DOSAGES FOR TREATMENT WITH ANTI-ErbB2 ANTIBODIES

Abstract: The present invention concerns the treatment of disorders characterized by the overexpression of ErbB2. More specifically, the invention concerns the treatment of human patients susceptible to or diagnosed with cancer overexpressing ErbB2 with anti-ErbB2 antibody.

Description: DOSAGES FOR TREATMENT WITH ANTI-ErbB2 ANTIBODIES Field of the Invention The present invention concerns the treatment of disorders characterized by the overexpression of ErbB2 or disorders expressing epidermal growth factor receptor (EGFR), comprising administering to a human or animal presenting the disorders a therapeutically effective amount of an antibody that binds ErbB2 More specifically, the invention concerns the treatment of human patients susceptible to or diagnosed with cancer overexpressing ErbB2 or expressing EGFR, where the treatment is with an antι-ErbB2 antibody administered by front loading the dose of antibody duπng treatment by intravenous and/or subcutaneous administration The invention optionally includes treatment of cancer in a human patient with a combination of an antι-ErbB2 antibody and a chemotherapeutic agent, such as, but not limited to, a taxoid The taxoid may be, but is not limited to paclitaxel or docetaxel The invention further includes treatment of cancer in a human patient with a combination of antι-ErbB2 antibody and a chemotherapeutic agent, such as, but not limited to, an anthracycline derivative Optionally, treatment with a combination of antι-ErbB2 and an anthracycline derivative includes treatment with an effective amount of a cardioprotectant The present invention further concerns infrequent dosing of antι-ErbB2 antibodies Background of the Invention Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer It has been found that the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2), which encodes a 185-kd transmembrane HFR2 glycoprotein receptor (p 185 ) related to the epidermal growth factor receptor (EGFR), is overexpressed in about 25% to 30% of human breast cancer (Slamon et al , Science 235 177-182 [1987], Slamon et al , Science 244 707- 712 [1989]) Several lines of evidence support a direct role for ErbB2 in the pathogenesis and clinical aggressiveness of ErbB2-overexpressιng tumors The introduction of ErbB2 into non-neoplastic cells has been shown to cause their malignant transformation (Hudziak etal , Proc Natl Acad Set USA 84 7159-7163 [1987], DiFiore etal , Science 237 78-182 [1987]) Transgenic mice that express HER2 were found to develop mammary tumors (Guy etal , Proc Natl Acad Sci USA 89 10578-10582 [1992]) Antibodies directed against human erbB2 protein products and proteins encoded by the rat equivalent of the erbB2 gene (neu) have been described Drebin et al , Cell 41 695-706 (1985) refer to an IgG2a monoclonal antibody which is directed against the rat neu gene product This antibody called 7 164 causes down-modulation of cell surface pi 85 expression on B 104-1-1 cells (NIH-3T3 cells transfected with the neu proto-oncogene) and inhibits colony formation of these cells In Drebin et al PNAS (USA) 83 9129-9133 (1986), the 7 16 4 antibody was shown to inhibit the tumoπgenic growth of neu-transformed NIH-3T3 cells as well as rat neuroblastoma cells (from which the neu oncogene was initially isolated) implanted into nude mice Drebin et al in Oncogene 2 387- 394 (1988) discuss the production of a panel of antibodies against the rat neu gene product All of the antibodies were found to exert a cytostatic effect on the growth of «ej/-transformed cells suspended in soft agar Antibodies of the IgM, IgG2a and IgG2b isotypes were able to mediate significant in vitro lysis of new-transformed cells in the presence of complement, whereas none of the antibodies were able to mediate high levels of antibody-dependent cellular cytotoxicity (ADCC) of the new-transformed cells Drebin et al Oncogene 2 273-277 (1988) report that mixtures of antibodies reactive with two distinct regions on the pi 85 molecule result in synergistic anti-tumor effects on new-transformed NIH-3T3 cells implanted into nude mice Biological effects of anti-new antibodies are reviewed in Myers et al , Meth Enzym 198 277-290 (1991 ) See also W094/22478 published October 13, 1994 Hudziak et al , Mol Cell Biol 9(3) 1165-1172 (1989) describe the generation of a panel of antι-ErbB2 antibodies which were characterized using the human breast tumor cell line SKBR3 Relative cell proliferation of the SKBR3 cells following exposure to the antibodies was determined by crystal violet staining of the monolayers after 72 hours Using this assay, maximum inhibition was obtained with the antibody called 4D5 which inhibited cellular proliferation by 56% Other antibodies in the panel, including 7C2 and 7F3, reduced cellular proliferation to a lesser extent in this assay Hudziak et al conclude that the effect of the 4D5 antibody on SKBR3 cells was cytostatic rather than cytotoxic, since SKBR3 cells resumed growth at a nearly normal rate following removal of the antibody from the medium The antibody 4D5 was further found to sensitize p 185 er B2 -overexpressmg breast tumor cell lines to the cytotoxic effects of TNF-α See also WO89/06692 published July 27, 1989 The antι-ErbB2 antibodies discussed in Hudziak et al are further characterized in Fendly et al Cancer Research 50 1550-1558 (1990), Kotts et al In Vitro 26(3) 59A (1990), Sarup et al Growth Regulation 1 72-82 (1991), Shepard et al J Clin Immunol 1 1(3) 117-127 (1991), Kumar et al Mol Cell Biol 1 1(2) 979-986 (1991 ), Lewis et al Cancer Immunol Immunother 37 255-263 (1993), Pietras et al Oncogene 9 1829-1838 (1994), Vitetta et al Cancer Research 54 5301-5309 (1994), Shwkowski et al J Biol Chem 269(20) 14661-14665 (1994), Scott etal J Biol Chem 266 14300-5 (1991), and D'souza et al Proc Natl Acad Set 91 7202-7206 (1994) Taghabue et al Int J Cancer Al 933-937 (1991 ) describe two antibodies which were selected for their reactivity on the lung adenocarcinoma cell line (Calu-3) which overexpresses ErbB2 One of the antibodies called MGR3, was found to internalize, induce phosphorylation of ErbB2, and inhibit tumor cell growth in vitro McKenzie et al Oncogene 4 543-548 (1989) generated a panel of antι-ErbB2 antibodies with varying epitope specificities, including the antibody designated TA1 This TA1 antibody was found to induce accelerated endocytosιs ofErbB2 (see Maιer /a/ CancerRes 51 5361-5369 [1991]) Bacus efα/ Molecular Carcinogenesis 3 350-362 (1990) reported that the TA1 antibody induced maturation of the breast cancer cell lines AU-565 (which overexpresses the erbB2 gene) and MCF-7 (which does not) Inhibition of growth and acquisition of a mature phenotype in these cells was found to be associated with reduced levels of ErbB2 receptor at the cell surface and transient increased levels in the cytoplasm Stancovski f α/ PNAS (USA) 88 8691-8695 (1991) generated a panel of antι-ErbB2 antibodies injected them l p into nude mice and evaluated their effect on tumor growth of muπne fibroblasts transformed by overexpression of the erbB2 gene Various levels of tumor inhibition were detected for four of the antibodies, but one of the antibodies (N28) consistently stimulated tumor growth Monoclonal antibody N28 induced significant phosphorylation of the ErbB2 receptor, whereas the other four antibodies generally displayed low or no phosphorylation-inducing activity The effect ofthe antι-ErbB2 antibodies on proliferation of SKBR3 cells was also assessed In this SKBR3 cell proliferation assay, two of the antibodies (N12 and N29) caused a reduction in cell proliferation relative to control The ability of the various antibodies to induce cell lysis in vitro via complement- dependent cytotoxicity (CDC) and antibody-mediated cell-dependent cytotoxicity (ADCC) was assessed, with the authors of this paper concluding that the inhibitory function of the antibodies was not attributed significantly to CDC or ADCC Bacus e/α/ Cancer Research 52 2580-2589 (1992) furthercharacteπzedthe antιbodιesdescπbedιnBacus et al (1990) and Stancovski et al of the preceding paragraphs Extending the l p studies of Stancovski et al , the effect of the antibodies after l v injection into nude mice harboring mouse fibroblasts overexpressing human ErbB2 was assessed As observed in their earlier work, N28 accelerated tumor growth, whereas N12 and N29 significantly inhibited growth of the ErbB2-expressιng cells Partial tumor inhibition was also observed with the N24 antibody Bacus et al also tested the ability of the antibodies to promote a mature phenotype in the human breast cancer cell lines AU-565 and MDA-MB453 (which overexpress ErbB2) as well as MCF-7 (containing low levels of the receptor) Bacus et al saw a correlation between tumor inhibition in vivo and cellular differentiation, the tumor-stimulatory antibody N28 had no effect on differentiation, and the tumor inhibitory action of the N12, N29 and N24 antibodies correlated with the extent of differentiation they induced Xu et al hit J Cancer 53 401-408 (1993) evaluated a panel of antι-ErbB2 antibodies for their epitope binding specificities, as well as their ability to inhibit anchorage independent and anchorage-dependent growth of SKBR3 cells (by individual antibodies and in combinations), modulate cell-surface ErbB2, and inhibit ligand stimulated anchorage-independent growth See also WO94/00136 published Jan 6, 1994 and Kasprzyk et al Cancer Research 52 2771 -2776 ( 1992) concerning antι-ErbB2 antibody combinations Other antι-ErbB2 antibodies are discussed in Hancock etal CancerRes 51 4575-4580 (1991), Shawvere?α/ CancerRes 54 1367-1373 (1994), Arteaga e/ α/ Cancer Res 54 3758-3765 (1994), and Harwerth et al J Biol Chem 267 15160-15167 (1992) A recombinant humanized antι-ErbB2 monoclonal antibody (a humanized version ofthe muπne antι-ErbB2 antibody 4D5, referred to as rhuMAb HER2, HERCEPTIN®, or HERCEPTIN® antι-ErbB2 antibody) has been clinically active in patients with ErbB2-overexpressιng metastatic breast cancers that had received extensive prior anti-cancer therapy (Baselga ef α/ , J Clin Oncol 14 737-744 [1996]) The recommended initial loading dose for HERCEPTIN® is 4mg/kg administered as a 90-mιnute infusion The recommended weekly maintenance dose is 2mg/kg and can be administered as a 30-mιnute infusion if the initial loading dose is well tolerated ErbB2 overexpression is commonly regarded as a predictor of a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (Slamon et al , [1987] and [1989], supra, Ravdin and Chamness, Gene 159 19-27 [1995], and Hynes and Stern, Biochim Biophys Ada 1198 165-184 [1994]), and has been linked to sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyc nes (Baselga etal , Oncology 11(3 Suppl 1) 43- 48 [1997]) However, despite the association of ErbB2 overexpression with poor prognosis, the odds of HER2- positive patients responding clinically to treatment with taxanes were greater than three times those of HER2- negative patients (Ibid) rhuMab HER2 was shown to enhance the activity of paclitaxel (TAXOL ) and doxorubicin against breast cancer xenografts in nude mice injected with BT-474 human breast adenocarcinoma cells, which express high levels of HER2 (Baselga et al , Breast Cancer, Proceedings ofASCO, Vol 13, Abstract 53 [1994]) Summary of the Invention The present invention concerns the discovery that an early attainment of an efficacious target trough serum concentration by providing an initial dose or doses of antι-ErbB2 antibodies followed by subsequent doses of equal or smaller amounts of antibody (greater front loading) is more efficacious than conventional treatments The efficacious target trough serum concentration is reached in 4 weeks or less, preferably 3 weeks or less, more preferably 2 weeks or less, and most preferably 1 week or less, including 1 day or less The target serum concentration is thereafter maintained by the administration of maintenance doses of equal or smaller amounts for the remainder of the treatment regimen or until suppression of disease symptoms is achieved The invention further concerns a method for the treatment of a human patient susceptible to or diagnosed with a disorder characterized by overexpression of ErbB2 receptor compπsing administering a therapeutically effective amount of an antι-ErbB2 antibody subcutaneously Preferably, the initial dose (or doses) as well as the subsequent maintenance dose or doses are administered subcutaneously Optionally, where the patient's tolerance to the antι-ErbB2 antibody is unknown, the initial dose is administered by intravenous infusion, followed by subcutaneous administration of the maintenance doses if the patient's tolerance for the antibody is acceptable According to the invention, the method of treatment involves administration of an initial dose of anti- ErbB2 antibody of more than approximately 4 mg/kg, preferably more than approximately 5 mg/kg The maximum initial dose or a subsequent dose does not exceed 50 mg/kg, preferably does not exceed 40 mg/kg, and more preferably does not exceed 30 mg/kg Administration is by intravenous or subcutaneous administration, preferably intravenous infusion or bolus injection, or more preferably subcutaneous bolus injection The initial dose may be one or more administrations of drug sufficient to reach the target trough serum concentration in 4 weeks or less, preferably 3 weeks or less, more preferably 2 weeks or less, and most preferably 1 week or less, including one day or less According to the invention, the initial dose or doses is/are followed by subsequent doses of equal or smaller amounts of antibody at intervals sufficiently close to maintain the trough serum concentration of antibody at or above an efficacious target level Preferably, an initial dose or subsequent dose does not exceed 50 mg/kg, and each subsequent dose is at least 0 01 mg kg Preferably the amount of drug administered is sufficient to maintain the target trough serum concentration such that the interval between administration cycles is at least one week Preferably the trough serum concentration does not exceed 2500 g/ml and does not fall below 0 01 μg/ml during treatment The front loading drug treatment method of the invention has the advantage of increased efficacy by reaching a target serum drug concentration early in treatment The subcutaneous delivery of maintenance doses according to the invention has the advantage of being convenient for the patient and health care professionals, reducing time and costs for drug treatment Preferably, the initial dose (or the last dose within an initial dose series) is separated in time from the first subsequent dose by 4 weeks or less, preferably 3 weeks or less, more preferably 3 weeks or less, most preferably 1 week or less In an embodiment of the invention, the initial dose of antι-ErbB2 is 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered by intravenous or subcutaneous administration, such as intravenous infusion or subcutaneous bolus injection The subsequent maintenance doses are 2 mg/kg delivered once per week by intravenous infusion, intravenous bolus injection, subcutaneous infusion, or subcutaneous bolus injection The choice of delivery method for the initial and maintenance doses is made according to the ability of the animal or human patient to tolerate introduction of the antibody into the body Where the antibody is well-tolerated, the time of infusion may be reduced The choice of delivery method as disclosed for this embodiment applies to all drug delivery regimens contemplated according to the invention In another embodiment, the invention includes an initial dose of 12 mg/kg antι-ErbB2 antibody, followed by subsequent maintenance doses of 6 mg/kg once per 3 weeks In still another embodiment, the invention includes an initial dose of 8 mg/kg antι-ErbB2 antibody, followed by 6 mg/kg once per 3 weeks In yet another embodiment, the invention includes an initial dose of 8 mg/kg antι-ErbB2 antibody, followed by subsequent maintenance doses of 8 mg/kg once per week or 8 mg/kg once every 2 to 3 weeks In another embodiment, the invention includes initial doses of at least 1 mg/kg, preferably 4 mg/kg, anti- ErbB2 antibody on each of days 1 , 2 and 3, followed by subsequent maintenance doses of 6 mg/kg once per 3 weeks In another embodiment, the invention includes an initial dose of 4 mg/kg antι-ErbB2 antibody, followed by subsequent maintenance doses of 2 mg/kg twice per week, wherein the maintenance doses are separated b 3 days In still another embodiment, the invention includes a cycle of dosing in which delivery of antι-ErbB2 antibody is 2-3 times per week for 3 weeks In one embodiment of the invention, each dose is approximately 25 mg/kg or less for a human patient, preferably approximately 10 mg/kg or less This 3 week cycle is preferably repeated as necessary to achieve suppression of disease symptoms In another embodiment, the invention includes a cycle of dosing in which delivery of antι-ErbB2 antibody is daily for 5 days According to the invention, the cycle is preferably repeated as necessary to achieve suppression of disease symptoms The disorder preferably is a benign or malignant tumor characterized by the overexpression of the ErbB2 receptor, e g a cancer, such as, breast cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, ghoblastoma, cervical cancer, ovarian cancer, liver cancer bladder cancer, hepatoma, colon cancer, colorectal cancer, endometπal carcinoma salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer The method of the invention may further comprise administration of a chemotherapeutic agent other than an anthracycline, e g doxorubicin or epirubicin The chemotherapeutic agent preferably is a taxoid, such as TAXOL® (paclitaxel) or a TAXOL® derivative Preferred antι-ErbB2 antibodies bind the extracellular domain of the ErbB2 receptor, and preferably bind to the epitope 4D5 or 3H4 within the ErbB2 extracellular domain sequence More preferably, the antibody is the antibody 4D5, most preferably in a humanized form Other preferred ErbB2-bιndιng antibodies include, but are not limited to, antibodies 7C2, 7F3, and 