4) basic: his, Lys, Arg;

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

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions will require the exchange of one member of one of these classes for another.

Any cysteine residue not involved in maintaining the correct conformation of the HER2 antibody may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred class of substitutional variants involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were produced. One convenient method of generating such surrogate variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., e6-7 sites) were mutated to generate all possible amino acid substitutions at each site. The antibody variants so produced are displayed in monovalent form on filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues which contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and human HER 2. The contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

Another class of amino acid variants of antibodies alters the original glycosylation pattern of the antibody. An alteration means the deletion of one or more carbohydrate moieties found in the antibody, and/or the addition of one or more glycosylation sites not present in the antibody.

Glycosylation of antibodies is usually either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate module to an asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

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

If the antibody comprises an Fc region, the structure of any oligosaccharides attached thereto may be altered. For example, antibodies having a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody are described in U.S. patent application No. US2003/0157108A1(Presta, L.). See also US2004/0093621a1 (kyowahakkokokgyoco, Ltd.). Antibodies having an aliquot of N-acetylglucosamine (GlcNAc) in the oligosaccharide structure attached to the Fc region of the antibody are mentioned in WO03/011878(Jean-Mairet et al) and U.S. Pat. No.6,602,684 (Umana et al). Antibodies having at least one galactose residue in the oligosaccharide structure attached to the Fc region of the antibody are reported in WO97/30087(Patel et al). For antibodies with altered carbohydrate attachment to their Fc region see also WO98/58964(Raju, S.) and WO99/22764(Raju, S.). Also contemplated herein are antibody compositions comprising a major species of antibody having such carbohydrate structures attached to one or both heavy chains of an Fc region

Nucleic acid molecules encoding amino acid sequence variants of the HER2 antibody can be prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants), or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or non-variant version of HER2 antibody.

(vii) Screening for antibodies with desired Properties

Techniques for generating antibodies have been described above. Antibodies having certain biological characteristics may also be selected, if desired.

To identify an antibody that blocks ligand activation of a HER receptor, the ability of the antibody to block HER ligand binding to a cell expressing a HER receptor (e.g., coupled to another HER receptor that forms a HER heteromer with the HER receptor of interest) can be determined. For example, cells that naturally express or are transfected to express HER receptors of HER hetero-oligomers may be incubated with antibodies and then exposed to labeled HER ligands. The HER2 antibody can then be evaluated for its ability to block ligand binding to HER receptors in HER heteromers.

For example, inhibition of HRG binding to MCF7 breast tumor cell lines by HER2 antibody can be performed on ice using a monolayer of MCF7 culture in a 24-well plate format essentially as described in WO 01/00245. The HER2 monoclonal antibody can be added to individual wells andincubate for 30 minutes. Then can be added¹²⁵I-labelled rHRG β 1_177-224(25pm) and incubation can be continued for 4-16 hours. Dose-response curves can be plotted and the IC of the antibody of interest can be calculated₅₀The value is obtained. In one embodiment, an antibody that blocks ligand activation of HER receptor inhibits the IC of HRG binding to MCF7 cells in this assay₅₀About 50nM or less, more preferably 10nM or less. IC for inhibiting HRG binding to MCF7 cells in this assay when the antibody is an antibody fragment such as a Fab fragment₅₀May be, for example, about 100nM or less, more preferably 50nM or less.

Alternatively, or in addition, the ability of the HER2 antibody to block HER ligand-stimulated tyrosine phosphorylation of HER receptors present in HER heteromers can be assessed. For example, cells endogenously expressing or transfected to express HER receptor can be incubated with the antibody and then HER ligand-dependent tyrosine phosphorylation activity measured using an anti-phosphotyrosine monoclonal, optionally conjugated with a detectable label. The kinase receptor activation assay described in us patent 5,766,863 can also be used to determine HER receptor activation and blocking of this activity by antibodies.

In one embodiment, antibodies that inhibit HRG-stimulated p180 tyrosine phosphorylation in MCF7 cells can be screened essentially as described in WO01/00245, for example, MCF7 cells can be plated in 24-well plates and a monoclonal antibody to HER2 can be added to each well, incubated at room temperature for 30 minutes, and then rHRG β 1 can be added to each well_177-244To a final concentration of 0.2nM and incubation can be continued for 8 minutes. The medium was aspirated from each well and the reaction was stopped by adding 100. mu.l of SDS sample buffer (5% SDS, 25mM DTT, 25mM Tris-HCl, pH 6.8). Each sample (25. mu.l) can be electrophoresed on a 4-12% gradient gel (Novex) and then transferred to a polyvinylidene fluoride membrane by electrophoresis. Immunoblots of anti-phosphotyrosine (at 1. mu.g/ml) were developed and the intensity of the major reactive band with an Mr of about 180,000 was quantified by reflection densitometry. In this assay, the selected antibody preferably significantly inhibits HRG-stimulated p180 tyrosine phosphorylation to about 0-35% of the control. Can be measured by reflection density methodDetermining the dose-response curve for inhibition of HRG-stimulated p180 tyrosine phosphorylation and calculating the IC of the antibody of interest₅₀. In one embodiment, an antibody that blocks ligand activation of HER receptor inhibits HRG-stimulated IC of p180 tyrosine phosphorylation in this assay₅₀About 50nM or less, more preferably 10nM or less. IC for inhibiting HRG stimulation of p180 tyrosine phosphorylation in this assay when the antibody is an antibody fragment such as a Fab fragment₅₀May be, for example, about 100nM or less, more preferably 50nM or less.

