HK1091391B - Treatment with anti-erbb2 antibodies - Google Patents

Description

Treatment with anti-ErbB 2 antibodies

The present application is a divisional application having application number 988120976, filed 10/12/1998 and entitled "treatment with anti-ErbB 2 antibodies".

Technical Field

The present invention relates to methods of treating diseases characterized by overexpression of ErbB 2. More particularly, the invention relates to the treatment of patients susceptible to or diagnosed with cancer overexpressing ErbB2 with a combination of an anti-ErbB 2 antibody and a chemotherapeutic agent other than an anthracycline, such as doxorubicin (toxorubicin) or epirubicin (epirubicin).

Background

Protooncogenes encoding growth factors and growth factor receptors have been identified to play an important role 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 the 185kd transmembrane glycoprotein receptor (p 185) associated with the Epidermal Growth Factor Receptor (EGFR), is overexpressed in about 25-30% of human breast cancer patients^HER2) (Slamon et al, Science, 235: 177-182[1987](ii) a Slamon et al, Science, 244: 707-712[1989])。

Several lines of evidence confirm that ErbB2 plays a direct role in the pathogenesis and clinical invasiveness of ErbB2 overexpressing tumors. It has been shown that introduction of ErbB2 into non-tumor cells causes them to undergo malignant transformation (Hudziak et al, Proc. Natl. Acad. Sci. USA 84: 7159-7163[1987 ]; DiFiore et al, Science 237: 78-82[1987 ]). It was found that transgenic mice expressing HER2 developed mammary tumors (Guy et al, Proc. Natl. Acad. Sci. USA 89: 10578-10582[1992 ]).

Antibodies against the rat equivalent of the erbB2 gene (neu) encoding human erbB2 protein products and proteins have been described. Drebin et al, Cell 41: 695-706(1985) reported an IgG2a monoclonal antibody directed against the rat neu gene product. This antibody, designated 7.16.4, down-regulated cell surface p185 expression on B104-101 cells (NIH-3T 3 cells transfected with the neu proto-oncogene) and inhibited colony formation by these cells. In Drebin et al, PNAS (USA) 83: 9129-9133(1986) showed that the 7.16.4 antibody inhibited the tumorigenic growth of neu-transformed NIH-3T3 cells and rat neuroblastoma cells from which the neu oncogene was first isolated, which were implanted in nude mice. Drebin et al in Oncogene 2: 387-394(1988) describe methods for producing a panel of antibodies against the murine neu gene product. All these antibodies were found to have a cytostatic effect on the growth of neu transformed cells suspended in soft agar. Antibodies of the IgM, IgG2a and IgG2b isotype apparently mediate lysis of neu-transformed cells in vitro in the presence of complement, but none of the antibodies mediate high levels of antibody-dependent cellular cytotoxicity (ADCC) of neu-transformed cells. Drebin et al Oncogene 2: 273-277(1988) reported that an antibody mixture reacting with two different regions on the p185 molecule had a synergistic antitumor effect on neu-transformed NIH-3T3 cells implanted in nude mice. Myers et al, meth.enzym.198: the biological effects of anti-neu antibodies were reviewed in 277-290 (1991). See also WO94/22478, published 10/13 in 1994.

Hudziak et al, mol.cell.biol.9 (3): 1165-1172(1989) describe the generation of a panel of anti-ErbB 2 antibodies characterized by the human breast tumor cell line SKBR 3. The relative proliferation of SKBR3 cells after exposure to antibody was determined by crystal violet staining of the cell monolayer after 72 hours. By this assay, antibody 4D5 was found to have the greatest inhibitory effect, inhibiting cell proliferation by 56%. Other antibodies in this panel (including 7C2 and 7F3) reduced cell proliferation to a lesser extent in this assay. Hudziak et al concluded that the effect of the 4D5 antibody on SKBR3 cells was a cytostatic effect rather than cytotoxic, as SKBR3 cells returned to near normal growth rates after removal of the antibody from the culture medium. It was also found that antibody 4D5 makes p185^erbB2The overexpressed breast tumor cell line is sensitive to the TNF-alpha cytotoxic effect. See also WO89/06692, published 1989 on 7/27. anti-ErbB 2 antibodies discussed in the Hudziak et al reference are also described in: fendly et al, Cancer Research 50: 1550 and 1558 (1990); kotts et al, In Vitro 26 (3): 59A (1990); sarup et al, Growth Regulation 1: 72-82 (1991); shepard et al, j.clin.immunol.11 (3): 117-127 (1991); kumar et al, mol.cell.biol.11 (2): 979-; lewis et al, Cancer immunol.immunoher.37: 255-; pietras et al, Oncogene 9: 1829-1838 (1994); vitetta et al, Cancer Research 54: 5301-5309 (1994); sliwkowski et al, j.biol.chem.269 (20): 14661-14665 (1994); scott et al, j.biol.chem.266: 14300-5 (1991); and D' souza et al, proc.natl.acad.sci.91: 7202-7206(1994).