2C4, preferably in a humanized form The method of the present invention is particularly suitable for the treatment of breast or ovarian cancer, characterized by the overexpression of the ErbB2 receptor The present application also provides a method of therapy involving infrequent dosing of an antι-ErbB2 antibody In particular, the invention provides a method for the treatment of cancer (e g cancer characterized by overexpression of the ErbB2 receptor) in a human patient comprising administering to the patient a first dose of an antι-ErbB2 antibody followed by at least one subsequent dose ofthe antibody, wherein the first dose and subsequent dose are separated from each other in time by at least about two weeks (e g from about two weeks to about two months), and optionally at least about three weeks (e g from about three weeks to about six weeks) For instance, the antibody may be administered about every three weeks, about two to about 20 times, e g about six times The first dose and subsequent dose may each be from about 2mg/kg to about 16mg/kg, e g from about 4mg/kg to about 12mg/kg, and optionally from about 6mg/kg to about 12mg/kg Generally, two or more subsequent doses (e g from about two to about ten subsequent doses) ofthe antibody are administered to the patient, and those subsequent doses are preferably separated from each other in time by at least about two weeks (e g from about two weeks to about two months), and optionally at least about three weeks (e g from about three weeks to about six weeks) The two or more subsequent doses may each be from about 2mg/kg to about 16mg/kg, or from about 4mg/kg to about 12mg/kg, or from about 6mg/kg to about 12mg/kg The invention additionally provides an article of manufacture, comprising a container, a composition within the container compπsing an antι-ErbB2 antibody, and a package insert containing instructions to administer the antibody according to such methods The presently described dosing protocols may be applied to other anti-ErbB antibodies such as anti- epidermal growth factor receptor (EGFR), antι-ErbB3 and anti ErbB4 antibodies Thus, the invention provides a method for the treatment of cancer in a human patient, comprising administering an effective amount of an anti-ErbB antibody to the human patient, the method compπsing administering to the patient an initial dose of at least approximately 5 mg/kg of the anti-ErbB antibody, and administering to the patient a plurality of subsequent doses of the antibody in an amount that is approximately the same or less than the initial dose Alternatively, or additionally, the invention pertains to a method for the treatment of cancer in a human patient comprising administering to the patient a first dose of an anti-ErbB antibody followed by at least one subsequent dose of the antibody, wherein the first dose and subsequent dose are separated from each other in time by at least about two weeks The invention additionally provides an article of manufacture, compπsing a container, a composition within the container compπsing an anti-ErbB antibody, and a package insert containing instructions to administer the antibody according to such methods In another aspect, the invention concerns an article of manufacture, comprising a container, a composition within the container comprising an antι-ErbB2 antibody, optionally a label on or associated with the container that indicates that the composition can be used for treating a condition characterized by overexpression of ErbB2 receptor, and a package insert containing instructions to avoid the use of anthracychne-type chemotherapeutics in combination with the composition According to the invention, the package insert further includes instructions to administer the antι-ErbB2 antibody at an initial dose of 5 mg/kg followed by the same or smaller subsequent dose or doses In another embodiment of the invention, the package insert further includes instructions to administer the antι-ErbB2 antibody subcutaneously for at least one of the doses, preferably for all of the subsequent doses following the initial dose, most preferably for all doses In a further aspect, the invention provides a method of treating ErbB2 expressing cancer in a human patient comprising administering to the patient effective amounts of an antι-ErbB2 antibody and a chemotherapeutic agent In one embodiment of the invention, the chemotherapeutic agent is a taxoid including, but not limited to, paclitaxel and docetaxel In another embodiment, the chemotherapeutic agent is an anthracy ne derivative including, but not limited to, doxorubicin or epirubicin In still another embodiment of the invention, treatment with an antι-ErbB2 antibody and an anthracycline derivative further includes administration of a cardioprotectant to the patient In still another embodiment, an anthracycline derivative is not administered to the patient with the antι-ErbB2 antibody One or more additional chemotherapeutic agents may also be administered to the patient The cancer is preferably characterized by overexpression of ErbB2 The invention further provides an article of manufacture comprising a container, a composition within the container compπsing an antι-ErbB2 antibody and a package insert instructing the user of the composition to administer the antι-ErbB2 antibody composition and a chemotherapeutic agent to a patient In another embodiment, ® the chemotherapeutic agent is other than an anthracycline, and is preferably a taxoid, such as TAXOL In still another embodiment, the chemotherapeutic agent is an anthracycline, including but not limited to, doxorubicin or epirubicin In yet another embodiment, the chemotherapeutic agent is an anthracycline and the package insert further instructs the user to administer a cardioprotectant The methods and compositions of the invention comprise an antι-ErbB2 antibody and include a humanized antι-ErbB2 antibody Thus, the invention further pertains to a composition comprising an antibody that binds ErbB2 and the use of the antibody for treating ErbB2 expressing cancer, e g , ErbB2 overexpressing cancer, in a human The invention also pertains to the use of the antibody for treating EGFR expressing cancer Preferably the antibody is a monoclonal antibody 4D5, e g , humanized 4D5 (and preferably huMAb4D5-8 (HERCEPTIN® antι-ErbB2 antibody) , or monoclonal antibody 2C4, e g , humanized 2C4 The antibody may be an intact antibody (e g , an intact IgG, antibody) or an antibody fragment (e g , a Fab, F(ab ) 2 , diabody, and the like) The variable light chain and variable heavy chain regions of humanized antι-ErbB2 antibody 2C4 are shown in Figs 5A and 5B Brief Description of the Drawings Fig 1 shows epitope-mapping of the extracellular domain of ErbB2 as determined by truncation mutant analysis and site-directed mutagenesis (Nakamura et al J of Virology 67(10) 6179-6191 [Oct 1993], Renz et al J Cell Biol 125(6) 1395-1406 [Jun 1994]) The anti-prohferative MAbs 4D5 and 3H4 bind adjacent to the transmembrane domain The various ErbB2-ECD truncations or point mutations were prepared from cDNA using polymerase chain reaction technology The ErbB2 mutants were expressed as gD fusion proteins in a mammalian expression plasmid This expression plasmid uses the cytomegalovirus promoter/enhancer with SV40 termination and polyadenylation signals located downstream of the inserted cDNA Plasmid DNA was transfected into 293S cells One day following transfection, the cells were metabohcally labeled overnight in methionine and cysteine- free, low glucose DMEM containing 1 % dialyzed fetal bovine serum and 25 μCi each of 35 S methionine and 3= S cysteine Supernatants were harvested either the ErbB2 MAbs or control antibodies were added to the supernatant and incubated 2-4 hours at 4 C C The complexes were precipitated, applied to a 10-20% Tπcine SDS gradient gel and electrophoresed at 100 V The gel was electroblotted onto a membrane and analyzed by autoradiography SEQ ID NOs 8 and 9 depict the 3H4 and 4D5 epitopes, respectively Fig 2 depicts with underlining the amino acid sequence of Domain 1 of ErbB2 (SEQ ID NO 1 ) Bold amino acids indicate the location of the epitope recognized by MAbs 7C2 and 7F3 as determined by deletion mapping, i e the "7C2/7F3 epitope" (SEQ ID NO 2) Fig 3 is a graph of antι-ErbB2 antibody (HERCEPTIN ) trough serum concentration (μg/ml, mean ± SE, dark circles) by week from week 2 through week 36 for ErbB2 overexpressing patients treated with HERCEPTIN ® anti-ErbB 2 antibody at 4 mg/kg initial dose, followed by 2 mg/kg weekly The number of patients at each time point is represented by "n" (white squares) Fig 4A is a linear plot of tumor volume changes over time in mice treated with HERCEPTIN ® antι-ErbB2 antibody Fig 4B is a semi-logarithmic plot of the same data as in Fig 4A such that the variation in tumor volume for the treated animals is observed more readily Figs 5A and 5B depict alignments of the amino acid sequences of the variable light V^ (Fig 5A) and variable heavy (V H ) (Fig 5B) domains of muπne monoclonal antibody 2C4 (SEQ ID Nos 10 and 11 , respectively), V L and V H domains of humanized Fab version 574 (SEQ ID Nos 12 and 13 , respectively), and human V L and V H consensus frameworks (hum κl , light kappa subgroup I, humlll, heavy subgroup III) (SEQ ID Nos 14 and 15, respectively) Asterisks identify differences between humanized Fab version 574 and muπne monoclonal antibody 2C4 or between humanized Fab version 574 and the human framework Complementarity Determining Regions (CDRs) are in brackets Humanized Fab version 574, with the changes ArgH71Val, AspH73Arg and IleH69Leu, appears to have binding restored to that of the original chimeπc 2C4 Fab fragment Additional FR and/or CDR residues, such as L2, L54, L55, L56, H35 and/or H48, may be modified (e g substituted as follows - IleL2Thr, ArgL54Leu, TyrL55Glu, ThrL56Ser, AspH35Ser, and ValH48Ile) in order to further refine or enhance binding of the humanized antibody Alternatively, or additionally, the humanized antibody may be affinity matured in order to further improve or refine its affinity and/or other biological activities Detailed Description of the Preferred Embodiments I. Definitions An "ErbB receptor" is a receptor protein tyrosine kinase which belongs to the ErbB receptor family and includes EGFR, HER2, ErbB3 and ErbB4 receptors as well as TEGFR (US Patent No 5,708, 156) and other members of this family to be identified in the future The ErbB receptor will generally comprise an extracellular domain, which may bind an ErbB ligand, a hpophilic transmembrane domain, a conserved intracellular tyrosine kinase domain, and a carboxyl-terminal signaling domain harboring several tyrosine residues which can be phosphorylated The ErbB receptor may be a native sequence ErbB receptor or an amino acid sequence variant thereof Preferably the ErbB receptor is native sequence human ErbB receptor The terms "ErbBl ", "epidermal growth factor receptor" and "EGFR" are used interchangeably herein and refer to native sequence EGFR as disclosed, for example, in Carpenter e/α/ Ann Rev Biochem 56 881-914 (1987), including variants thereof (e g a deletion mutant EGFR as in Humphrey et al PNAS (USA) 87 4207-421 1 (1990)) erbB 1 refers to the gene encoding the EGFR protein product Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No 4,943, 533, Mendelsohn etal ) and variants thereof, such as chimeπzed 225 (C225) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc ) "ErbB 3' and "HER3" refer to the receptor polypeptide as disclosed, for example, in US Pat Nos 5,183,884 and 5,480,968 as well as Kraus et al PNAS (USA) 86 9193-9197 (1989), including variants thereof Examples of antibodies which bind HER3 are described in US Patent No 5,968,51 1 (Akita and Shwkowski), e g the 8B8 antibody (ATCC HB 12070) or a humanized variant thereof The terms "ErbB4" and "HER4" herein refer to the receptor polypeptide as disclosed for example, in EP Pat Appln No 599,274, Plowman et al , Proc Natl Acad Sci USA, 90 1746-1750 (1993), and Plowman et al Nature, 366 473-475 (1993), including variants thereof such as the HER4 isoforms disclosed in WO 99/19488 The terms "HER2", "ErbB2" "c-Erb-B2" are used interchangeably Unless indicated otherwise, the terms ' ErbB2" "c-Erb-B2" and "HER2" when used herein refer to the human protein, and "erbB2," "c-erb-B2," and "her2" refer to human gene The human erbB2 gene and ErbB2 protein are, for example, described in Semba et al , PNAS (USA) 82 6497-6501 (1985) and Yamamoto f α/ Nature 3 \9 230-234 ( 1986) (Genebank accession number X03363) ErbB2 comprises four domains (Domains 1-4) The "epitope 4D5" is the region in the extracellular domain of ErbB2 to which the antibody 4D5 (ATCC CRL 10463) binds This epitope is close to the transmembrane region of ErbB2 To screen for antibodies which bind to the 4D5 epitope, a routine cross-blocking assay such as that described in Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed Alternatively, epitope mapping can be performed (see Fig 1 ) to assess whether the antibody binds to the 4D5 epitope of ErbB2 (J e any one or more residues in the region from about residue 529, e g about residue 561 to about residue 625, inclusive) The ' epitope 3H4" is the region in the extracellular domain of ErbB2 to which the antibody 3H4 binds This epitope is shown in Fig 1 , and includes residues from about 541 to about 599, inclusive, in the amino acid sequence of ErbB2 extracellular domain The "epitope 7C2/7F3" is the region at the N-terminus of the extracellular domain of ErbB2 to which the 7C2 and/or 7F3 antibodies (each deposited with the ATCC, see below) bind To screen for antibodies which bind to the 7C2/7F3 epitope, a routine cross-blocking assay such as that described in Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed Alternatively, epitope mapping can be performed to establish whether the antibody binds to the 7C2/7F3 epitope on ErbB2 (; e any one or more of residues in the region from about residue 22 to about residue 53 of ErbB2, SEQ ID NO 2) The term "induces cell death" or "capable of inducing cell death" refers to the ability of the antibody to make a viable cell become nonviable The "cell" here is one which expresses the ErbB2 receptor, especially where the cell overexpresses the ErbB2 receptor A cell which "overexpresses" ErbB2 has significantly higher than normal ErbB2 levels compared to a noncancerous cell of the same tissue type Preferably, the cell is a cancer cell, e g a breast, ovarian, stomach, endometπal, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell In vitro, the cell may be a SKBR3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3 cell Cell death in vitro may be determined in the absence of complement and immune effector cells to distinguish cell death induced by antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) Thus, the assay for cell death may be performed using heat inactivated serum (i e in the absence of complement) and in the absence of immune effector cells To determine whether the antibody is able to induce cell death, loss of membrane integrity as evaluated by uptake of propidium iodide (PI), trypan blue (see Moore et al Cytotechnology 17 1-11 [1995]) or 7AAD can be assessed relative to untreated cells Preferred cell death-inducing antibodies are those which induce PI uptake in the "PI uptake assay in BT474 cells" The phrase "induces apoptosis" or "capable of inducing apoptosis" refers to the ability of the antibody to induce programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shπnkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies) The cell is one which overexpresses the ErbB2 receptor Preferably the "cell" is a tumor cell, e g a breast, ovaπan, stomach, endometπal, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell In vitro, the cell may be a SKBR3, BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or SKOV3 cell Various methods are available for evaluating the cellular events associated with apoptosis For example, phosphatidyl seπne (PS) translocation can be measured by annexin binding, DNA fragmentation can be evaluated through DNA laddering as disclosed in the example herein, and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells Preferably, the antibody which induces apoptosis is one which results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an "annexin binding assay using BT474 cells" (see below) Sometimes the pro-apoptotic antibody will be one which blocks HRG binding/activation of the ErbB2/ErbB3 complex (e g 7F3 antibody) In other situations, the antibody is one which does not significantly block activation of the ErbB2/ErbB3 receptor complex by HRG (e g 7C2) Further, the antibody may be one like 7C2 which, while inducing apoptosis, does not induce a large reduction in the percent of cells in S phase (e g one which only induces about 0-10% reduction in the percent of these cells relative to control) The antibody of interest may be one like 7C2 which binds specifically to human ErbB2 and does not significantly cross-react with other proteins such as those encoded by the erbB l , erbB3 and/or erbB4 genes Sometimes, the antibody may not significantly cross-react with the rat new protein, e g , as described in Schecter et al Nature 312 513 (1984) and Drebin et al , Nature 312 545-548 (1984) In such embodiments, the extent of binding of the antibody to these proteins (e g , cell surface binding to endogenous receptor) will be less than about 10% as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA) "Hereguhn" (HRG) when used herein refers to a polypeptide which activates the ErbB2-ErbB3 and ErbB2-ErbB4 protein complexes (i e induces phosphorylation of tyrosine residues in the complex upon binding thereto) Various hereguhn polypeptides encompassed by this term are disclosed in Holmes et al Science, 256 1205-1210 (1992), WO 92/20798, Wen etal , Mol Cell Biol , U(3) 1909-1919 (1994), and Marchionni etal , Nature, 362 312-318 (1993), for example The term includes biologically active fragments and/or variants of a naturally occurring HRG polypeptide, such as an EGF-hke domain fragment thereof (e g HRGβl 177 744 ) The "ErbB2-ErbB3 