The growth inhibitory effect of the antibodies on MDA-MB-175 cells can also be assessed, for example, essentially as described in schaeferet, Oncogene 15: 1385-1394 (1997). According to this assay, MDA-MB-175 cells may be treated with HER2 monoclonal antibody (10. mu.g/mL) for 4 days and stained with crystal violet. Incubation with the HER2 antibody may show similar growth inhibitory effects on this cell line as with monoclonal antibody 2C 4. In yet another embodiment, exogenous HRG does not significantly reverse this inhibitory effect. Preferably, the antibody will be capable of inhibiting cellular proliferation of MDA-MB-175 cells to a greater extent than monoclonal antibody 4D5 (and optionally to a greater extent than monoclonal antibody 7F 3), regardless of the presence or absence of exogenous HRG.

In one embodiment, the HER2 antibody of interest can block heregulin-dependent HER2 binding to HER3 in MCF7 and SK-BR-3 cells substantially more potent than monoclonal antibody 4D5, and preferably substantially more potent than monoclonal antibody 7F3, as determined by co-immunoprecipitation experiments, such as described in WO 01/00245.

To identify growth inhibitory HER2 antibodies, antibodies can be screened that inhibit the growth of cancer cells that overexpress HER 2. In one embodiment, the growth inhibitory antibody is selected to inhibit the growth of SK-BR-3 cells in cell culture by about 20-100%, preferably about 50-100%, at an antibody concentration of about 0.5-30 μ g/ml. To identify such antibodies, the SK-BR-3 assay described in U.S. Pat. No.5,677,171 can be performed. According to this assay, 1: 1 in DMEM medium supplemented with 10% fetal bovine serum, glutamine and penicillin streptomycin1 culturing SK-BR-3 cells in the mixed solution. SK-BR-3 cells were plated in 35mm cell culture dishes (2ml/35mm dish) with 20,000 cells. Each dish was loaded with 0.5-30. mu.g/ml HER2 antibody. After 6 days, the electronic COULTER was used^TMThe cell counter counts the number of cells compared to untreated cells. Those 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. For assays used to screen for growth inhibitory antibodies, such as 4D5 and 3E8, see U.S. patent 5,677,171.

To select antibodies that induce apoptosis, an annexin binding assay using BT474 cells can be used. BT474 cells were cultured and seeded in petri dishes as discussed in the previous paragraph. The medium was then removed and replaced with fresh medium alone or medium containing 10. mu.g/ml monoclonal antibody. After a 3 day incubation period, the cell monolayer was washed with PBS and detached by trypsinization. The cells were then centrifuged and resuspended in Ca as discussed above for the cell death assay²⁺Combined in buffer and aliquoted into tubes. Labelled annexin (e.g.annexin V-FTIC) (1. mu.g/ml) was then added to the tubes. The FACSCAN can be used^TMFlow cytometer and FACSCOPERT^TMCellQuest software (Becton Dickinson) was used to analyze the samples. Those antibodies that induced a statistically significant level of annexin binding relative to the control were selected as apoptosis-inducing antibodies. In addition to the annexin binding assay, a DNA staining assay using BT474 cells can also be utilized. To perform this assay, BT474 cells, which had been treated with the antibody of interest as described in the two preceding paragraphs, were incubated with 9. mu.g/ml HOECHST33342^TMIncubation at 37 ℃ for 2 hours followed by MODFILT^TMSoftware (VeritySoftware House) at EPICSELITE^TMAnalysis was performed on a flow cytometer (Coulter corporation). Antibodies that induce a 2-fold or greater (preferably 3-fold or greater) change in the percentage of apoptotic cells in this assay as compared to untreated cells (up to 100% apoptotic cells) may be selected as pro-apoptotic antibodies. Assays for screening for antibodies that induce apoptosis, such as 7C2 and 7F3, are described in WO 98/17797.