Tagliabue et al, int.j. cancer 47: 933-937(1991) describe two antibodies selected for reactivity to a lung adenocarcinoma cell line (Calu-3) that overexpresses ErbB 2. One of the antibodies (designated MGR3) was found to internalize, induce phosphorylation of ErbB2, and inhibit tumor cell growth in vitro.

McKenzie et al Oncogene 4: 543 548(1989) produced a panel of anti-ErbB 2 antibodies with different epitope specificities, including the antibody designated TA 1. The TA1 antibody was found to cause acceleration of endocytosis of ErbB2 (see Maier et al, Cancer Res.51: 5361-5369 (1991)). Bacillus et al Molecular Carcinogenesis 3: 350-362(1990) reported that the TA1 antibody induced maturation of the breast cancer cell lines AU-565 (cell line overexpressing the erbB2 gene) and MCF-7 (cell line not overexpressing the erbB2 gene). The growth and acquisition of the mature phenotype of these cells was found to be inhibited and was associated with a decrease in the levels of ErbB2 receptor on the cell surface and a transient increase in the levels of receptor in the cytoplasm.

Stancovski et al PNAS (USA) 88: 8691-8695(1991) obtained a panel of antibodies against ErbB2, which were injected intraperitoneally into nude mice and evaluated for their effect on tumor growth in mouse fibroblasts transformed by overexpression of the erbB2 gene. Different levels of tumor inhibition were detected for the four antibodies, but one antibody (N28) continuously stimulated tumor growth. Monoclonal antibody N28 significantly induced phosphorylation of the ErbB2 receptor, while the other four antibodies generally exhibited low or no phosphorylation-inducing activity. The effect of anti-ErbB 2 antibodies on the proliferation of SKBR3 cells was also evaluated. In this SKBR3 cell proliferation assay, two antibodies (N12 and N29) reduced cell proliferation compared to controls. The ability of various antibodies to induce cell lysis in vitro by complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated, and the authors of this paper concluded that the inhibitory function of the antibodies was apparently not due to CDC or ADCC.

Bacus et al in Cancer Research 52: 2580 (1992) further characterisation of the antibodies described previously in Bacus et al (1990) and Srancovski et al. This identification extended the study of Stancovski et al, evaluating the effect of intravenous injection of these antibodies in nude mice carrying mouse fibroblasts overexpressing human ErbB 2. In their early work, it was found that N28 accelerated tumor growth, while N12 and N29 significantly inhibited growth of cells expressing ErbB 2. The N24 antibody was also found to have partial tumor suppression. Bacus et al also tested the ability of these antibodies to promote mature phenotypes in human breast cancer cell lines AU565 and MDA-MB453 (overexpressing ErbB2) and MCF-7 (containing low levels of this receptor). Bacus et al found that in vivo inhibition of tumors was associated with cell differentiation; tumor stimulating antibody N28 had no effect on differentiation; the tumor suppression effects of the N12, N29, and N24 antibodies correlated with the degree of differentiation they induced.

Xu et al int.j.cancer 53: 401-408(1993) evaluated the epitope binding specificity of a panel of anti-ErbB 2 antibodies and their ability to inhibit anchorage-independent and anchorage-dependent growth of SKBR3 cells (by single antibody and combination), to modulate cell surface ErbB2, and to inhibit ligand-stimulated anchorage-independent growth. See also WO94/00136, published on month 1 and 6 of 1994, and Kasprzyk et al, Cancer Research 52: 2771-2776(1992) for compositions of anti-ErbB 2 antibodies. In addition, in Cancer Res.51 of Hancock et al: 4575-4580 (1991); shawver et al, Cancer Res.54: 1367-; aretag et al Cancer Res.54: 3758-3765(1994) and Harwerth et al J.biol.chem.267: 15160 other anti-ErbB 2 antibodies are discussed in 15167 (1992).

A recombinant humanized anti-ErbB 2 monoclonal antibody (humanized version of murine anti-ErbB 2 antibody 4D5, designated rhuMAb HER2 or) Clinical activity was shown in patients with metastatic breast cancer overexpressing ErbB2, who had received extensive anti-cancer therapy (Baselga et al, j.clin.oncol.14: 737-744[1996])。