protein complex" and "ErbB2-ErbB4 protein complex" are noncovalently associated oligomers of the ErbB2 receptor and the ErbB3 receptor or ErbB4 receptor, respectively The complexes form when a cell expressing both of these receptors is exposed to HRG and can be isolated by lmmunoprecipitation and analyzed by SDS-PAGE as described in Shwkowski et al , ] Biol Chem , 269(20) 14661-14665 (1994) "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody- ke molecules which lack antigen specificity Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas "Native antibodies" and "native immunoglobulins" are usually heterotetrameπc glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different lmmunoglobulin isotypes Each heavy and light chain also has regularly spaced intrachain disulfide bridges Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains Each light chain has a variable domain at one end (V ) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen However, the variability is not evenly distributed throughout the variable domains of antibodies It is concentrated in three segments called complementarity determining regions (CDRs) or hypervaπable regions both in the light-chain and the heavy-chain variable domains The more highly conserved portions of variable domains are called the framework region (FR) The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of. the β-sheet structure The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al , NlH Publ No 91-3242, Vol I, pages 647-669 [1991]) The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH 1 ) of the heavy chain Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine resιdue(s) of the constant domains bear a free thiol group F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino acid sequences of their constant domains Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgGl , IgG2, IgG3, IgG4, IgA, and IgA2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known The term "antibody" is used 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 "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody Examples of antibody fragments include Fab, Fab', F(ab ) 2 , and Fv fragments, diabodies , linear antibodies (Zapata etal , Protein Eng 8(10) 1057- 1062 [ 1995] ) , single-chain antibody molecules , and multispecific antibodies formed from antibody fragments The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, * e , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigenic site Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized b> the hybπdoma culture, 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, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybπdoma method first described by Kohler et al , Nature, 256 495 (1975), or may be made by recombinant DNA methods (see, e g , U S Patent No 4,816,567) The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al , Nature, 352 624-628 ( 1991) and Marks et al . J Mol Biol , 222 581-597 (1991), for example The monoclonal antibodies herein specifically include "chimeπc" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with 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 chaιn(s) is identical with 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 Patent No 4,816,567, Morrison et al , Proc Natl Acad Sci USA, 81 6851-6855 [1984]) "Humanized" forms of non-human (e g , muπne) antibodies are chimeπc immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences These modifications are made to further refine and maximize antibody performance In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc) typically that of a human immunoglobulin For further details, see Jones et al , Nature, 321 522-525 (1986), Reichmann et al , Nature, 332 323-329 (1988), and Presta, Curr Op Struct Biol , 2 593-596 ( 1992) The humanized antibody includes a PRIMATTZED™ antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest "Single-chain Fv" or "sFv" antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain Preferably, the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol 1 13, Rosenburg and Moore eds , Springer-Verlag, New York, pp 269-315 (1994) The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H - 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 another chain and create two antigen-binding sites Diabodies are described more fully in, for example, EP 404,097, WO 93/1 1 161 , and Hol nger et al , Proc Natl Acad Sci USA, 90 6444-6448 (1993) An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present Ordinarily, however, isolated antibody will be prepared by at least one purification step As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e g , IgG ] , IgG 2 , IgG^, or IgG 4 ) that is responsible for increasing the in vivo serum half-life of the IgG molecule "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc Preferably, the mammal is human A "disorder" is any condition that would benefit from treatment with the antι-ErbB2 antibody This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question Non-limiting examples of disorders to be treated herein include benign and malignant tumors, leukemias and lymphoid malignancies, neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoehc disorders, and inflammatory, angiogenic and lmmunologic disorders The term "therapeutically effective amount" is used to refer to an amount having antiprohferative effect Preferably, the therapeutically effective amount has apoptotic activity, or is capable of inducing cell death, and preferably death of benign or malignant tumor cells, in particular cancer cells Efficacy can be measured in conventional ways, depending on the condition to be treated For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression ( IP), or determining the response rates (RR) (see Example 1 , below) Therapeutically effective amount also refers to a target serum concentration, such as a trough serum concentration, that has been shown to be effective in suppressing disease symptoms when maintained for a period of time 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, sarcoma, and leukemia More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, ghoblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometπal carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells The term is intended to include radioactive isotopes (e g I , I " , Y and Re ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™), alkyl sulfonates such as busulfan, improsulfan and piposulfan, aziπdines such as benzodopa, carboquone, meturedopa, and uredopa, ethylenimines and methylamelamines including altretamine, tπethylenemelamine, tπetylenephosphoramide, tπethylenethiophosphaoramide and tπmethylolomelamine, nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, lfosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesteπne, prednimustine, trofosfamide, uracil mustard, nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, rammustine, antibiotics such as aclacinomysins, actinomycin, authramycin, azaseπne, bleomycins, cactinomycin, cahcheamicin, carabicin, carminomycin, carzinophihn, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-dιazo-5-oxo-L-norleucme, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenohc acid, nogalamycin, o vomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgπn, streptozocm, tubercidin, ubemmex, zinostatin, zorubicin, anti-metabolites such as methotrexate and 5-fluorouracιl (5-FU), fo c acid analogues such as denopteπn, methotrexate, pteropteπn, tπmetrexate, puπne analogs such as fludarabine, 6- mercaptopuπne, thiamipπne, thioguanine, pyrimidine analogs such as ancitabine, azacitidine, 6-azauπdιne, carmofur, cytarabine, dideoxyuπdine, doxifluπdine, enocitabine, floxuπdine, 5-FU, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, anti-adrenals such as aminoglutethimide, mitotane, tπlostane, fohc acid replenisher such as fro nic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacπne, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elhptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracπne, pentostatin, phenamet, pirarubicin, podophylhnic acid, 2-ethylhydrazιde, procarbazine, PSK®, razoxane, sizofiran, spirogermanium, tenuazonic acid, tπaziquone, 2, 2',2 '- tπchlorotπethylamine, urethan, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxanes, e g paclitaxel (TAXOL ® , Bristol Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhδne-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thιoguanιne, mercaptopuπne, methotrexate, platinum analogs such as cisplatin and carboplatin, vinblastine, platinum, etoposide (VP-16), lfosfamide, mitomycin C, mitoxantrone, vincπstine, vinorelbine, navelbine, novantrone, temposide, daunomycin, aminopteπn, xeloda, ibandronate, CPT-1 1 , topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoic acid, esperamicins, capecitabine, and pharmaceutically acceptable salts, acids or derivatives of any ofthe above Also included in this definition are anti- hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-ιmιdazoles, 4-hydroxytamoxιfen, tπoxifene, keoxifene, LY 1 17018, onapπstone, and toremifene (Fareston), and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprohde, and goserelm, and pharmaceutically acceptable salts, acids or derivatives of any of the above A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially an ErbB2-overexpressιng cancer cell either in vitro or in vivo Thus, the growth inhibitory agent is one which significantly reduces the percentage of ErbB2 overexpressing cells in S phase Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce GI arrest and M-phase arrest Classical M-phase blockers include the vincas (vincπstine and vinblastine), TAXOL®, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin Those agents that arrest GI also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracιl, and ara-C Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds , Chapter 1 , entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami etal (WB Saunders Philadelphia, 1995), especially p 13 The 4D5 antibody (and functional equivalents thereof) can also be employed for this purpose "Doxorubicin" is an athracychne antibiotic The full chemical name of doxorubicin is (8S-cιs)-10-[(3- amιno-2,3,6-tπdeoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8, l l-tπhydroxy-8-(hydroxyacetyl)-l- methoxy-5,12-naphthacenedιone The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones Included among the cytokines are growth hormone such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and -β, mulleπan-inhibiting substance, mouse gonadotropin- associated peptide, inhibin, activin, vascular endothelial growth factor, lntegπn, thrombopoietin (TPO), nerve growth factors such as NGF-β, platelet-growth factor, transforming growth factors (TGFs) such as TGF-α and TGF- β, insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factors, interferons such as mterferon- α -β, and -γ, colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (ILs) such as IL- 1 , IL- 1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11 , IL-12, a tumor necrosis factor 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 The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form See, e g , Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp 375-382, 615th Meeting Belfast (1986) and Stella et al , "Prodrugs A Chemical Approach to Targeted Drug Delivery," Directed Drug Deliver , Borchardt et al , (ed ), pp 247-267, Humana Press (1985) The prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide- containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosιne and other 5-fluorouπdιne prodrugs which can be converted into the more active cytotoxic free drug Examples of cytotoxic drugs that can be deπvatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above By "solid phase" is meant a non-aqueous matrix to which the antibodies used in accordance with the present invention can adhere Examples of solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysacchaπdes (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and sihcones In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate, in others it is a purification column (e g ,an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275,149 A "hposome" is a small vesicle composed of various types of lipids, phosphohpids and/or surfactant which is useful for delivery of a drug (such as the antι-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal The components of the hposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products The term "serum concentration," "serum drug concentration, ' or "serum HERCEPTIN antι-ErbB2 antibody concentration" refers to the concentration of a drug, such as HERCEPTIN antι-ErbB2 antibody, in the ® blood serum of an animal or human patient being treated with the drug Serum concentration of HERCEPTIN antι-ErbB2 antibody, for example, is preferably determined by lmmunoassay Preferably, the immunoassay is an ELISA according to the procedure disclosed herein The term "peak serum concentration" refers to the maximal serum drug concentration shortly after delivery of the drug into the animal or human patient, after the drug has been distributed throughout the blood system, but before significant tissue distribution, metabolism or excretion of drug by the body has occurred The term "trough serum concentration" refers to the serum drug concentration at a time after delivery of a previous dose and immediately prior to delivery of the next subsequent dose of drug in a series of doses Generally, the trough serum concentration is a minimum sustained efficacious drug concentration in the series of drug administrations Also, the trough serum concentration is frequently targeted as a minimum serum concentration for efficacy because it represents the serum concentration at which another dose of drug is to be administered as part of the treatment regimen If the delivery of drug is by intravenous administration, the trough serum concentration is most preferably attained within 1 day of a front loading initial drug delivery If the delivery of drug is by subcutaneous administration, the peak serum concentration is preferably attained in 3 days or less According to the invention, the trough serum concentration is preferably attained in 4 weeks or less, preferably 3 weeks or less, more preferably 2 weeks or less, most preferably in 1 week or less, including 1 day or less using any of the drug delivery methods disclosed herein The term "intravenous infusion" refers to introduction of a drug into the vein of an animal or human patient over a period of time greater than approximately 5 minutes, preferably between approximately 30 to 90 minutes, although, according to the invention, intravenous infusion is alternatively administered for 10 hours or less The term "intravenous bolus" or "intravenous push" refers to drug administration into a vein of an animal or human such that the body receives the drug in approximately 15 minutes or less, preferably 5 minutes or less The term "subcutaneous administration" refers to introduction of a drug under the skin of an animal or human patient, preferable within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle The pocket may be created by pinching or drawing the skin up and away from underlying tissue The term "subcutaneous infusion" refers to introduction of a drug under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle for a period of time including, but not limited to, 30 minutes or less, or 90 minutes or less Optionally, the infusion may be made by subcutaneous implantation of a drug delivery pump implanted under the skin of the animal or human patient, wherein the pump deli ers a predetermined amount of drug for a predetermined period of time, such as 30 minutes, 90 minutes, or a time period spanning the length of the treatment regimen The term "subcutaneous bolus" refers to drug administration beneath the skin of an animal or human patient, where bolus drug delivery is preferably less than approximately 15 minutes, more preferably less than 5 minutes, and most preferably less than 60 seconds Administration is preferably within a pocket between the skin and underlying tissue, where the pocket is created, for example, by pinching or drawing the skin up and away from underlying tissue The term "front loading" when referring to drug administration is meant to describe an initially higher dose followed by the same or lower doses at intervals The initial higher dose or doses are meant to more rapidly increase the animal or human patient's serum drug concentration to an efficacious target serum concentration According to the present invention, front loading is achieved by an initial dose or doses delivered over three weeks or less that causes the animal's or patient's serum concentration to reach a target serum trough concentration Preferably, the initial front loading dose or series of doses is administered in two weeks or less, more preferably in 1 week or less, including 1 day or less Most preferably, where the initial dose is a single dose and is not followed by a subsequent maintenance dose for at least 1 week, the initial dose is administered in 1 day or less Where the initial dose is a series of doses, each dose is separated by at least 3 hours, but not more than 3 weeks or less, preferably 2 weeks or less, more preferably 1 week or less, most preferably 1 day or less To avoid adveise immune ( reaction to an antibody drug such as an antι-ErbB2 antibody (e g , HERCEPTIN antι-ErbB2 antibody) in an animal or patient who has not previously been treated with the antibody, it may be preferable to deliver initial doses of the antibody by intravenous infusion The present invention includes front loading drug delivery of initial and maintenance doses by infusion or bolus administration, intravenously or subcutaneously Published information related to anti-ErbB 2 antibodies includes the following issued patents and published applications PCT/US89/00051, published January 5, 1989, PCT/US90/02697, published May 18, 1990, EU 0474727 issued July 23, 1997, DE 69031 120 6, issued July 23, 1997, PCT US97/18385, published October 9, 1997, SA 97/9185, issued October 14, 