To screen for Antibodies that bind to an epitope bound by an antibody of interest on HER2, a conventional cross-blocking assay, such as that described in Antibodies, acquired manual, cold spring harbor laboratory, EdHarlow and DavidLane, 1988, can be performed to assess whether the Antibodies cross-block the binding of an antibody, such as 2C4 or Pertuzumab, to HER 2. Alternatively, or in addition, epitope mapping can be performed by methods known in the art, and/or the antibody-HER 2 structure (Franklinetal, cancer cell 5: 317-328(2004)) can be studied to understand which domain/domains of HER2 the antibody binds.

(viii) Immunoconjugates

The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a toxin (e.g., a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof), or a radioisotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Conjugates of the antibody with one or more small molecule toxins, such as calicheamicin, maytansine (U.S. Pat. No.5,208,020), trichothecene, and CC1065 are also contemplated herein.

In a preferred embodiment of the invention, the antibody is conjugated to one or more maytansine molecules (e.g., about 1 to about 10 maytansine molecules per antibody molecule). For example, maytansine can be converted to May-SS-Me, which can be reduced to May-SH3 and reacted with a modified antibody (Charieral., cancer research 52: 127-131(1992)) to produce a maytansinoid-antibody immunoconjugate.

Another immunoconjugate of interest comprises an HER2 antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Structural analogs of calicheamicin that can be used include, but are not limited to, gamma₁ ^I、α₂ ^I、α₃ ^IN-acetyl-gamma₁ ^IPSAG and θ^I ₁(Hinmanet., cancer research 53: 3336-. See also U.S. Pat. nos. 5,714,586; 5,712,374; 5,264,586, respectively; and 5,773,001, expressly incorporated herein by reference.

Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii toxalbumin, Dianthus caryophyllus toxicoprotein, Phytolacca americana toxoprotein (PAPI, PAPII and PAP-S), Momordica charantia (momordia) inhibitor, Jatropha curcin (curcin), crotin (crotin), Saponaria officinalis (saparofia ficilalis) inhibitor, gelonin (gelonin), lincomycin (mitogellin), tricin (triphenomycin), crotin (triphenomycin), trichothecin (triomycin), and trichothecin (enomycin). See, for example, WO93/21232, published on month 10 and 28, 1993.

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

There are a variety of radioisotopes available for use in the generation of radioconjugated HER2 antibody. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²And radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein-coupling agents, such as N-succinimidyl 3- (2-pyridyldithiol) propionate (SPDP), succinimidyl 4- (N-maleimidomethyl) cyclohexylamine-1-carboxylate, Iminothiolane (IT), imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), bifunctional derivatives of aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-nitrogen derivatives (such as bis (p-azidobenzoyl) hexanediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, the methods can be described as Vitettaetaetaetaetatal, Science 238: 1098(1987) ricin immunotoxin was prepared as described in (1098). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, dimethyl linkers, or disulfide-containing linkers can be used (Charieral., cancer research 52: 127-.

Alternatively, a fusion protein comprising the HER2 antibody and a cytotoxic agent may be prepared, e.g. by recombinant techniques or peptide synthesis.

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

(ix) Other antibody modifications

Other modifications of the antibodies are also contemplated herein. For example, the antibody can be linked to one of a variety of non-proteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol. The antibody may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's pharmaceutical sciences, 16 th edition, Osol, A. eds, 1980.

It may be desirable to modify an antibody of the invention with respect to effector function, e.g., to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or in addition, cysteine residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody so produced may have improved internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See caronet, j.exp.med.176: 1191-1195(1992) and shop, B., J.Immunol.148: 2918-2922(1992). Homodimeric antibodies with enhanced anti-tumor activity can also be used as described in wolfffetal, cancer research 53: 2560, 2565 (1993). Alternatively, the antibody may be engineered to have dual Fc regions, and thus may have enhanced complement lysis and ADCC capabilities. See stevensonetal, Anti-cancer great design 3: 219-230(1989).

WO00/42072(Presta, L.) describes antibodies with improved ADCC function in the presence of human effector cells, wherein the antibodies comprise amino acid substitutions in their Fc region. Preferably, the antibody with improved ADCC comprises a substitution at position 298, 333 and/or 334 of the Fc region. Preferably, the altered Fc region is a human IgG1Fc region comprising, replacing, or consisting of one, two, or three of these positions.

Antibodies with altered C1q binding and/or Complement Dependent Cytotoxicity (CDC) are described in WO99/51642, U.S. patent 6,194,551B1, U.S. patent 6,242,195B1, U.S. patent 6,528,624B1, and U.S. patent 6,538,124 (iduogie et al). These antibodies comprise amino acid substitutions at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333 and/or 334 of their Fc region.