ErbB2 overexpression is generally considered to be a predictor of poor prognosis, particularly in patients with primary diseases involving axillary lymph nodes (Slamon et al, 1987]And [ 1989)]The same as above; ravdin and Chamness, Gene 159: 19-27[1995](ii) a And Hynes and Stern, Biochim biophysicaca 1198: 165-184[1994]) And are associated with sensitivity and/or resistance to hormonal and chemotherapeutic agents, including CMF (cyclophosphamide, methotrexate and fluorouracil) and anthracyclines (Baselga et al, Oncology 11(3Suppl 1): 43-48[1997]). However. Although ErbB2 overexpression is associated with poor prognosis, HER 2-positive patients have a 3-fold better likelihood of clinical response to treatment with taxans than HER 2-negative patients (3-fold)Ibid). rhuMab HER2 was shown to potentiate paclitaxel (paclitaxel) (paclitaxel)) And Adriamycin Activity against Breast Cancer xenografts in nude mice injected with human Breast adenocarcinoma cells expressing high levels of HER2 (Baselga et al, Breast Cancer, Proceedings of ASCO, Vol 13, Abstract 53[1994 ]])。

Summary of The Invention

The present invention relates to the treatment of diseases characterized by overexpression of ErbB2, based on the recognition that, although treatment with anti-ErbB 2 antibodies can significantly enhance the clinical effects of conventional chemotherapeutic agents, administration of anti-ErbB 2 antibodies exacerbates the myocardial dysfunction syndrome that has been considered as a side effect of anthracycline derivatives.

Accordingly, the present invention relates to a method of treating a patient susceptible to or diagnosed with a disease characterized by overexpression of the ErbB2 receptor, comprising administering to the patient a therapeutically effective amount of a combination of an anti-ErbB 2 antibody and a chemotherapeutic agent that is not an anthracycline derivative (e.g., doxorubicin or epirubicin) in the absence of the anthracycline derivative.

The disease to be treated is preferably a benign or malignant tumour characterised by overexpression of the ErbB2 receptor, for example cancer such as breast cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, intestinal gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrial cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatocellular carcinoma and various head and neck cancers. The chemotherapeutic agent is preferably tacrolid, e.g. sodium tartrate(paclitaxel) orAnd (3) derivatives.

Although the anti-proliferative effects of anti-ErbB 2 antibodies are sufficient in the preferred embodiment, anti-ErbB 2 antibodies induce cell death or apoptosis. Preferred anti-ErbB 2 antibodies bind to the extracellular domain of ErbB2 receptor, preferably to epitope 4D5 or 3H4 in the sequence of the extracellular domain of ErbB 2. More preferably, the antibody is antibody 4D5, most preferably in humanized form.

The methods of the invention are particularly suitable for treating breast or ovarian cancer characterized by overexpression of the ErbB2 receptor.

In another aspect, the invention relates to an article of manufacture comprising a container having therein a composition comprising an anti-ErbB 2 antibody, optionally together with or attached to the container a label indicating that the composition can be used to treat a condition characterized by overexpression of the ErbB2 receptor, and a package insert indicating that the use of an anthracycline chemotherapeutic agent in combination with the composition is avoided.

Brief Description of Drawings

FIG. 1 shows an epitope map 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-. Antiproliferative monoclonal antibodies 4D5 and 3H4 bound to the vicinity of the transmembrane domain. Various ErbB2-ECD truncates or point mutations were prepared from cDNA using polymerase chain reaction techniques. The ErbB2 mutant was expressed as a gD fusion protein in a mammalian expression plasmid. The expression plasmid employs the cytomegalovirus promoter/enhancer as well as the SV40 terminator and polyadenylation signal located downstream of the inserted cDNA. Plasmid DNA was transfected into 293S cells. 1 day after transfection, cells were plated in methionine and cysteine free, 1% dialyzed fetal bovine serum and 25. mu. Ci each³⁵S methionine and³⁵labeling by metabolism was carried out overnight in S-cysteine low glucose DMEM. The supernatant was collected, and the ErbB2 monoclonal antibody or control antibody was added to the supernatant, and the supernatant was collected after 2-4 hours of incubation at 4 ℃. The complexes were pelleted, applied to a 10-20% Tricine SDS gradient gel, and electrophoresed at 100V. Gels were electrophoretically blotted onto membranes and analyzed by autoradiography. SEQ ID NO: epitopes 3H4 and 4D5 are shown at 8 and 9, respectively.

FIG. 2 shows the amino acid sequence of domain 1 of ErbB2 (SEQ ID NO: 1). The bold amino acids represent the epitope positions recognized by mAbs 7C2 and 7F3 as determined by deletion mapping, i.e., the "7C 2/7F3 epitope" (SEQ ID NO: 2).

Detailed description of the preferred embodiments

I. Definition of

The terms "HER 2", "ErbB 2", "c-Erb-B2" are used interchangeably. As used herein, unless otherwise indicated, the terms "ErbB 2", "c-Erb-B2" and "HER 2" refer to human proteins and "HER 2", "erbB 2" and "c-Erb-B2" refer to human genes. Human erbB2 gene and erbB2 protein are described, for example, in Semba et al pnas (usa) 82: 6497-: 230-. ErbB2 comprises four domains (domains 1-4).