1997, US 5677171 , issued October 14, 1997, US 5720937 issued February 24, 1998, US 5720954, issued February 24, 1998, US 5725856, issued March 10, 1998, US 5770195, issued June 23, 1998, US 5772997, issued June 30, 1998, PCT/US98/2626, published December 10, 1998, and PCT/US99/06673, published March 26, 1999, each of which patents and publications is herein incorporated by reference in its entirety II. Production of anti-ErbB2 Antibodies A descπption follows as to exemplary techniques for the production of the antibodies used in accordance with the present invention The ErbB2 antigen to be used for production of antibodies may be, e g , a soluble form of the extracellular domain of ErbB2 or a portion thereof, containing the desired epitope Alternatively cells expressing ErbB2 at their cell surface (e g NIH-3T3 cells transformed to overexpress ErbB2, or a carcinoma cell line such as SKBR3 cells, see Stancovski et al , PNAS (USA) 88 8691-8695 [1991]) can be used to generate antibodies Other forms of ErbB2 useful for generating antibodies will be apparent to those skilled in the art (i) Polyclonal antibodies Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (lp) injections of the relevant antigen and an adjuvant It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e g , keyhole limpet hemocyanin, serum albumin, bovine thyroglobuhn, or soybean trypsin inhibitor using a bifunctional or deπvatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R 2 N=C=NR, where R and R 1 are different alkyl groups Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e g , 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites Seven to 14 days later the animals are bled and the serum is assayed for antibody titer Animals are boosted until the titer plateaus Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent Conjugates also can be made in recombinant cell culture as protein fusions Also, aggregating agents such as alum are suitably used to enhance the immune response (u) Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, ; e , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler etal , Nature, 256 495 (1975), or may be made by recombinant DNA methods (U S Patent No 4,816,567) In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as hereinabove described 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 Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies Principles and Practice, pp 59-103 [Academic Press, 1986]) The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybπdomas typically will include hypoxanthine, aminopteπn, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells Prefeπed myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium Among these, preferred myeloma cell lines are muπne myeloma lines, such as those derived from MOPC-21 and MPC-1 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J Immunol , 133 3001 (1984), Brodeur et al , Monoclonal Antibody Production Techniques and Applications, pp 51-63 [Marcel Dekker, Inc , New York, 1987]) Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by lmmunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al , Anal Biochem , 107 220 (1980) After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies Principles and Practice, pp 59-103 [Academic Press, 1986]) Suitable culture media for this purpose include, for example, D-MEM or RPMI- 1640 medium In addition, the hybπdoma cells may be grown in vivo as ascites tumors in an animal The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of muπne antibodies) The hybridoma cells serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E coli cells, simian COS cells Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al , Curr Opinion in Immunol 5 256-262 (1993) and Pluckthun, Immunol Revs , 130 151 -188 ( 1992) In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al , Nature, 348 552-554 (1990) Clackson et al , Nature, 352 624-628 (1991 ) and Marks et al , J Mol Biol , 222 581 -597 (1991) describe the isolation of muπne and human antibodies, respectively, using phage libraries Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks etal , Bio/Technology, 10 779-783 [1992]), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al , Nuc Acids Res , 21 2265-2266 [1993]) Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies The DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous muπne sequences (U S Patent No 4,816,567, Morrison, etal , Proc NatlAcad Sci USA, 81 6851 [1984]), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobuhn polypeptide Typically such non-immunoglobuhn polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeπc bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen- combining site having specificity for a different antigen (in) Humanized and human antibodies Methods for humanizing non-human antibodies are well known in the art Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human These non- human amino acid residues are often refeπed to as "import" residues, which are typically taken from an "import" variable domain Humanization can be essentially performed following the method of Winter and co-workers (Jones et al . Nature, 321 522-525 (1986), Riechmann et al Nature, 332 323 327 (1988) Verhoeyen et al , Science, 239 1534- 1536 [ 1988]), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such "humanized" antibodies are chimeπc antibodies (U S Patent No 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity According to the so-called "best-fit" method, the sequence ofthe variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences The human sequence which is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al , J Immunol , 151 2296 (1993), Chothia et al , J Mol Biol , 196 901 [ 1987]) Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains The same framework may be used for several different humanized antibodies (Carter et al , Proc Natl Acad Sci USA, 89 4285 (1992), Presta et al , J Immnol , 151 2623 [1993]) It is further important that antibodies be humanized with retention of high affinity tor the antigen and other favorable biological properties To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, t e , the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antιgen(s), is achieved In general, the CDR residues are directly and most substantially involved in influencing antigen binding Alternatively, it is now possible to produce transgenic animals (e g , mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J H ) gene in chimeπc and germ-line mutant mice results in complete inhibition of endogenous antibody production Transfer of the human germ-line immunoglobulin gene array in such germ-hne mutant mice will result in the production of human antibodies upon antigen challenge See, e g , Jakobovits et al , Proc Natl Acad Sci USA, 90 2551 ( 1993), Jakobovits et al Nature, 362 255-258 (1993), Bruggermann et al , Year in lmmuno , 7 33 (1993) Human antibodies can also be derived from phage-display libraries (Hoogenboom et al , J Mol Biol , 227 381 (1991), Marks et al , J Mol Biol , 222 581-597 [1991]) (iv) Antibody fragments Various techniques have been developed for the production of antibody fragments Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e g , Morimoto et al , Journal of Biochemical and Biophysical Methods 24 107-117 (1992) and Brennan et al , Science, 229 81 [1985]) However, these fragments can now be produced directly by recombinant host cells For example the antibody fragments can be isolated from the antibody phage libraries discussed above Alternatively, Fab'-SH fragments can be directly recovered from£ coli and chemically coupled to form F(ab fragments (Carter et al , Bio/Technology 10 163-167 [1992]) According to another approach, F(ab τ ) 2 fragments can be isolated directly from recombinant host cell culture Other techniques for the production of 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 (v) Bispec c antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes Exemplary bispecific antibodies may bind to two different epitopes of the ErbB2 protein For example, one arm may bind an epitope in Domain 1 of ErbB2 such as the 7C2/7F3 epitope, the other may bind a different ErbB2 epitope, e g the 4D5 epitope Other such antibodies may combine an ErbB2 binding site with binding sιte(s) for EGFR, ErbB3 and/or ErbB4 Alternatively, an antι-ErbB2 arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e g CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD 16) so as to focus cellular defense mechanisms to the ErbB2-expressιng cell Bispecific antibodies may also be used to localize cytotoxic agents to cells which express ErbB2 These antibodies possess an ErbB2-bιndιng arm and an arm which binds the cytotoxic agent (e g sapoπn, anti-interferon-α, vinca alkaloid, πcin A chain, methotrexate or radioactive isotope hapten) Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e g F(ab') 2 bispecific antibodies) Methods for making bispecific antibodies are known in the art Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al , Nature, 305 537-539 [1983]) Because of the random assortment of immunoglobulin heavy and light chains, these hybπdomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low Similar procedures are disclosed in WO 93/08829, and in Traunecker et al , EMBO 7 , 10 3655-3659 (1991) According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide the optimum yields It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain- light chain pair (providing a second binding specificity) in the other arm It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation This approach is disclosed in WO 94/04690 For further details of generating bispecific antibodies see, for example, Suresh et al , Methods in Enzymology , 121 210 (1986) According to another approach described in W096/2701 1 , the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture The preferred interface comprises at least a part of the C H 3 domain of an antibody constant domain In this method, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e g tyrosine or tryptophan) Compensatory "cavities" of identical or similar size to the large side chaιn(s) are created on the interface ofthe 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 the heterodimer over other unwanted end-products such as homodimers Bispecific antibodies include cross-linked or "heteroconjugate" antibodies For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (US Patent No 4,676 980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089) Heteroconjugate antibodies may be made using any convenient cross-linking methods Suitable cross-linking agents are well known in the art, and are disclosed in US Patent No 4,676,980, along with a number of cross-linking techniques Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature For example, bispecific antibodies can be prepared using chemical linkage Brennan et al , Science, 229 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes Recent progress has facilitated the direct recovery of Fab'-SH fragments from E coli, which can be chemically coupled to form bispecific antibodies Shalaby et al , J Exp Med , 175 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab ) 2 molecule Each Fab' fragment was separately secreted from E coli and subjected to directed chemical coupling in vitro to form the bispecific antibody The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described For example, bispecific antibodies have been produced using leucine zippers Kostelny et al . J Immunol , 148(5) 1547-1553 (1992) The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers This method can also be utilized for the production of antibody homodimers The "diabody" technology described by Holhnger etal , Proc Natl Acad Sci USA, 90 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments The fragments comprise a heavy-chain variable domain (V H ) connected to a light- chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported See Gruber et al , J Immunol , 152 5368 (1994) Antibodies with more than two valencies are contemplated For example, tπspecific antibodies can be prepared Tutt et al , J Immunol \M 60 (1991 ) (vi) Screening for antibodies with the desired properties Techniques for generating antibodies have been described above Those antibodies having the characteristics described herein are selected To select for antibodies which induce cell death, loss of membrane integrity as indicated by, e g , PI, trypan blue or 7AAD uptake is assessed relative to control The preferred assay is the "PI uptake assay using BT474 cells" According to this assay, BT474 cells (which can be obtained from the American Type Culture Collection [Rockville. MD]) are cultured in Dulbecco's Modified Eagle Medium (D-MEM) Ham's F-12 (50 50) supplemented with 10% heat-inactivated FBS (Hyclone) and 2 mM L-glutamine (Thus, the assay is performed in the absence of complement and immune effector cells) The BT474 cells are seeded at a density of 3 x 10 per dish in 100 x 20 mm dishes and allowed to attach overnight The medium is then removed and replaced with fresh medium alone or medium containing lOμg/ml of the appropriate MAb The cells are incubated for a 3 day time period Following each treatment, monolayers are washed with PBS and detached by trypsinization Cells are then centrifuged at 1200rpm for 5 minutes at 4"C, the pellet resuspended in 3 ml ice cold Ca "+ binding buffer (10 mM Hepes, pH 7 4, 140 mM NaCl, 2 5 mM CaCl 2 ) and ahquoted into 35 mm strainer-capped 12 x 75 tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps Tubes then receive PI (lOμg/ml) Samples may be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson) Those antibodies which induce statistically significant levels of cell death as determined by PI uptake are selected In order to select for antibodies which induce apoptosis, an "annexin binding assay using BT474 cells" is available The BT474 cells are cultured and seeded in dishes as discussed in the preceding paragraph The medium is then removed and replaced with fresh medium alone or medium containing lOμg/ml of the MAb Following a three day incubation period, monolayers are washed with PBS and detached by trypsinization Cells are then centrifuged, resuspended in Ca + binding buffer and ahquoted into tubes as discussed above for the cell death assay Tubes then receive labeled annexin (e g annexin V-FTIC) (1 μg/ml) Samples may be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson) Those antibodies which induce statistically significant levels of annexin binding relative to control are selected as apoptosis-inducing antibodies In addition to the annexin binding assay, a "DNA staining assay using BT474 cells" is available In order to perform this assay, BT474 cells which have been treated with the antibody of interest as described in the preceding two paragraphs are incubated with 9μg/ml HOECHST 33342™ for 2 hr at 37°C, then analyzed on an EPICS ELITE™ flow cytometer (Coulter Corporation) using MODFIT LT™ software (Verity Software House) Antibodies which induce a change in the percentage of apoptotic cells which is 2 fold or greater (and preferably 3 fold or greater) than untreated cells (up to 100% apoptotic cells) may be selected as pro-apoptotic antibodies using this assay To screen for antibodies which bind to an epitope on ErbB2 bound by an antibody of interest a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane ( 1988), can be performed Alternatively, epitope mapping can be performed by methods known in the art To identify anti ErbB2 antibodies which inhibit growth of SKBR3 cells in cell culture by 50-100%, the SKBR3 assay described in WO 89/06692 can be performed According to this assay, SKBR3 cells are grown in a 1 1 mixture of F12 and DMEM medium supplemented with 10% fetal bovine serum, glutamine and penicilhnstreptomycin The SKBR3 cells are plated at 20,000 cells in a 35mm cell culture dish (2 mls/35 mm dish) 2 5μg/ml of the antι-ErbB2 antibody is added per dish After six days, the number of cells, compared to untreated cells are counted using an electronic COULTER™ cell counter Those antibodies which inhibit growth of the SKBR3 cells by 50-100% are selected for combination with the apoptotic antibodies as desired (vu) Effector function engineering It may be desirable to modify the antibody of the invention with respect to effector function so as to enhance the effectiveness of the antibody in treating cancer, for example For example, cysteine resιdue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region The homodimeric antibody thus generated may have improved lnternahzation capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See Caron et al , J Exp Med 176 1191 - 1195 ( 1992 ) and Shopes, B J Immunol 148 2918-2922 (1992) Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al Cancer Research 53 2560-2565 (1993) Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities See Stevenson et al Anti-Cancer Drug Design 3 219-230 (1989) (vui) Immunoconjugates The invention also pertains to immunoconjugates comprising the antibody described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i e a radioconjugate) Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), πcin A chain, abπn A chain, modeccm A chain, alpha-sarcin, Aleuntes fordu proteins, dianthin proteins, Phytolaca amencana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaπa officinahs inhibitor, gelonin, mitogelhn, restπctocin, phenomycin, enomycin and the tπcothecenes A variety of radionuchdes are available for the production of radioconjugated antι-ErbB2 antibodies Examples include Bi, I, In, Y and Re Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succιnιmιdyl-3-(2-pyπdyldιthιol) propionate (SPDP), liriinofhiolane (IT), bifunctional derivatives of lmidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazomum derivatives (such as bιs-(p-dιazonιumbenzoyl)-efhylenedιamιne), dnsocyanates (such as tolyene 2,6-dnsocyanate), and bis-active fluorine compounds (such as 1 ,5-dιfluoro-2,4-dιnιtrobenzene) For example, a πcin lmmunotoxin can be prepared as described in Vitetta et al Science 238 1098 (1987) Carbon- 14-labeled l-ιsothιocyanatobenzyl-3- methyldiethylene tπaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody See WO 94/1 1026 In another embodiment, the antibody may be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e g avidin) which is conjugated to a cytotoxic agent (e g a radionucleotide) (ix) Immunoliposomes The antι-ErbB2 antibodies disclosed herein may also be formulated as immunoliposomes Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al , Proc Natl Acad Sci USA, 82 3688 ( 1985), Hwang et al , Proc Natl Acad Sci USA, 11 4030 (1980), and U S Pat Nos 4,485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U S Patent No 5 013,556 Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylchohne, cholesterol and PEG-deπ vatized phosphatidylefhanolamine (PEG-PE) Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al , J Biol Chem 257 286-288 (1982) via a disulfide interchange reaction A chemotherapeutic agent is optionally contained within the hposome See Gabizon et al , J National Cancer Inst 81 (19)1484 (1989) (x) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT) The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e g a peptidyl chemotherapeutic agent, see WO 81/01 145) to an active anti-cancer drug See, for example, WO 88/07378 and U S Patent No 4,975,278 The enzyme component ofthe lmmunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs, arylsulfatase useful for converting sulfate - containing prodrugs into free drugs, cytosine deaminase useful for converting non-toxic 5-fluorocytosιne into the anti-cancer drug, 5-fluorouracιl, proteases, such as serratia protease, thermolysin, subtihsin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs, D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate- cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs, β-lactamase useful for converting drugs deπvatized with β-lactams into free drugs, and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs deπvatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, e g , Massey, Nature 328 457-458 [1987]) Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population The enzymes of this invention can be covalently bound to the antι-ErbB2 antibodies by techniques well known in the art such as the use of the heterobifunctional crosshnking reagents discussed above Alternatively, fusion proteins compπsing at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e g , Neuberger et al , Nature, 312 604-608 [1984]) (xi) Antibody-salvage receptor binding epitope fusions In certain embodiments of the invention, it may be desirable to use an antibody fragment, rather than an intact antibody, to increase tumor penetration, for example In this case, it may be desirable to modify the antibody fragment in order to increase its serum half life This may be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e g by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e g , by DNA or peptide synthesis) A systematic method for preparing such an antibody variant having an increased in \ ι\o half-life comprises several steps The first involves identifying the sequence and conformation of a salvage receptor binding epitope of an Fc region of an IgG molecule Once this epitope is identified, the sequence of the antibody of interest is modified to include the sequence and conformation of the identified binding epitope Alter the sequence is mutated, the antibody variant is tested to see if it has a longer in vivo half-life than that of the original antibody If the antibody variant does not have a longer in vivo half-life upon testing, its sequence is further altered to include the sequence and conformation of the identified binding epitope The altered antibody is tested for longer in vivo half-life, and this process is continued until a molecule is obtained that exhibits a longer in vivo half- life The salvage receptor binding epitope being thus incorporated into the antibody of interest is any suitable such epitope as defined above, and its nature will depend, e g , on the type of antibody being modified The transfer is made such that the antibody of interest still possesses the biological activities described herein The epitope preferably constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment Even more preferably, three or more residues from one or two loops of the Fc domain are transferred Still more preferred, the epitope is taken from the CH2 domain of the Fc region (e g , of an IgG) and transferred to the CHI , CH3, or V H region, or more than one such region, of the antibody Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the C L region or V L region, or both, of the antibody fragment In one most preferred embodiment, the salvage receptor binding epitope comprises the sequence (5 to 3') PKNSSMISNTP (SEQ ID NO 3), and optionally further comprises a sequence selected from the group consisting of HQSLGTQ (SEQ ID NO 4), HQNLSDGK (SEQ ID NO 5), HQNISDGK (SEQ ID NO 6), or VISSHLGQ (SEQ ID NO 7), particularly where the antibody fragment is a Fab or F(ab') 2 . In another most preferred embodiment, the salvage receptor binding epitope is a polypeptide containing the sequence(s)(5' to 3^ HQNLSDGK (SEQ ID NO 5), HQNISDGK (SEQ ID NO 6), or VISSHLGQ (SEQ ID NO 7) and the sequence PKNSSMISNTP (SEQ ID NO 3) (xn) Purification of antι-ErbB2 antibody When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration Carter et al , Bio/Technology 10 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of £ coli Briefly, cell paste is thawed in the presence of sodium acetate (pH 3 5), EDTA, and phenylmethylsulfonylfluoπde (PMSF) over about 30 min Cell debris can be removed by centrifugation Where the antibody is secreted into the medium, supernatants from such expression systems are preferably first concentrated using a commercially available protein concentration filter, for example, an Amicon or Milhpore Pelhcon ultrafiltration unit A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody Protein A can be used to purify antibodies that are based on human γl , γ2, or γ4 heavy chains (Lindmark et al , J Immunol Meth 62 1-13 [1983]) Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al , EMBO J 5 15671575 [1986]) The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available Mechanically stable matrices such as controlled pore glass or poly(styrenedιvιnyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose Where the antibody comprises a C H 3 domain, the Bakerbond ABX™ resin (J T Baker, Philhpsburg, NJ) is useful for purification Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on hepaπn SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2 5-4 5, preferably performed at low salt concentrations (e g from about 0-0 25M salt) III. Determination of anti-ErbB2 antibody concentration in serum The following non-limiting assay is useful for determining the presence of and to quantitate the amount of specific rhuMAb HER2 (humanized antι-pl 85 HER2 monoclonal antibody, including HERCEPTIN ® antι-ErbB2 antibody) in a body fluid of a mammal including, but not limited to, serum, ammotic fluid, milk, umbilical cord serum, ocular aqueous and vitreous liquids, and ocular vitreous gel Plate Binding Activity Assay for rhuMAb HER2 (Humanized Antι-pl 85 HER2 Monoclonal Antibody The method of assaying rhuMAb HER2 described herein is meant as an example of such a method and is not meant to be limiting A standardized preparation of rhuMAb HER2 (Genentech, Inc , South San Francisco, CA), controls, and serum samples were diluted with Assay Diluent (PBS/05% BSA/005% Polysorbate 20/0 01% Thimerosal) The dilutions of standardized rhuMAb HER2 were prepared to span a range of concentrations useful for a standard curve The samples were diluted to fall within the standard curve HFR2 An aliquot of Coat Antigen in Coating buffer (recombinant p 185 (Genentech, Inc ) in 0 05 M sodium carbonate buffer) was added to each well of a microtiter plate and incubated at 2-8 °C for 12-72 hours The coating solution was removed and each well was washed six times with water, then blotted to remove excess water An aliquot of Assay Diluent was added to each well and incubated for 1 -2 hours at ambient temperature with agitation The wells were washed as in the previous step Aliquots of diluted standard, control and sample solutions were added to the wells and incubated at ambient temperature for 1 hour with agitation to allow binding of the antibody to the coating antigen The wells are washed again with water as in previous steps Horse radish peroxidase conjugate (HRP-conjugate, Goat anti-human IgG Fc conjugated to horseradish peroxidase, Organon Teknika catalog #55253 or equivalent) was diluted with Assay Diluent to yield an appropriate optical density range between the highest and lowest standards An aliquot ofthe HRP conjugate solution was added to each well and incubated at ambient temperature for 1 hour with agitation The wells were washed with water as in previous steps An aliquot of Substrate Solution (o-phenylenediamine (OPD) 5 mg tablet (Sigma P6912 or equivalent) in 12 5 ml 4 mM H 2 0, in PBS) was added to each well and incubated for a sufficient period of time (approximately 8-10 minutes) in the dark at ambient temperature to allow color development The reaction was stopped with an aliquot of 4 5 N sulfuπc acid Optical density was read at 490-492 nm for detection absorbance and 405 nm for reference absorbance The standard curve data are plotted and the results for the controls and samples are determined from the standard curve IV. Pharmaceutical Formulations Therapeutic formulations of the antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington 's Pharmaceutical Sciences 16th edition, Osol, A Ed [1980]), in the form of lyophilized formulations or aqueous solutions Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl 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, disacchaπdes, and other carbohydrates including glucose, mannose, or dextπns, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt- forming counter-ions such as sodium, metal complexes (e g Zn-protein complexes), and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG) Preferred lyophilized antι-ErbB2 antibody formulations are described in WO 97/04801, expressly incorporated herein be reference The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other For example, it may be desirable to further provide antibodies which bind to EGFR, ErbB 2 (e g an antibody which binds a different epitope on ErbB2), ErbB3, ErbB4, or vascular endothelial growth factor (VEGF) in the one formulation Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended 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, nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington 's Pharmaceutical Sciences 16th edition, Osol, A Ed (1980) The formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes Sustained-release preparations may 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 (for example, poly(2-hydroxyethyl-methacrylate), or poly(vιnylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate), and poly-D-(-)-3-hydroxybutyπc acid While polymers such as ethylene- vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C. resulting in a loss of biological activity and possible changes in lmmunogenicity Rational strategies can be devised for stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through fhio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophihzing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions V. Treatment with the Anti-ErbB2 Antibodies It is contemplated that, according to the present invention, the antι-ErbB2 antibodies may be used to treat various conditions characterized by overexpression and/or activation of the ErbB2 receptor Exemplary conditions or disorders include benign or malignant tumors (e g renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas, sarcomas, ghoblastomas, and various head and neck tumors), leukemias and lymphoid malignancies, other disorders such as neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoehc disorders, and inflammatory, angiogenic and lmmunologic disorders The antibodies of the invention are administered to a human patient, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, mtrasynovial, intrathecal, oral, topical, or inhalation routes Intravenous or subcutaneous administration of the antibody is preferred The treatment of the present invention involves the administration of an antι-ErbB2 antibody to an animal or human patient, followed at intervals by subsequent doses of equal or smaller doses such that a target serum concentration is achieved and maintained during treatment Preferably, maintenance doses are delivered by bolus delivery, preferably by subcutaneous bolus administration, making treatment convenient and cost-effective for the patient and health care professionals Where combined administration of a chemotherapeutic agent (other than an antracychne) is desired, the combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed , M C Perry, Williams & Wilkins, Baltimore, MD (1992) The chemotherapeutic agent may precede, or follow administration ofthe antibody or may be given simultaneously therewith The antibody may be combined with an anti-estrogen compound such as tamoxifen or an anti-progesterone such as onapπstone (see, EP 616 812) in dosages known for such molecules It may be desirable to also administer antibodies against other tumor associated antigens, such as antibodies which bind to the EGFR, ErbB3, ErbB4, or vascular endothelial growth factor (VEGF) Alternatively, or additionally, two or more antι-ErbB2 antibodies may be co-administered to the patient Sometimes, it may be beneficial to also administer one or more cytokines to the patient The ErbB2 antibody may be co-administered with a growth inhibitory agent For example, the growth inhibitory agent may be administered first, followed by the ErbB2 antibody However, simultaneous administration, or administration of the ErbB2 antibody first is also contemplated Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and anti-ErbB 2 antibody In addition to the above therapeutic regimens, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy For the prevention or treatment of disease, the appropπate dosage of antι-ErbB2 antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician The antibody is suitably administered to the patient at one time or over a series of treatments Where the treatment involves a series of treatments, the initial dose or initial doses are followed at daily or weekly intervals by maintenance doses Each maintenance dose provides the same or a smaller amount of antibody compared to the amount of antibody administered in the initial dose or doses Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e g 0 l-20mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs The progress of this therapy is easily monitored by conventional techniques and assays According to the invention, dosage regimens may include an initial dose of antι-ErbB2 of 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered by intravenous or subcutaneous infusion, followed by subsequent weekly maintenance doses of 2 mg/kg by intravenous infusion, intravenous bolus injection, subcutaneous infusion, or subcutaneous bolus injection Where the antibody is well-tolerated by the patient, the time of infusion may be reduced Alternatively, the invention includes an initial dose of 12 mg/kg antι-ErbB2 antibody, followed by subsequent maintenance doses of 6 mg/kg once per 3 weeks Another dosage regimen involves an initial dose of 8 mg/kg antι-ErbB2 antibody, followed by 6 mg/kg once per 3 weeks Still another dosage regimen involves an initial dose of 8 mg/kg antι-ErbB2 antibody, followed by subsequent maintenance doses of 8 mg/kg once per week or 8 mg/kg once every 2 to 3 weeks As an alternative regimen, initial doses of 4 mg/kg antι-ErbB2 antibody may be administered on each of days 1 , 2 and 3, followed by subsequent maintenance doses of 6 mg/kg once per 3 weeks An additional regimen involves an initial dose of 4 mg/kg antι-ErbB2 antibody, followed by subsequent maintenance doses of 2 mg/kg twice per week, wherein the maintenance doses are separated by 3 days Alternatively, the invention may include a cycle of dosing in which delivery of antι-ErbB2 antibody is 2-3 times per week for 3 weeks The 3 week cycle is preferably repeated a necessary to achieve suppression of disease symptoms The invention further includes a cyclic dosage regimen in which delivery of antι-ErbB2 antibody is daily for 5 days According to the invention, the cycle is preferably repeated as necessary to achieve suppression of disease symptoms Further information about suitable dosages is provided in the Examples below VI. Articles of Manufacture In another embodiment of the invention, an article of manufacture containing materials useful for the treatment of the disorders described above is provided The article of manufacture comprises a container, a label and a package insert Suitable containers include, for example, bottles, vials, syringes, etc The containers may be formed from a variety of materials such as glass or plastic The container holds a composition which is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) At least one active agent in the composition is an anti-ErbB 2 antibody The label on, or associated with, the container indicates that the composition is used for treating the condition of choice The article of manufacture may further comprise a second container compπsing a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes In addition, the article of manufacture may comprise a package inserts with instructions for use, including, e g , a warning that the composition is not to be used in combination with anthacychne-type chemotherapeutic agent, e g doxorubicin or epirubicin Deposit of Materials The following hybridoma cell lines have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA (ATCC) Antibody Designation ATCC No. Deposit Date 7C2 ATCC HB-12215 October 17, 1996 7F3 ATCC HB-12216 October 17, 1996 4D5 ATCC CRL 10463 May 24, 1990 2C4 ATCC HB- 12697 April 8, 1999 Further details of the invention are illustrated by the following