To increase the serum half-life of the antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly in U.S. Pat. No.5,739,277, for example)Which is an antibody fragment). As used herein, the term "salvage receptor binding epitope" refers to an IgG molecule (e.g., IgG)₁、IgG₂、IgG₃Or IgG₄) The Fc region is the epitope responsible for increasing the serum half-life of the IgG molecule in vivo. Antibodies with substitutions in their Fc region and increased serum half-life are also described in WO00/42072(Presta, L.).

Engineered antibodies having three or more (preferably four) functional antigen binding sites are also contemplated (U.S. patent application US2002/0004587A1, Miller et al).

The HER2 antibodies disclosed herein can also be formulated as liposomes. Antibody-containing liposomes can be prepared by methods known in the art, such as epsteinetinal, proc.natl.acad.sci.usa82: 3688 (1985); hwang et al, proc.natl.acad.sci.usa77: 4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published 1997, month 10 and 23. Liposomes with extended circulation time are disclosed in U.S. patent No.5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter having a set pore size to produce liposomes having the desired diameter. As can be seen in martineteal, j.biol.chem.257: 286-288(1982), the Fab' fragments of the antibodies of the invention were coupled to liposomes via a disulfide exchange reaction. Optionally, a chemotherapeutic agent is included in the liposomes. See gabizonet, j.national cancer inst.81 (19): 1484(1989).

Pharmaceutical formulations

Therapeutic formulations of the compositions of the present invention are prepared by mixing the compositions with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's pharmaceutical sciences, 16 th edition, Osol, eds., 1980) and storing in lyophilized formulations or as aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, lemon, and the likeAcid salts and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG). Lyophilized HER2 antibody formulations are described in WO 97/04801. Particularly preferred formulations for the composition of the invention are described in US 20006/088523.

The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those compounds whose activities are complementary and do not adversely affect each other. For example, it may also be desirable to provide antibodies that bind EGFR, HER2 (e.g., an antibody that binds a different epitope on HER 2), HER3, HER4, or Vascular Endothelial Growth Factor (VEGF) in the formulation. Alternatively or additionally, chemotherapeutic agents, cytotoxic agents, cytokines, growth inhibitory agents, anti-hormonal agents, EGFR-targeting drugs, anti-angiogenic agents, tyrosine kinase inhibitors and/or cardioprotective agents may also be included in the formulations. Such molecules are suitably present in combination in amounts effective for the desired purpose.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's pharmaceutical sciences, 16 th edition, Osol, A. eds, 1980.

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and L-glutamic acid gamma-ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRONDEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid.

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

Treatment with HER2 antibody

According to the invention, the HER2 antibody is useful for the treatment of cancer. The cancer is typically HER2 positive, such that the HER2 antibody herein is capable of binding to the cancer cell. In one embodiment, the cancer expresses low HER3 (e.g. ovarian cancer) or has an elevated HER 2: HER3 ratio (e.g. ovarian cancer). The above definitions list cancers that may be treated with the compositions of the present invention.

Preferred cancers to be treated herein include: breast cancer, including HER2 positive breast cancer, optionally in combination with trastuzumab and a taxane-like such as docetaxel, and including neoadjuvant therapy of breast cancer; ovarian cancer (including platinum-resistant and platinum-sensitive ovarian cancer) (see, e.g., US 2006/0013819); lung cancer (including non-small cell lung cancer, NSCLC), optionally in combination with an EGFR inhibitor (see, e.g., US 2007/0020261); colorectal cancer, and the like.