non-limiting Examples EXAMPLES Example 1: Preparation and Efficacy of HERCEPTIN ® Anti-ErbB2 Antibody Materials and Methods Antι-ErbB2 monoclonal antibody The antι-ErbB2 IgG,κ muπne monoclonal antibody 4D5, specific for the extracellular domain of ErbB2, was produced as described in Fendly et al , Cancer Research 5o 1550-1558 (1990) and WO89/06692 Briefly, NIH 3T3/HER2-3 400 cells (expressing approximately 1 x 10 ErbB2 molecules/cell) produced as described in Hudziak et al , Proc Natl Acad Sci (USA) 84 7159 ( 1987) were harvested with phosphate buffered saline (PBS) containing 25mM EDTA and used to immunize BALB/c mice The mice were given injections l p of 10 7 cells in 0 5 ml PBS on weeks, 0, 2, 5 and 7 The mice with antisera that immunoprecipitated 32 P-labeled ErbB2 were given l p injections of a wheat germ agglutinin-Sepharose (WGA) purified ErbB2 membrane extract on weeks 9 and 13 This was followed by an l v injection of 0 1 ml ofthe ErbB2 preparation and the splenocytes were fused with mouse myeloma line X63-Ag8 653 Hybridoma supernatants were screened for ErbB2-bιndιng by ELISA and radioimmunoprecipitation MOPC-21 (IgG l), (Cappell, Durham, NC), was used as an isotype-matched control The treatment was performed with a humanized version ofthe muπne 4D5 antibody (HERCEPTIN anti- ErbB 2 antibody) The humanized antibody was engineered by inserting the complementarity determining regions of the muπne 4D5 antibody into the framework of a consensus human immunoglobulin IgG l (IgG j ) (Carter et al Proc Natl Acad Sci USA 89 4285-4289 [1992]) The resulting humanized antι-ErbB2 monoclonal antibody has HFR high affinity for pi 85 " (Dillohiation constant [K d ]=0 1 nmol/L), markedly inhibits, in vitro and in human HFR2 xenografts, the growth of breast cancer cells that contain high levels of pi 85 , induces antibody-dependent cellular cytotoxicity (ADCC), and has been found clinically active, as a single agent, in patients with ErbB2 overexpressing metastatic breast cancers that had received extensive pπor therapy HERCEPTIN" antι-ErbB2 antibody is produced by a genetically engineered Chinese Hamster Ovary (CHO) cell line, grown in large scale, that secretes the antibody into the culture medium The antibody is purified from the CHO culture media using standard chromatographic and filtration methods Each lot of antibody used in this study was assayed to verify identity, purity, and potency, as well as to meet Food and Drug Administration requirements for sterility and safety Eligibility Criteria Patients had to fulfill all of the following criteria to be eligible for study admission - Metastatic breast cancer - Overexpression of the ErbB 2 (HER2) oncogene (2+ to 3+ as determined by immunohistochemistry or fluorescence in situ hybridization (FISH) [Tumor expression of ErbB2 can be determined by immunohistochemical analysis, as previously described (Slamon et al , [1987] and [1989], supra), of a set of thin sections prepared from the patient's paraffin-archived tumor blocks The primary detecting antibody used is murine 4D5 MAb, which has the same CDRs as the humanized antibody used for the treatment Tumors are considered to overexpress ErbB 2 if HFR2 at least 25% of tumor cells exhibit characteristic membrane staining for pi 85 ] - Bidimensionally measurable disease (including lytic bone lesions) by radiographic means, physical examination, or photographs Measurable disease was defined as any mass reproducibly measurable in two perpendicular diameters by physical examination, X-ray (plain films), computerized tomography (CT), magnetic resonance imaging (MRI), ultrasound, or photographs Osteoblastic metastases, pleural effusions, or ascites were not considered to be measurable Measurable lesions must be at least 1 cm in greatest dimension Enumeration of e valuable sites of metastatic disease and number of lesions in an evaluable site (e g lung) had to be recorded on the appropriate Case Report Form (CRF) If a large number of pulmonary or hepatic lesions were present, the six largest lesions per site were followed - The ability to understand and willingness to sign a written informed consent form - Women > 18 years - Suitable candidates for receiving concomitant cytotoxic chemotherapy as evidenced by screening laboratory assessments of hematologic, renal, hepatic, and metabolic functions Exclusion Criteria Patients with any of the following were excluded from study entry - Prior cytotoxic chemotherapy for metastatic breast cancer - Patients may have received prior hormonal therapy (e g tamoxifen) for metastatic disease or cytotoxic therapy in the adjuvant setting - Concomitant malignancy that has not been curatively treated - A performance status of <60% on the Karnofsky scale - Pregnant or nursing women, women of childbeaπng potential, unless using effective contraception as determined by the investigator - Bilateral breast cancer (either both primary tumors must have 2+ to 3+ HER2 overexpression, or the metastatic site must have 2+ to 3+ HER2 overexpression) - Use of investigational or unlicensed agents within 30 days prior to study entry - Clinically unstable or untreated metastases to the brain (e g requiring radiation therapy) Based upon the foregoing criteria, 469 patients were chosen, and enrolled in the study Half the patients ( (ssttrraattiiffiued by chemotherapy) were randomized to additionally receive the HERCEPTIN antι-ErbB2 antibody (see below) Administration and Dosage Antι-ErbB2 Antibody On day 0, a 4 mg/kg dose of humanized antι-ErbB2 antibody (HERCEPTIN , H) was administered intravenously, over a 90-mιnute period Beginning on day 7, patients received weekly administration of 2 mg/kg antibody (1 v ) over a 90-mιnute period Chemotherapy The patients received one of two chemotherapy regimens for a minimum of six cycles, provided their disease was not progressing a) cyclophosphamide and doxorubicin or epirubicin (AC), if patients have not received anthracycline therapy in the adjuvant setting, or b) paclitaxel (T, TAXOL ), if patients have received any anthracycline therapy in the adjuvant setting The initial dose of the HERCEPTIN antι-ErbB2 antibody preceded the first cycle of either chemotherapy regimen by 24 hours Subsequent doses of the antibody were given immediately before chemotherapy administration, if the initial dose of the antibody was well tolerated If the first dose of the antibody was not well tolerated, subsequent infusions continued to precede chemotherapy administration by 24 hours Patients were permitted to continue receiving chemotherapy beyond six cycles if, in the opinion of the treating physician, they were continuing to receive treatment benefit Cyclophosphamide (600 mg/m ) was given either by IV push over a minimum period of 3 minutes or by infusion over a maximum period of 2 hours 2 2 Doxorubicin (60 mg/m ) or epirubicin (75 mg/m " ) were given either by slow iv push over a minimum period of 3-5 minutes or by infusion over a maximum period of 2 hours, according to institutional protocol Paclitaxel (TAXOL ) was given at a dose of 175 mg/m" over 3 hours by intravenous administration All patients receiving paclitaxel were premedicated with dexamethasone (or its equivalent) 20 mg x 2, administered orally 12 and 6 hours pπor to paclitaxel, diphenhydramine (or its equivalent) 50 mg, iv, administered 30 minutes prior to paclitaxel, and dimetidine (or another H 2 blocker) 300 mg, iv, administered 30 minutes prior to paclitaxel Response Criteria Progressive Disease Objective evidence of an increase of 25% or more in any measurable lesion Progressive disease also includes those instances when new lesions have appeared For bone lesions, progression is defined as a 25% increase in objective measurement by plain film, CT, MRI, symptomatic new lesions not due to fracture, or requirement for palliative radiotherapy Complete Response Disappearance of all radiographically and/or visually apparent tumor for a minimum of 4 weeks Skin and chest wall complete responses had to be confirmed by biopsy Partial Response A reduction of at least 50% in the sum of the products of the perpendicular diameters of all measurable lesions for a minimum period of 4 weeks No new lesions may have appeared, nor may any lesions have progressed in size Minor Response A reduction of 25% to 49% in the sum of the products of the perpendicular diameters of all measurable lesions No new lesions may have appeared, nor may any lesions have progressed in size Stable Disease No change of greater than 25% in the size of measurable lesions No lesions may have appeared Time to disease progression (TTP) was calculated Irom the beginning of therapy to progression Confidence limits for response rates were calculated using the exact method for a single proportion (Fleiss, JL, Statistical Methods for Rates and Proportions (ed 2), New York, NY, Wiley, 1981 , pp 13-17) Results At a median follow-up of 10 5 months, assessments of time to disease progression (TTP in months) and ® response rates (RR) showed a significant augmentation of the chemotherapeutic effect by HERCEPTIN anti- ErbB2 antibody, without increase in overall severe adverse events (AE) Table 1 HERCEPTIN ® Antι-ErbB2 Antibody Efficacy Enrolled TTP(months) RR(%) AE(%) CRx 234 5 5 36 2 66 CRx +H 235 8 6* 62 00** 69 AC 145 6 5 42 1 71 AC+H 146 9 0 64 9 68 T 89 4 2 25 0 59 T+H 89 7 1 57 3 70 * p<0 001 by log-rank test, ** p<0 01 by X 2 test, CRx chemotherapy, AC anthracychne/cyclophosphamide treatment, H HERCEPTIN® antι-ErbB2 antibody, T TAXOL® A syndrome of myocardial dysfunction similar to that observed with anthracychnes was reported more commonly with a combined treatment of AC+H (18% Grade 3/4) than with AC alone (3%) T (0%), or T+H (2%) These data indicate that the combination of antι-ErbB2 antibody treatment with chemotherapy markedly increases the clinical benefit, as assessed by response rates and the evaluation of disease progression However, due to the increased cardiac side-effects of doxorubicin or epirubicin, the combined use of anthracychnes with anti- ErbB2 antibody therapy is contraindicated The results, taking into account risk and benefit, favor treatment with HERCEPTIN antι-ErbB2 antibody and paclitaxel (TAXOL ) where a combined treatment regimen is desired Example 2: Pharmacokinetic and Pharmacodvnamic Properties of Anti-ErbB2 Antibody (HERCEPTIN 8 ) ® HERCEPTIN antι-ErbB2 antibody was administered by intravenous infusion to human patients selected according to the criteria provided in Example 1 An initial dose of 4 mg/kg HERCEPTIN " antι-ErbB2 ® antibody was delivered by intravenous infusion, followed by subsequent l v infusions of 2 mg/kg HERCEPTIN antι-ErbB2 antibody weekly for several weeks Two hundred thirteen patients began this treatment regimen and serum drug concentration was obtained beyond 8 weeks for fewer than 90 patients as selective discontinuation of patients with rapidly progressing disease occurred Of the 213 patients who began treatment, serum trough concentration data were available for 80 patients at Week 12, for 77 patients at Week 16 for 44 patients at Week 20, for 51 patients at Week 24, for 25 patients at Week 28, for 23 patients at Week 32, and for 37 patients at Week 36 ® HERCEPTIN antι-ErbB2 antibody trough serum concentrations for Weeks 0-36 ® The HERCEPTIN antι-ErbB2 antibody trough serum concentrations (μg/ml, mean ± SE) from Week 2 through Week 36 are plotted in Fig 3 (dark circles) The number of patients was fairly constant because data from patients discontinued from the program due to rapidly progressing disease were excluded from this analysis Trough serum concentrations tended to increase through Week 12 and tended to plateau after that time ® HERCEPTIN antι-ErbB2 antibody trough and peak serum concentrations for Weeks 1-8 ® Some HERCEPTIN antι-ErbB2 antibody serum concentration data were available for 212 of the original ® 213 patients Trough and peak serum concentration data reflecting the first HERCEPTIN antι-ErbB2 antibody infusion were available for 195 ofthe 212 patients For the seventh infusion, trough serum concentration data were available for 137/212 patients and peak serum concentration data were available for 1 14/212 patients Table 2 presents a summary of statistics from trough and peak serum concentrations for the first 8 weeks of treatment Peak ® samples were drawn shortly after the end of HERCEPTIN antι-ErbB2 antibody administration, trough samples were drawn prior to the subsequent dose (l e , 1 week later) Serum concentrations of HERCEPTIN antι-ErbB2 antibody were determined as disclosed herein Table 2 HERCEPTIN ® Anti-ErbB 2 Antibody Trough and Peak Serum Concentrations for the First 8 Weeks of Treatment (μg/ml) The data in Table 2 suggest that there was an increase in trough serum concentration over time Of the many patients studied, there were 18 patients for whom the trough concentrations did not exceed 20 μg/ml from ® Week 2 through Week 8 A HERCEPTIN antι-ErbB2 antibody trough serum concentration of 20 μg/ml was nominally targeted for these studies based on prior pharmacologic studies in animals and exploratory analyses in clinical trials ® Patient response status was evaluated relative to serum concentration of HERCEPTIN antι-ErbB2 antibody For this purpose, mean serum concentration (an average of troughs and peaks) was calculated for various times and patient response status (where the patient response status was determined by an independent Response Evaluation Committee) The increase in serum concentration between Weeks 2 and 8 appeared to be greater in responders than in nonresponders, suggesting that there is a relationship between response status and ® HERCEPTIN antι-ErbB2 antibody serum concentration A statistical analysis (analysis of variance) of trough serum concentration values at Week 2 and an average of Weeks 7 and 8 in relation to response status indicated a highly significant relationship between response status and average trough of Weeks 7 and 8 (p < 0 001 ) The results indicated that there was a significant difference between the trough serum concentration (average troughs of Weeks 7 and 8) in the responders and nonresponders trough concentrations were 60 ± 20 μg/ml in the responders versus 44 ± 25 μg/ml in the nonresponders (mean ± SD) HER2 overexpression level and type of metastatic sites were associated with significant differences in trough serum concentrations At Week 2, patients with 2+ HER2 overexpression had significantly higher trough serum concentrations (n = 40, mean = 28 8 μg/ml, SD = 10 4) compared with patients with 3+ HER2 overexpression (n = 155, mean = 24 1 μg/ml, SD = 13 1 ) This difference in the average trough serum concentrations for Weeks 7 and 8 was no longer statistically significant Further, at Week 2, patients with superficial disease had significantly higher trough serum concentrations (n = 12, mean 34 1 μg/ml, SD = 12 0) compared with patients with visceral disease (n = 183, mean = 24 4 μg/ml, SD = 12 6) This difference in the average trough serum concentrations for Weeks 7 and 8 was significant These data indicate that the rise in trough serum concentrations between Weeks 2 and 7/8 occurs for human patients with various disease profiles In a subsequent, similarly designed study, human breast cancer patients were treated with a loading dose of 8 mg/kg followed by maintenance doses of 4 mg/kg weekly The results of this preliminary human study indicated that an 8mg/kg load 4mg/kg weekly maintenance regimen was efficacious in reducing tumor volume in the patients The data disclosed in this Example indicate that front loading of antibody, such that a target serum concentration is reached more quickly, may be associated with improved outcomes Example 3: LV. Bolus Delivery and Subcutaneous Infusion of HERCEPTIN ® Anti-ErbB2 Antibody Effectively Decrease Tumor Volume in the Mouse ® The efficacy of infusion or bolus delivery of humanized antι-ErbB2 antibody (HERCEPTIN, see Example 1 for preparation), either by intravenous injection or subcutaneous injection, was examined The purpose ofthe study was to ask whether subcutaneous delivery was feasible and whether the convenient subcutaneous bolus delivery was useful in treating metastatic breast cancer in animals inoculated with a cell line that overexpresses the HER2 gene The results, detailed below, show that l v and s c infusion and bolus delivery are feasible treatment methodologies A study in a nude mouse xenograft model, which incorporates a human breast cancer cell line that naturally overexpresses the HER2 gene (BT-474M1 , derived from BT-474 cells, ATCC Accession number HTB-20), comparing tumor volume as a function of l v bolus versus s c infusion was performed as follows In the first study athymic nude nu nu 7 -9 week old female mice were obtained from Taconic Inc (Germantown, NY) To initiate tumor development, each mouse was inoculated subcutaneously with 3 x 10 BT474M1 cells suspended in Matπgel™ When tumor nodules reached a volume of approximately 100 mm , animals were randomized to 4 treatment groups The groups were treated according to Table 3 Table 3 Animal Groups and Doses for Comparison of I V Bolus and S C. Infusion Serum Cone = concentration in serum LD = loading dose MD = maintenance dose Infusate concentration was calculated to achieve targeted serum concentration using Alzet® osmotic minipumps (Alza Corp , Palo Alto, CA) Animals were exposed to estrogen by subcutaneous sustained release estrogen pellet 9 days before the start of dosing to promote growth of grafted tumor cells The animals were inoculated with the BT474M 1 cells 8 days before the beginning of treatment and tumors were allowed to grow The animals were then treated with nonrelevant antibody E25 (non-specific for HER2 receptor, but a member of the monoclonal IgG class) or test antibody HERCEPTIN antι-ErbB2 anitbody as indicated in Table 3 The dosage levels were selected to achieve target serum concentrations of HERCEPTIN , either 1 μg ml or 20 μg/ml, by subcutaneous pump infusion or by l v bolus delivery The study groups were treated until day 35 The serum concentration of HERCEPTIN " antι-ErbB2 antibody was measured weekly (just pπor to dosing