The HER2 antibodies herein are useful for treating various non-malignant diseases or disorders, such as autoimmune diseases (e.g. psoriasis); endometriosis; scleroderma; restenosis; polyps, such as colon polyps, nasal polyps, or gastrointestinal polyps; fibroadenoma; respiratory diseases; cholecystitis (cholecystitis); neurofibromatosis; polycystic kidney disease; inflammatory diseases; skin disorders including psoriasis and dermatitis; vascular disease; conditions involving abnormal proliferation of vascular epithelial cells; gastrointestinal ulcers; meniere's disease, secretory adenoma or protein loss syndrome (proteinlosssyndrome); renal disorders; angiogenic disorders; ocular diseases such as age-related macular degeneration, presumed ocular histoplasmosis syndrome (preshadoophoroplasmosis syndrome), retinal neovascularization from proliferative diabetic retinopathy, retinal angiogenesis, diabetic retinopathy or age-related macular degeneration; bone-related pathologies such as osteoarthritis, rickets and osteoporosis; injury following a cerebral ischemic event; fibrotic or edematous (edema) diseases such as cirrhosis of the liver, pulmonary fibrosis, carpoidosis, throiditis, systemic hyperviscosity syndrome, Osler-Weber-Rendu disease, chronic obstructive pulmonary disease, or edema following burns, trauma, radiation, stroke, tissue hypoxia or ischemia; hypersensitivity of the skin; diabetic retinopathy and diabetic nephropathy; Guillain-Barre syndrome (Guillain-Barre); graft versus host disease or transplant rejection; paget's (Paget) disease; inflammation of bones or joints; photoaging (e.g., caused by UV irradiation of human skin); benign prostatic hyperplasia; certain microbial infections, including microbial pathogens selected from the group consisting of adenovirus, hantaviruses (hantaviruses), borrelia burgdorferi (borrelia burgdorferi), yersinia (yersinia spp.) and bordetella pertussis (bordetella pertussis); thrombosis caused by platelet aggregation; reproductive disorders such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding or menorrhagia; synovitis; (ii) atheroma; acute and chronic kidney diseases (including proliferative glomerulonephritis and diabetes-induced nephropathy); eczema; hypertrophic scarring; endotoxic shock and fungal infections; familial adenomatous polyposis; neurodegenerative diseases (such as Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration); myelodysplastic syndrome (myelodysplastic syndrome); aplastic anemia; ischemic injury; fibrosis of the lung, kidney or liver; t cell mediated hypersensitivity disorders; hypertrophic pyloric stenosis in infants; urinary obstruction syndrome; psoriatic arthritis; and hashimoto's thyroiditis. Preferred non-malignant indications for treatment herein include psoriasis, endometriosis, scleroderma, vascular disease (such as restenosis, atherosclerosis, coronary artery disease or hypertension), colonic polyps, fibroadenomas or respiratory disease (such as asthma, chronic bronchitis, bronchiectasis or cystic fibrosis).

Treatment with the HER2 antibody will result in an improvement in the signs or symptoms of the disease. For example, if the disease being treated is cancer, such therapy may result in improved survival (overall survival and/or progression-free survival) and/or may result in objective clinical response (partial or complete).

Preferably, the HER2 antibody in the administered composition is a naked antibody. However, the HER2 antibody administered may be conjugated to a cytotoxic agent. Preferably, the immunoconjugate and/or HER2 protein to which it binds is/are subject to cellular internalization resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds. In a preferred embodiment, the cytotoxic agent targets or interferes with nucleic acid in cancer cells. Examples of such cytotoxic agents include maytansinoids, calicheamicin, ribonucleases and DNA endonucleases.

The HER2 antibody is administered to a human patient according to known methods, such as intravenous administration, e.g. bolus injection or continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes. Intravenous administration of the antibody composition is preferred.

For the prevention or treatment of disease, the appropriate dosage of the HER2 antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the HER2 antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the HER2 antibody, and the discretion of the attending physician. The HER2 antibody is suitably administered to the patient at a time or by a series of treatments. Depending on the type and severity of the disease, the initial candidate dose administered to the patient is about 1 μ g/kg to 50mg/kg (e.g., 0.1-20mg/kg) of HER2 antibody, whether by one or more separate administrations, for example, or by continuous infusion. In one embodiment, the initial infusion time of the HER2 antibody may be longer than the subsequent infusion time, e.g. the initial infusion is about 90 minutes, and the subsequent infusion is about 30 minutes (if the initial infusion is well tolerated). Preferred doses of HER2 antibody will range from about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or 10mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g., weekly or every three weeks (e.g., such that the patient receives from about 2 doses to about 20 doses, e.g., about 6 doses of HER2 antibody). A higher loading dose may be administered first, followed by a lower dose or doses. In one embodiment, the HER2 antibody is first administered at a loading dose of about 840mg, followed by about 420mg about every three weeks. In another embodiment, the HER2 antibody is first administered at a loading dose of about 1050mg, followed by about 525mg about every three weeks.

Other therapeutic agents may be combined with the HER2 antibody. Such combined administration includes co-administration or simultaneous administration using separate formulations or a single pharmaceutical formulation, as well as sequential administration in either order, wherein preferably all two (or more) active agents exert their biological activities simultaneously over a period of time. Thus, the other therapeutic agent may be administered before or after the administration of the HER2 antibody. In this embodiment, the timing between the administration of the at least one additional therapeutic agent and the administration of the at least one HER2 antibody is preferably about 1 month or less, and most preferably about 2 weeks or less. Alternatively, the other therapeutic agent and the HER2 antibody are administered to the patient simultaneously in a single formulation or separate formulations.

Other treatments that may be combined with HER2 antibodyExamples of therapeutic agents include any one or more of the following: chemotherapeutic agents, such as antimetabolites, such as gemcitabine; a second, different HER2 antibody (e.g. a growth inhibitory HER2 antibody such as Trastuzumab or a HER2 antibody such as 7C2, 7F3 or humanized variants thereof that induces apoptosis of HER2 overexpressing cells); a second antibody targeting another tumor associated antigen such as EGFR, HER3, HER 4; anti-hormonal compounds, for example anti-estrogenic compounds such as tamoxifen, or aromatase inhibitors; cardioprotective agents (to prevent or alleviate any myocardial dysfunction associated with treatment); a cytokine; EGFR targeting drugs (such as Erlitonib, Gefitinib, or Cetuximab); anti-angiogenic agents (especially Bevacizumab sold by Genentech under the trademark AVASTIN^TM) (ii) a Tyrosine kinase inhibitors; COX inhibitors (e.g., COX-1 or COX-2 inhibitors); nonsteroidal anti-inflammatory drugs, CelecoxibFarnesyl transferase inhibitors (e.g., available from Johnson and Johnson)R115777 or LonafamibSCH66336 available from Schering-Plough); antibodies that bind oncofetal protein CA125, such as oregovmab (moabb 43.13); HER2 vaccine (such as Pharmexia's HER2AutoVac vaccine, or Dendreon's APC8024 protein vaccine, or GSK/Corixa's HER2 peptide vaccine); other HER targeted therapies (e.g., trastuzumab, cetuximab, gefitinib, erlotinib, CI1033, GW2016, etc.); raf and/or ras inhibitors (see, e.g., WO 2003/86467); doxil; topotecan (Topetecan); a taxane; GW 572016; TLK 286; EMD-7200; drugs for the treatment of nausea, such as serotonin antagonists, steroids or benzodiazepines (benzodiazepiens); drugs to prevent or treat skin rash or standard acne therapy including topical or oral antibiotics; hypothermic drugs such as acetaminophen (acetaminophen), diphenhydramine (diphenhydramine), or meperidine (meperidine); hematopoietic growth factors, and the like.

Suitable dosages for any of the above co-administered agents are those presently used and may be reduced by the combined effect (synergy) of the agent and the HER2 antibody. Treatment with the HER2 antibody composition in combination with other therapeutic agents may result in synergistic, or greater than additive, therapeutic benefit to the patient.

If a chemotherapeutic agent is administered, it is usually administered at a dose known for this, or optionally reduced by the combined effect of the drugs or by adverse side effects attributable to the administration of the chemotherapeutic agent. The preparation and dosing regimen of the chemotherapeutic agent may be carried out in accordance with the manufacturer's instructions or determined empirically by the skilled practitioner. See also "chemotherapy service", ed., m.c. perry, Williams & Wilkins, Baltimore, MD (1992).

In addition to the above treatment regimens, the patient may also be subjected to surgical removal of cancer cells and/or radiation therapy.

Collection of materials

The following hybridoma cell lines have been deposited with the American type culture Collection (American type culture Collection, ATCC, 10801university boulevard, Manassas, VA20110-2209, USA):

name of antibody	ATCC accession number	Date of storage
			7C2	ATCC HB-12215	10 month and 17 days 1996
7F3	ATCC HB-12216	10 month and 17 days 1996
			4D5	ATCC CRL 10463	24 days 5 month in 1990
2C4	ATCC HB-12697	4/8/1999

The following non-limiting examples illustrate further details of the invention. The contents of all cited documents in the specification are expressly incorporated herein by reference.

Example 1

This example describes the characterization of a composition comprising the main species HER2 antibody (Pertuzumab) that binds domain II of HER2 and acidic variants thereof.

Pertuzumab is a recombinant humanized monoclonal antibody produced on the basis of the human IgG1(κ) framework. It comprises two heavy chains (448 residues) and two light chains (214 residues). The two heavy chains are connected by two interchain disulfide bonds, and each light chain is attached to a heavy chain by one interchain disulfide bond. There is an N-linked glycosylation site at Asn-299 of the two heavy chains in the Fc region of Pertuzumab. Pertuzumab and(Trastuzumab) differs in the epitope-binding regions of the light chain (12 amino acid differences) and heavy chain (30 amino acid differences). Due to these differences, Pertuzumab binds to a completely different epitope on the HER2 receptor. Binding of Pertuzumab to HER2 receptor on human epithelial cells prevents it from forming complexes with other members of the HER receptor family (agricultural, cancer cell 2: 12)7-137(2002)). By blocking the formation of the complex, Pertuzumab prevents the growth stimulatory effect of the ligands of the complex (e.g., EGF and heregulin). In vitro experiments demonstrated that both Pertuzumab and Pertuzumab-Fab inhibit Heregulin (HRG) binding to MCF7 cells, and HRG-stimulated phosphorylation of the HER2-HER3 complex is inhibited by both Pertuzumab and Pertuzumab-Fab (Agusetal, cancer cell 2: 127-137 (2002)). Furthermore, it was found that the inhibitory effect of Pertuzumab and polyethylene glycol-derivatized Fab of Pertuzumab on tumor growth in vivo was comparable in a murine prostate cancer xenograft model (Agusetal, cancer cell 2: 127-. These data indicate that the Fc region of the antibody is not required for tumor growth inhibition, and that the bivalent and Fc-mediated effector functions are not required for biological activity in vivo or in vitro.