for Group 4) using 3 mice/group/time point The antι-ErbB2 antibody concentration was determined according to the method disclosed herein involving standard techniques Tumor volumes were measured two days before dosing began and twice per week from day 6 to day 35 in the study for which data is tabulated below Tumors were measured in three dimensions and volumes were expressed in mm . Efficacy was determined by a statistical comparison (ANOVA) of tumor volumes of test animals relative to untreated control animals As shown in Table 4, below, treatment of the BT474M1 tumor-bearing mice with HERCEPTIN anti- ErbB2 antibody by the indicated dosage methods significantly inhibited the growth of the tumors All H HEERRCCEEPPTTIINN ® --ttrreeaatteedd , groups showed similar inhibition of tumor growth relative to the control group No dose response was observed Table 4 Comparison of S C Infusion and I V Bolus Delivery s c = subcutaneous delivery, l v = intravenous delivery * 4 0 mg/kg Loading Dose and 2 0 mg/kg/week Maintenance Dose ** at predose (trough serum concentration immediately prior to a maintenance dose) The results tabulated above indicate that maintenance of a serum concentration of approximately 2 μg/ml was as effective as a concentration of 20 μg/ml in this study The results indicated that dosing by subcutaneous infusion was as effective as intravenous bolus dosing and achieved similar trough serum concentrations The results also indicate that the dose levels studied are at the top of the dose-response curve in this model and that subcutaneous dosing is effective in treating breast cancer tumors Thus, subcutaneous administration of maintenance doses is feasible as part of a HERCEPTIN ® anti-ErbB 2 antibody treatment regimen Example 4: 1.V. Bolus and Subcutaneous Bolus Deliveries of HERCEPTIN ® Anti-ErbB2 Antibody Effectively Decrease Tumor Volume in the Mouse Subcutaneous bolus delivery is convenient and cost-effective for the patient and health care professionals The results of the study disclosed in this example indicate that subcutaneous bolus delivery was as effective as intravenous bolus delivery in reducing breast cell tumor size in a mouse This study was set up as disclosed herein in Example 3 for the comparison of intravenous bolus and subcutaneous infusion delivery A sustained release estrogen implant was inserted subcutaneously one day before tumor cell innoculation as described in Example 3 Six days after tumor cell innoculation, the initial tumor measurement was performed Seven days after tumor cell innoculation, the first dose of control antibody or HERCEPTIN antι-ErbB2 antibody was delivered The animal groups, type of delivery, loading dose and maintenance doses are provided in Table 4 Animals were dosed once weekly for 4 weeks Table 5 Animal Groups and Doses for Comparison of I V Bolus and S C Bolus Delivery IV= intraveneous, SC = subcutaneous, n=number of animals per group The mice were treated according to the information in Table 4 and using the techniques disclosed in ® Example 3 The serum concentration of HERCEPTIN antι-ErbB2 antibody was measured weekly before each weekly l v maintenance dose according to the procedure described herein and using standard techniques The control E25 antibody serum concentration was determined according to standard immunoassay techniques Table ® 6 shows the increase in HERCEPTIN antι-ErbB2 antibody serum concentrations with time Table 6 IV versus SC Bolus Delivery Serum HERCEPTIN ® Antι-ErbB2 Antibody Concentration Serum Concentration, μg/ml n=10 for time points Days 0, 7 and 14 N=9 for Day 21 Table 7 shows the relative efficacy of intravenous bolus delivery and subcutaneous bolus delivery for Groups 1 -5 having achieved the serum antibody concentrations presented in Table 6 For this study, efficacy was measured as a decrease in tumor volume Tumor volume was measured twice weekly Table 7 Efficacy of HERCEPTIN ® Antι-ErbB2 Antibody Measured as a Change in Tumor Volume Comparing Intravenous Bolus and Subcutaneous Bolus Delivery, Mean (SD) N= 10 for each data point TM=tumor measurement IV=ιntra venous SC=subcutaneous MD=maιntenance dose Tumor Vol =tumor volume, mm 3 *Day 17 excluded due to measurement error Tumor growth rate calculated on Day 21 -Day 31 Log(TM + 1) Area under the curve is the area beneath a plot of tumor volume versus time Figs 4A and 4B are graphical plots of changes in tumor volume over time, some of which data is found in 1 able 7 Fig 4A is a linear plot of tumor volume versus time Fig 4B is a semilogaπthmic plot of the same data, allowing the test points be viewed more clearly The data in Table 7 and Figs 4A and 4B indicate that, although a dose-related response was not observed between HERCEPTIN-treated groups, dosing by subcutaneous bolus was as effective as intravenous bolus dosing and achieved similar trough serum concentrations Example 5: Regimens for Intravenous and Subcutaneous Delivery of Anti-ErbB2 Antibody According to the invention, methods of antι-ErbB2 antibody (e g , HERCEPTIN ) delivery comprise greater front loading of the drug to achieve a target serum concentration in approximately 4 weeks or less, preferably 3 weeks or less, more preferably 2 weeks or less, and most preferably 1 week or less, including one day or less According to the invention, this initial dosing is followed by dosing that maintains the target serum concentration by subsequent doses of equal or smaller amount An advantage ofthe methods ofthe invention is that the maintenance dosing may be less frequent and/or delivered by subcutaneous injection, making the treatment regimens of the invention convenient and cost effective for the patient and medical professionals administering the antibody In addition, a subcutaneous maintenance dose regimen may be interrupted by intravenous dosing (such as infusion) when the patient's chemotherapy requires delivery of other drugs by intravenous injection To test the following dosage regimens, human subjects are selected according to the criteria disclosed in Example 1 , above The number of initial doses is one or more doses sufficient to achieve an efficacious target serum concentration in approximately 4 weeks or less, preferably 3 weeks or less, more preferably 2 weeks or less, and most preferably 1 week or less, including 1 day or less The number of maintenance doses may be one or more doses sufficient to achieve suppression of disease symptoms, such as a decrease in tumor volume The maintenance doses are equal to or smaller than the initial dose or doses, consistent with an object of the invention of administering HERCEPTIN ® antι-ErbB2 antibody by regimens providing greater front loading The specific drug delivery regimens disclosed herein are representative of the invention and are not meant to be limiting ® In one trial, an initial dose of 6 mg/kg, 8 mg/kg, or 12 mg/kg of HERCEPTIN anti-ErbB 2 antibody is delivered to human patients by intravenous or subcutaneous injection Initial doses (loading doses) are delivered by intravenous infusion or bolus injection or preferably subcutaneous bolus injection Preferably a target trough serum ® concentration of HERCEPTIN antι-ErbB2 antibody of approximately 10-20 μg/ml is achieved (averaged for all patients in the treatment group) and maintained by subsequent doses of antι-ErbB2 antibody that are equal to or smaller than the initial dose In one method, a target trough serum concentration is achieved and maintained by ® once-per-week deliveries of 2 mg/kg HERCEPTIN anti ErbB2 antibody by intravenous or subcutaneous injection for at least eight weeks Alternatively, for this or any dosage regimen disclosed herein, subcutaneous continuous infusion by subcutaneous pump is used to delivery subsequent maintenance doses ® In another method, an initial (front loading) dose of 8 mg/kg HERCEPTIN anti-ErbB 2 antibody is delivered by intravenous injection (infusion or bolus injection) or by subcutaneous bolus injection This is followed by intravenous bolus injections, intravenous infusion, subcutaneous infusion, or subcutaneous bolus injection of 6 mg/kg at 3- week intervals to maintain a trough serum concentration of approximately 10-20 μg/ml, averaged for an entire treatment group ® In another method, an initial (front loading) dose of 12 mg/kg HERCEPTIN antι-ErbB2 antibody is delivered by intravenous injection (infusion or bolus injection) or by subcutaneous bolus injection This is followed by intravenous bolus injections, intravenous infusion, subcutaneous infusion, or subcutaneous bolus injection of 6 mg/kg at 3-week intervals to maintain a trough serum concentration of approximately 10-20 μg/ml ® In yet another method, an initial (front loading) dose of 8 mg/kg HERCEPTIN antι-ErbB2 antibody is delivered by intravenous infusion or bolus injection, or preferably by subcutaneous bolus injection or infusion This is followed by administration of 8 mg/kg per week or 8 mg/kg per 2-3 weeks to maintain a trough serum ® concentration of HERCEPTIN antι-ErbB2 antibody of approximately 10-20 μg/ml Maintenance doses are delivered by intravenous infusion or bolus injection, or preferably by subcutaneous infusion or bolus injection In another method, the front loading initial dose is a series of intravenous or subcutaneous injections, for example, one on each of days 1, 2, and 3 of at least 1 mg/kg for each injection (where the amount of antι-ErbB2 antibody delivered by the sum of initial injections is more than 4 mg/kg) , followed by maintenance doses of 6 mg/kg once each 3 week interval to maintain a target trough serum concentration (for example, approximately 10-20 ® μg/ml) of HERCEPTIN antι-ErbB2 antibody The maintenance doses are delivered by intravenous infusion or bolus injection or by subcutaneous infusion or subcutaneous bolus injection In yet another method, the front loading is by intravenous infusion of at least 1 mg/kg, preferably 4 mg/kg on each of five consecutive days, followed by repeats of this cycle a sufficient number of times to achieve suppression of disease symptoms Following the initial dose or doses, subsequent doses may be delivered by subcutaneous infusion or bolus injection if tolerated by the patient Such subcutaneous delivery is convenient and cost-effective for the patient and administering health care professionals ® In still another method, HERCEPTIN anti-ErbB 2 antibody is delivered initially as at least 2 intravenous infusions per week for three weeks, followed by repeats of this cycle to maintain an efficacious trough serum ® concentration of HERCEPTIN antι-ErbB2 antibody The dose is at least 4 mg/kg of antι-ErbB2 antibody, preferably at least 5 mg/kg The maintenance drug deliveries may be intravenous or subcutaneous Where the animal or patient tolerates the antibody during and after an initial dose, delivery of subsequent doses may be subcutaneous, thereby providing greater convenience and cost-effectiveness for the patient and health caie professionals In animal studies, an initial dose of more than 4 mg/kg, preferably more than 5 mg/kg delivered by intravenous or subcutaneous injection, is followed by subcutaneous bolus injections of 2 mg/kg twice per week (separated by 3 days) to maintain a trough serum concentration of approximately 10-20 μg/ml In addition, where ® the animal or patient is known to tolerate the antibody, an initial dose of HERCEPTIN antι-ErbB2 antibody is optionally and preferably deliverable by subcutaneous bolus injection followed by subcutaneous maintenance injections While target serum concentrations are disclosed herein for the purpose of comparing animal studies and human trials, target serum concentrations in clinical uses may differ The disclosure provided herein guides the user in selecting a front loading drug delivery regimen that provides an efficacious target trough serum concentration ® The methods ofthe invention disclosed herein optionally include the delivery of HERCEPTIN antι-ErbB2 antibody in combination with a chemotherapeutic agent (other than an anthrocychne derivative) to achieve ® suppression of disease symptoms The chemotherapeutic agent may be delivered with HERCEPTIN antι-ErbB2 antibody or separately and according to a different dosing schedule For example, subcutaneous delivery of ® ® HERCEPTIN antι-ErbB2 antibody with TAXOL is included in the invention In addition, intravenous or ® subcutaneous injection of 8 mg/kg HERCEPTIN antι-ErbB2 antibody, followed by intravenous or subcutaneous injection of 6 mg/kg HERCEPTIN ® antι-ErbB2 antibody every 3 weeks is administered in combination with a chemotherapeutic agent, such as a taxoid (e g paclitaxel 175mg/m2 every 3 weeks) or an anthracycline derivative (e g doxorubicin 60 mg/m2 or epirubicin 75 mg/m2 every 3 weeks) Optionally, where an anthracycline derivative is administered, a cardioprotectant (e g 600 mg/m2 cyclophosphamide every 3 weeks) is also administered In another combination therapy, antι-ErbB2 antibody is administered in a loading dose of more than 4 mg/kg, preferably more than 5 mg/kg, and more preferably at least 8 mg/kg The loading dose is followed by maintenance doses of at least 2 mg/kg weekly, preferably 6 mg/kg every 3 weeks The combination therapy includes administration of a taxoid during treatment with antι-ErbB2 antibody According to one embodiment of the invention, the taxoid is paclitaxel and is administered at a dose of 70-100 mg/m /week According to another embodiment of the invention, the taxoid is docetaxel and is administered at a dose of 30-70 mg/m"/week Example 6: HERCEPTIN® Administered Intravenously Every Three Weeks in Combination with Paclitaxel Currently, the recommended dose of HERCEPTIN® is 2mg/kg once weekly Patients will be administered HERCEPTIN® every three weeks instead of weekly, along with paclitaxel (175mg/m " every three weeks) Simulation of the proposed treatment regimen suggests that the trough serum concentrations will be 17 mcg/ml, in the range ( 10-20mcg/ml) ofthe targeted trough serum concentrations from previous HERCEPTIN® IV clinical trials After the first 12 patients the PK parameters will be assessed, if exposure is felt inadequate, then the dose will be increased to 8 mg/kg every three weeks for the remaining 12 patients Inclusion Criteria 1 ) Females ≥ 18 years of age 2) Histologically confirmed ErbB2 over-expressing metastatic breast cancer 3) Patients who have been newly diagnosed with metastatic disease 4) Have a Karnofsky performance status of > 70% 5) Give written informed consent prior to any study specific screening procedures with the understanding that the patient has the right to withdraw from the study at any time, without prejudice Exclusion Criteria 1 ) Pregnant or lactating women 2) Women of childbeaπng potential unless (1) surgically sterile or (2) using adequate measures of contraception such as oral contraceptive, lntra-uteπne device or bamer method of contraception in conjunction with spermicidal jelly 3) Clinical or radiologic evidence of CNS metastases 4) History of any significant cardiac disease 5) LVEF < 50% 6) No prior taxane therapy in any treatment setting 7) Any of the following abnormal baseline hematologic values - Hb less than 9 g/dl - WBC less than 3 0 x 10 9 /1 Granulocytes less than 1 5 x 10 /1 - Platelets less than 100 x 10 9 /1 8) Any of the following abnormal baseline liver function tests - Serum bihrubin greater than 1 5 x ULN (upper normal limit) - ALT and/or AST greater than 2 5 x ULN ( greater than 4 0 x ULN if liver or bone metastasis) - Alkaline phosphatase greater than 2 5 x ULN ( greater than 4 0 x ULN if liver or bone metastasis) 9) The following abnormal baseline renal function tests - serum creatinine greater than 1 5 x ULN 10) History of other serious medical conditions that would preclude patient participation in an investigational study HERCEPTIN ® Loading dose and schedule 8 mg/kg for first dose Maintenance dose and schedule 6mg/kg every 3 weeks Paclitaxel - 175 mg/m IV every 3 weeks x 6 cycles as a 3-hour infusion NOTE On the first cycle of treatment, paclitaxel will be dosed 8 hours prior to HERCEPTIN® to determine the PK of paclitaxel alone HERCEPTIN® will be administered 8 hours post-pachtaxel for the 1 st cycle only In subsequent treatment cycles, HERCEPTIN® will be administered prior to paclitaxel The total duration of this study is 18 weeks Study subjects will receive up to 6 total HERCEPTIN® doses After the last subject has received the last cycle of paclitaxel, data collection for safety and pharmacokinetic analysis will stop, and the study will close to protocol specified treatment Study subjects may continue to receive the HERCEPTIN® +/- paclitaxel at the discretion of the investigator It is believed that the above treatment regimen will be effective in treating metastatic breast cancer, despite the infrequency with which HERCEPTIN® is administered to the patient While the particular aspects and embodiments of the invention as herein shown and disclosed in detail is fully capable of obtaining the objects and providing the advantages herein before stated, it is to be understood that it is merely illustrative of some of the presently preferred embodiments of the invention and that no limitations are intended to the details of methods and articles of manufacture shown other than as described in the appended claims The disclosures of all citations in the specification are expressly incorporated herein by reference