In this example, the main peak of Pertuzumab was collected from a cation exchange column and incubated in cell culture media or processed using standard antibody purification procedures. Acidic variants are formed when the main peak is incubated with cell culture media components. Acidic variants of monoclonal antibodies are modified forms of the desired product that elute earlier than the main peak when separated by cation exchange chromatography. Minor differences in the amount and/or distribution of acidic variants are often observed before and after process changes and present challenges to demonstrating product comparability. The purification operation has less influence on the formation of acidic variants. The variants identified in the acidic variant fraction include glycated variants, deamidated variants, disulfide reduced variants, sialylated variants, and non-reducible variants. In conclusion, the acidic variant is fully potent.

The purpose of this study was: better understanding of the effect of cell culture and recovery processes on the formation of acidic variants of Pertuzumab, characterization of the major acidic variants of Pertuzumab, and evaluation of the effect of acidic variants on Pharmacokinetics (PK), etc.

Rat pharmacokinetic studies showed that the area under the curve for the acidic variant fraction and the main peak fraction was equivalent to that of Pertuzumab starting material (geometric mean ratios of 0.96 and 0.95, respectively). These results demonstrate that although the acidic variants are chemically distinct from the main peak, they have equivalent pharmacokinetics.

Method and results

Figure 10 depicts the experimental design of the isolation, cell culture, recovery, and PK (pharmacokinetics) evaluation and analytical testing of cation-exchanged MP (main peak) and AV (acidic variant). Fresh medium ═ standard medium; spent medium was used as standard medium after 12 days of cell culture, and cells were removed by centrifugation. Dissolved oxygen, pH, and other parameters were not controlled.

A. Separation of major peaks and acidic variants

Charge variants of Pertuzumab were separated on a 4.0x250mm dionexapacwcx-10J Cation Exchange (CEX) column using the following conditions:

and (3) buffer solution A: 20mM Tris, pH6.0

And (3) buffer solution B: 20mM Tris, 200mM NaCl, pH6.0

Gradient: 0.5% B/min, run at 1.0mL/min

Column temperature: 35 deg.C

And (3) detection: 280nm

FIG. 11 shows a typical chromatogram. The AV (acidic variant) and MP (main peak) fractions were collected.

Potency and monomer content were similar among pertuzmab starting material, main peak, and acidic variants (fig. 12). The purity of the main peak and acidic variant CEX fractions was acceptable for pharmacokinetic studies according to the 90% purity standard as determined by CEX (fig. 12).

B. Main peak incorporation experiment

The main peak of Pertuzumab isolated by CEX was spiked into fresh or spent cell culture medium (no cells) and incubated at 37 ℃ for 12 days as outlined in figure 10. Samples at each time point were analyzed by CEX either directly or after separation by protein a. The main peak was also incorporated into the medium +/-various medium components such as glucose and peptone. In addition, the main peak was processed for several cycles via standard recovery procedures such as protein a chromatography (ProA), low pH treatment, and spsepharose fastflow (spff) and analyzed by CEX.

The CEX patterns of the main peak were similar when incubated for 12 days in fresh or spent medium (fig. 13 and 14). The main peak was more reduced after incubation in fresh medium than in used medium. Elimination of various media components did not affect the reduction of the main peak. The percentage main peak in the incubated samples was the same with and without protein a separation. Incubation in medium buffer alone caused a loss of the main peak.

Separation of the main peak from media protein a did not affect the CEX plot, demonstrating that alterations during incubation did not affect protein a binding or elution. Recovery operations had little or no effect on the percent CEX main peak.

C. Characterization of acidic variants

Pertuzumab acidic variants were isolated by CEX from the main peak incubated in cell culture medium or Pertuzumab starting material. The isolated acidic variants were analyzed by the method listed in fig. 15. The acidic variants accounted for 21% of the total peak area, thus identifying about 80% (17% of 21%) of the acidic variants. The deamidated form cannot be quantified.

The forms identified in the acidic variants generated by incubation of the main peak with the medium are identical to those identified in Pertuzumab starting material. The following forms were detected: sialylation variants, disulfide reduction variants, glycation variants, non-reducible variants, and deamidation variants. The higher glycated forms were identified by electrospray ionization mass spectrometry (ESI-MS) after reduction and PNGase treatment.

D. Pharmacokinetic (PK) study

Single dose Intravenous (IV)10mg/kg, 12 rats per group, 3 groups (acidic variant, main peak, Pertuzumab starting material). Extensive PK sampling was performed for 35 days. Geometric mean ratios of AUC (days 0-14) between acidic variants, main peak, and Pertuzumab starting material. Geometric mean ratio GM sample/GMIgG 1 starting material. Pertuzumab concentration versus time curves for the Pertuzumab starting material, acidic variant, and main peak were similar (fig. 16 and 17). No significant differences in exposure were observed between the acidic variant, the main peak, and the Pertuzumab starting material. The GMR is about 1.0 and 90% CI is between 0.80 and 1.25.

Conclusion

Multiple cell culture factors contribute to acidic variant formation, but recovery has not been shown to affect acidic variant formation. Disulfide reduced variants, non-reducible variants, sialylated variants, saccharified variants, and deamidated variants are identified in the acidic fraction. The acidic fraction isolated from Pertuzumab starting material and those generated by incubation of the CEX main peak contained the same form. The acidic variant, main peak, and Pertuzumab starting material have the same pharmacokinetics.

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Application No.: PCT/US09/32220 file date: 01/28/2009

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Claims (15)

1. A composition comprising a main species HER2 antibody that binds domain II of HER2 and acidic variants thereof, wherein the main species HER2 antibody comprises light and heavy chain amino acid sequences as shown in seq id nos. 15 and 16, respectively, and wherein the acidic variants are glycated, deamidated, sialylated, disulfide reduced and non-reducible variants of the main species HER2 antibody, wherein disulfide reduced variant is one in which one or more disulfide-forming cysteines are chemically reduced to the free thiol form, and wherein non-reducible variant is one in which it cannot be chemically reduced to the heavy and light chains by treatment with a reducing agent.

2. The composition of claim 1, wherein the amount of the acidic variant is less than 25% of the composition.

3. The composition of claim 1 or 2 wherein both said main species HER2 antibody and said acidic variant are intact antibodies.

4. The composition of any one of claims 1-3, wherein said composition further comprises an amino-terminal leader extension variant of said main species HER2 antibody.

5. The composition of claim 4, wherein said amino-terminal leader extension comprises VHS-.

6. The composition of claim 5, wherein said amino-terminal leader extension consists of VHS-.

7. The composition of any one of claims 1-6, wherein said composition further comprises an amino acid sequence variant of said main species HER2 antibody selected from the group consisting of: antibodies comprising a C-terminal lysine residue on one or both of their heavy chains, and antibodies having one or more oxidized methionine residues.

8. The composition of any one of claims 1-7, further comprising Trastuzumab.

9. Use of a composition of any one of claims 1-7 in the manufacture of a medicament for treating cancer in a patient, wherein the cancer is breast cancer, gastric cancer, or ovarian cancer.

10. The use of claim 9, wherein the medicament is for use in combination with Trastuzumab in the treatment of cancer.

11. A pharmaceutical formulation comprising the composition of any one of claims 1-8 in a pharmaceutically acceptable carrier.

12. The pharmaceutical formulation of claim 11, which is sterile.

13. A composition comprising a main species HER2 antibody and acidic variants thereof, wherein the main species HER2 antibody comprises light and heavy chain amino acid sequences as shown in seq id nos. 15 and 16, respectively, and wherein the acidic variants are glycovariant, deamidated variant, disulfide reduced variant, sialylated variant and non-reducible variant, wherein the amount of acidic variant is less than 25% of the composition, and wherein the acidic variant has substantially the same pharmacokinetics as the main species HER2 antibody, wherein disulfide reduced variant is a main species antibody variant having one or more disulfide forming cysteines chemically reduced to the free thiol form, and wherein non-reducible variant is a main species antibody variant that cannot be chemically reduced to the heavy and light chains by treatment with a reducing agent.

14. A pharmaceutical formulation comprising the composition of claim 13 in a pharmaceutically acceptable carrier.

15. A pharmaceutical composition comprising:

(a) a main species HER2 antibody comprising light and heavy chain amino acid sequences as shown in seq id nos. 15 and 16, respectively;

(b) an acidic variant of the main species HER2 antibody, wherein the acidic variant is a glycovariant, a deamidated variant, a disulfide reduced variant, a sialylated variant, and a non-reducible variant, wherein the amount of the acidic variant is less than 25% of the composition, and wherein the acidic variant has substantially the same pharmacokinetics as the main species HER2 antibody; and

(c) a pharmaceutically acceptable carrier,

wherein the disulfide-reducing variant is a major species antibody variant having one or more disulfide-forming cysteines chemically reduced to the free thiol form, and wherein the non-reducible variant is a major species antibody variant that cannot be chemically reduced to heavy and light chains by treatment with a reducing agent.