Claims: Claims 1 A method for the treatment of a human patient susceptible to or diagnosed with a disorder characterized by overexpression of ErbB2 receptor, comprising administering an effective amount of an antι-ErbB2 antibody to the human patient, the method comprising administering to the patient an initial dose of at least approximately 5 mg/kg of the antι-ErbB2 antibody, and administering to the patient a plurality of subsequent doses of the antibody in an amount that is approximately the same or less than the initial dose 2 The method of claim 1 , wherein the initial dose is at least approximately 6 mg/kg 3 The method of claim 2, wherein the initial dose is at least approximately 8 mg/kg 4 The method of claim 3, wherein the initial dose is at least approximately 12 mg/kg 5 The method of claim 1 , wherein the subsequent doses are separated from each other in time by at least one week 6 The method of claim 1 , wherein the subsequent doses are separated in time fro each other by at least two weeks 7 The method of claim 1 , wheiein the subsequent doses are separated in time from each other by at least three weeks 8 The method of claim 1 , wherein the initial dose is administered by intravenous injection, and wherein at least one subsequent dose is administered by subcutaneous injection 9 The method of claim 1, wherein the initial dose is administered by intravenous injection, wherein at least two subsequent doses are administered, and wherein each subsequent dose is administered by a method selected from the group consisting essentially of intravenous injection and subcutaneous injection 10 The method of claim 1 , wherein the initial dose and at least one subsequent dose are administered by subcutaneous injection 1 1 The method of claim 1 , wherein the initial dose is selected from the group consisting essentially of approximately 6 mg/kg, 8 mg/kg, or 12 mg/kg, wherein the plurality of subsequent doses are at least approximately 2 mg/kg, and wherein the subsequent doses are separated in time from each other by at least one week 12 The method of claim 1 1 , wherein the plurality of subsequent doses are separated in time from each other by at least two weeks 13 The method of claim 12, wherein the plurality of subsequent doses are separated in time from each other by at least three weeks 14 The method of claim 13, wherein the initial dose is approximately 8 mg/kg, and wherein at least one subsequent dose is approximately 6 mg/kg 15 The method of claim 13, wherein the initial dose is approximately 12 mg/kg, and wherein at least one subsequent dose is approximately 6 mg/kg 16 The method of claim 1 1 , wherein the initial dose is approximately 8 mg/kg, and wherein at least one subsequent dose is approximately 8 mg/kg 17 The method of claim 1 1 , wherein the initial dose is approximately 8 mg/kg, wherein at least one subsequent dose is 8 mg/kg, and wherein administration of the initial dose and subsequent doses are separated in time by at least 2 weeks 18 The method of claim 17, wherein the initial dose and subsequent doses are separated in time by at least 3 weeks 19 The method of claim 1 , wherein the initial dose is a plurality of doses, wherein each ofthe plurality of initial doses is at least approximately 1 mg/kg and is administered on at least 2 consecutive days, wherein the at least one subsequent dose is at least approximately 2 mg/kg, and wherein administration of the last initial dose and the first subsequent and additional subsequent doses are separated in time by at least one week 20 The method of claim 19, wherein the initial dose is a plurality of doses, wherein each of the plurality of initial doses is at least approximately 1 mg/kg and is administered on at least 3 consecutive days, wherein the at least one subsequent dose is at least approximately 6 mg/kg, and wherein administration ofthe last initial dose and the first subsequent and additional subsequent doses are separated in time by at least 3 weeks 21 The method of claim 1 , wherein the initial dose is a plurality of doses, wherein each ofthe plurality of initial doses is at least approximately 1 mg/kg and is administered at least twice per week for 3 weeks as a first dosage cycle, wherein the dosage cycle is repeated, and wherein the doses of each cycle are separated in time by at least 1 day, and wherein the dosage cycles are separated in time by at least 1 week 22 The method of claim 1 , wherein said disorder is a benign or malignant tumor 23 The method of claim 1 , wherein said disorder is a cancer 24 The method of claim 23, wherein said cancer is selected from the group consisting of breast cancer, leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer 25 The method of claim 24, wherein said cancer is breast cancer 26 The method of claim 25, wherein said cancer is metastatic breast carcinoma 27 The method of claim 1 wherein said antibody binds to the extracellular domain of the ErbB 2 receptor 28 The method of claim 27, wherein said antibody binds to epitope 4D5 within the ErbB2 extracellular domain sequence 29 The method of claim 28, wherein said antibody is a humanized 4D5 antι-ErbB2 antibody 30 The method of claim 1 wherein the method further comprises administering an effective amount of a chemotherapeutic agent 31 The method of claim 30, wherein the chemotherapeutic agent is a taxoid 32 The method of claim 31 , wherein said taxoid is paclitaxel or docetaxel 33 The method of claim 30, wherein the effective amount ofthe antι-ErbB2 antibody and the effective amount of the chemotherapeutic agent as a combination is lower than the sum of the effective amounts of said anti- ErbB2 antibody and said chemotherapeutic agent, when administered individually, as single agents 34 The method of claim 30, wherein the chemotherapeutic agent is an anthrcychne derivative 35 The method of claim 34, wherein the anthracycline derivative is doxorubicin or epirubicin 36 The method of claim 34, wherein the method further comprises administration of a cardioprotectant 37 The method of claim 1, wherein efficacy is measured by determining the time to disease progression or the response rate 38 An article of manufacture, comprising a container, a composition within the container comprising an antι-ErbB2 antibody, and a package insert containing instructions to administer an initial dose of antι-ErbB2 antibody of at least 5 mg/kg, and at least one subsequent dose that is the same amount or less than the initial dose 39 The article of manufacture of claim 38, wherein the instructions are for administration of an initial dose by intravenous injection and at least one subsequent dose by subcutaneous injection 40 The article of manufacture of claim 38, wherein the initial dose is at least approximately 6 mg/kg 41 The article of manufacture of claim 40, wherein the initial dose is at least approximately 8 mg/kg 42 The article of manufacture of claim 41 , wherein the initial dose is at least approximately 12 mg/kg 43 The article of manufacture of claim 38, wherein the subsequent doses are separated from each other in time by at least one week 44 The article of manufacture of claim 38, wherein the subsequent doses are separated in time from each other by at least two weeks 45 The article of manufacture of claim 38, wherein the subsequent doses are separated in time from each other by at least three weeks 46 The article of manufacture of claim 38, wherein the initial dose and at least one subsequent dose are administered by subcutaneous injection 47 The article of manufacture of claim 38, wherein the initial dose is selected from the group consisting essentially of approximately 6 mg/kg, 8 mg/kg, or 12 mg/kg, wherein the plurality of subsequent doses are at least approximately 2 mg/kg, and wherein the subsequent doses are separated in time from each other by at least one week 48 The article of manufacture of claim 47, wherein the plurality of subsequent doses are separated in time from each other by at least two weeks 49 The article of manufacture of claim 48, wherein the plurality of subsequent doses are separated in time from each other by at least three weeks 50 The article of manufacture of claim 49, wherein the initial dose is approximately 8 mg/kg, and wherein at least one subsequent dose is approximately 6 mg/kg 51 The article of manufacture of claim 49, wherein the initial dose is approximately 12 mg/kg, and wherein at least one subsequent dose is approximately 6 mg/kg 52 The article of manufacture of claim 47, wherein the initial dose is approximately 8 mg/kg, and wherein at least one subsequent dose is approximately 8 mg/kg 53 The article of manufacture of claim 47, wherein the initial dose is approximately 8 mg/kg, wherein at least one subsequent dose is 8 mg/kg, and wherein administration of the initial dose and subsequent doses are separated in time by at least 2 weeks 54 The article of manufacture of claim 53, wherein the initial dose and subsequent doses are separated in time by at least 3 weeks 55 The article of manufacture of claim 38, wherein the initial dose is aplurahty of doses, wherein each of the plurality of initial doses is at least approximately 1 mg/kg and is administered on at least 2 consecutive days, wherein at least one subsequent dose is at least approximately 2 mg/kg, and wherein administration ofthe last initial dose and the first subsequent and additional subsequent doses are separated in time by at least one week 56 The article of manufacture of claim 55, wherein the initial dose is a plurality of doses, wherein each of the plurality of initial doses is at least approximately 1 mg/kg and is administered on at least 3 consecutive days, wherein the at least one subsequent dose is at least approximately 6 mg/kg, and wherein administration of the last initial dose and the first subsequent and additional subsequent doses are separated in time by at least 3 weeks 57 The article of manufacture of claim 38, wherein the initial dose is a plurality of doses, wherein each of the plurality of initial doses is at least approximately 1 mg/kg and is administered at least twice per week for 3 weeks as a first dosage cycle, wherein the dosage cycle is repeated, and wherein the doses of each cycle are separated in time by at least 1 day, and wherein the dosage cycles are separated in time by at least 1 week 58 The article of manufacture of claim 38, wherein the instructions are for administration of an initial dose by subcutaneous injection and at least one subsequent dose by subcutaneous injection 59 The article of manufacture of claim 58, wherein the initial dose is at least approximately 6 mg/kg 60 The article of manufacture of claim 59, wherein the initial dose is at least approximately 8 mg/kg 61 The article of manufacture of claim 60, wherein the initial dose is at least approximately 12 mg/kg 62 The article of manufacture of claim 58, wherein the subsequent doses are separated from each other in time by at least one week 63 The article of manufacture of claim 58, wherein the subsequent doses are separated in time from each other by at least two weeks 64 The article of manufacture of claim 58. wherein the subsequent doses are separated in time from each other by at least three weeks 65 The article of manufacture of claim 58, wherein the initial dose is selected from the group consisting essentially of approximately 6 mg/kg, 8 mg/kg, or 12 mg/kg, wherein the plurality of subsequent doses are at least approximately 2 mg/kg, and wherein the subsequent doses are separated in time from each other by at least one week 66 The article of manufacture of claim 58, wherein the plurality of subsequent doses are separated in time from each other by at least two weeks 67 The article of manufacture of claim 66, wherein the plurality of subsequent doses are separated in time from each other by at least three weeks 68 The article of manufacture of claim 67, wherein the initial dose is approximately 8 mg/kg, and wherein at least one subsequent dose is approximately 6 mg/kg 69 The article of manufacture of claim 67, wherein the initial dose is approximately 12 mg/kg, and wherein at least one subsequent dose is approximately 6 mg/kg 70 The article of manufacture of claim 65, wherein the initial dose is approximately 8 mg/kg, and wherein at least one subsequent dose is approximately 8 mg/kg 71 The article of manufacture of claim 65, wherein the initial dose is approximately 8 mg/kg, wherein at least one subsequent dose is 8 mg/kg, and wherein administration of the initial dose and subsequent doses are separated in time by at least 2 weeks 72 The article of manufacture of claim 71 , wherein the initial dose and subsequent doses are separated in time by at least 3 weeks 73 The article of manufacture of claim 58, wherein the initial dose is aplurahty of doses, wherein each of the plurality of initial doses is at least approximately 1 mg/kg and is administered on at least 2 consecutive days, wherein at least one subsequent dose is at least approximately 2 mg/kg, and wherein administration of the last initial dose and the first subsequent and additional subsequent doses are separated in time by at least one week 74 The article of manufacture of claim 73, wherein the initial dose is aplurahty of doses, wherein each of the plurality of initial doses is at least approximately 1 mg/kg and is administered on at least 3 consecutive days, wherein the at least one subsequent dose is at least approximately 6 mg/kg, and wherein administration of the last initial dose and the first subsequent and additional subsequent doses are separated in time by at least 3 weeks 75 The article of manufacture of claim 58, wherein the initial dose is a plurality of doses, wherein each of the plurality of initial doses is at least approximately 1 mg/kg and is administered at least twice per week for 3 weeks as a first dosage cycle, wherein the dosage cycle is repeated, and wherein the doses of each cycle are separated in time by at least 1 day, and wherein the dosage cycles are separated in time by at least 1 week 76 The article of manufacture of claim 38, wherein the instructions further include administration of a chemotherapeutic agent 77 The article of manufacture of claim 76, wherein the chemotherapeutic agent is a taxoid 78 The article of manufacture of claim 77, wherein the taxoid is paclitaxel or docetaxel 79 The article of manufacture of claim 76, wherein the chemotherapeutic agent is an anthracycline derivative 80 The article of manufacture of claim 79, wherein the instructions further include administration of a cardioprotectant 81 The article of manufacture of claim 38, further compπsing a label on or associated with the container that indicates that said composition can be used for treating a condition characterized by overexpression of ErbB 2 receptor 82 The article of manufacture of claim 81 , wherein said label indicates that said composition can be used for the treatment of breast cancer 83 The article of manufacture of claim 38, wherein said antι-ErbB2 antibody binds to the extracellular domain of the receptor 84 The article of manufacture of claim 83, wherein said antι-ErbB2 antibody binds to epitope 4D5 within the ErbB2 extracellular domain sequence 85 The article of manufacture of claim 84, wherein said antibody is a humanized 4D5 antι-ErbB2 antibody 86 A method for the treatment of cancer in a human patient comprising administering to the patient a first dose of an antι-ErbB2 antibody followed by at least one subsequent dose of the antibody, wherein the first dose and subsequent dose are separated from each other in time by at least about two weeks 87 The method of claim 86, wherein the first dose and subsequent dose are separated from each other in time by at least about three weeks 88 The method of claim 86, wherein the first dose and subsequent dose are each from about 2mg/kg to about 16mg/kg 89 The method of claim 88, wherein the first dose and subsequent dose are each from about 4mg/kg to about 12mg/kg 90 The method of claim 89, wherein the first dose and subsequent dose are each from about 6mg/kg to about 12mg/kg 91 The method of claim 86, wherein two or more subsequent doses ofthe antibody are administered to the patient 92 The method of claim 91 , wherein from about two to about ten subsequent doses of the antibody are administered to the patient 93 The method of claim 91 , wherein the two or more subsequent doses are separated from each other in time by at least about two weeks 94 The method of claim 93, wherein the two or more subsequent doses are separated from each other in time by at least about three weeks 95 The method of claim 91 , wherein the two or more subsequent doses are each from about 2mg/kg to about 16mg/kg 96 The method of claim 91 , wherein the two or more subsequent doses are each from about 4mg/kg to about 12mg/kg 97 The method of claim 91, wherein the two or more subsequent doses are each from about 6mg/kg to about 12mg/kg 98 The method of claim 86, wherein the method further comprises administering an effective amount of a chemotherapeutic agent to the patient 99 The method of claim 98, wherein the chemotherapeutic agent is a taxoid 100 An article of manufacture, comprising a container, a composition within the container comprising an antι-ErbB2 antibody, and a package insert containing instructions to dose the antibody according to any one of claims 86 to 99 101 A method for the treatment of cancer in a human patient, comprising administering an effective amount of an anti-ErbB antibody to the human patient, the method comprising administering to the patient an initial dose of at least approximately 5 mg/kg of the anti-ErbB antibody, and administering to the patient a plurality of subsequent doses of the antibody in an amount that is approximately the same or less than the initial dose 102 The method of claim 101 wherein the anti-ErbB antibody is selected from the group consisting of anti- epidermal growth factor receptor (EGFR), antι-ErbB3 and antι-ErbB4 103 A method for the treatment of cancer in a human patient comprising administering to the patient a first dose of an anti-ErbB antibody followed by at least one subsequent dose ofthe antibody, wherein the first dose and subsequent dose are separated from each other in time by at least about two weeks 104 The method of claim 103 wherein the anti-ErbB antibody is selected from the group consisting of anti- epidermal growth factor receptor (EGFR), antι-ErbB3 and antι-ErbB4 105 An article of manufacture, comprising a container, a composition within the container compπsing an anti-ErbB antibody, and a package insert containing instructions to dose the antibody according to any one of claims 101 to 104

Inventors: BAUGHMAN SHARON ANN, SHAK STEVEN, BAUGHMAN, SHARON, ANN, SHAK, STEVEN
Applicants: GENENTECH INC, GENENTECH, INC.
Application year: 2000
Main classification: A61K39
IPC codes: A61K31, A61K38, A61K39, A61P15, A61P35, A61P43, C07K16