"epitope 4D 5" is the region of the extracellular domain of ErbB2 that binds to antibody 4D5(ATCC CRL 10463). This epitope is adjacent to the transmembrane region of ErbB 2. For screening for antibodies that bind to the 4D5 epitope, a method such as "antibody: a conventional cross-blocking assay is described in the Experimental handbook, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988). Alternatively, an epitope map (see figure 1) can be made to assess whether the antibody binds to the 4D5 epitope of ErbB2 (i.e., about residue 529, such as any one or more residues in the region of residues 561 through 625).

The term "epitope 3H 4" refers to the region of the extracellular domain of ErbB2 that binds to antibody 3H 4. The epitope is shown in figure 1 and includes residues about 541 to 599 of the amino acid sequence of the extracellular domain of ErbB 2.

The term "epitope 7C2/7F 3" is the region N-terminal to the extracellular domain of ErbB2 that binds to the 7C2 and/or 7F3 antibodies (both deposited with the ATCC, see below). For screening for antibodies that bind to the epitope 7C2/7F3, antibodies such as "antibodies: a conventional cross-blocking assay is described in the Experimental handbook, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping can be used to determine whether the antibody binds to the 7C2/7F3 epitope on ErbB2 (i.e., any one or more residues in the region of approximately residues 22 to 53 in ErbB2 (SEQ ID NO: 2)).

The term "induces cell death" or "capable of inducing cell death" refers to the ability of an antibody to render a living cell non-viable. Herein, "cell" is a cell expressing the ErbB2 receptor, particularly a cell overexpressing the ErbB2 receptor. Cells overexpressing ErbB2 had significantly higher than normal levels of ErbB2 compared to non-cancerous cells of the same tissue type. Preferably, the cell is a cancer cell, such as a cancer cell of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas or bladder. In vitro, the cell may be an SKBR3, BT474, Calu3, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Cell death in vitro can be measured 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, cell death can be tested using heat inactivated serum (i.e., without complement) and in the absence of immune effector cells. To determine whether the antibody is capable of inducing cell death, the extent of loss of membrane integrity was assessed by uptake of Propidium Iodide (PI), trypan blue (see, Moore et al, Cytotechnology 17: 1-11(1995)), or 7AAD relative to untreated cells. Preferred antibodies that induce cell death are those that induce PI uptake in the PI uptake assay for BT474 cells.

The term "induce apoptosis" or "capable of inducing apoptosis" refers to the ability of an antibody to induce apoptosis, as determined by binding to annexin V, DNA fragmentation, cell shrinkage, endoplasmic reticulum swelling, cell lysis and/or formation of membrane vesicles known as apoptotic bodies. The cell is one that overexpresses the ErbB2 receptor. The "cell" is preferably a tumor cell, such as a cancer cell of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas or bladder. In vitro, the cell may be an SKBR3, BT474, Calu3 cell, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Various methods can be used to assess cellular activity associated with apoptosis. For example, Phosphatidylserine (PS) transport can be measured by annexin binding; DNA fragmentation can be assessed by a DNA ladder, as described in the examples herein; nuclear/chromatin condensation and DNA fragmentation can be assessed by the growth of hypodiploid cells. The antibody inducing apoptosis is preferably an antibody which induces about 2 to 50-fold (preferably 5 to 50-fold, and most preferably about 10 to 150-fold) of annexin binding as compared with untreated cells in the annexin binding assay using BT474 cells (see below).

Sometimes, the pro-apoptotic antibody is an antibody that blocks HRG binding to/activating the ErbB2/ErbB3 complex (e.g., the 7F3 antibody). In other instances, the antibody is an antibody that does not significantly block HRG activation of the ErbB2/ErbB3 receptor complex (e.g., 7C 2). Alternatively, the antibody may induce apoptosis without substantially decreasing the percentage of S phase cells (e.g., an antibody that decreases the percentage of such cells by about 0-10% relative to a control), as in 7C 2.

The antibody of interest binds specifically to human ErbB2 as does 7C2, and does not cross-react significantly with other proteins, such as those encoded by the ErbB1, ErbB3, and/or ErbB4 genes. Sometimes, this antibody may not significantly cross-react with rat neu protein (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 the proteins (e.g., binding of the cell surface to endogenous receptors) will be less than 10% (as measured by Fluorescein Activated Cell Sorting (FACS) analysis or Radioimmunoprecipitation (RIA)).

The term "genetic regulatory protein (HRG)" as used herein refers to a polypeptide that activates the ErbB2-ErbB3 and ErbB2-ErbB4 protein complexes (i.e., induces phosphorylation of tyrosine residues in the complexes when bound thereto). Various genetic regulatory protein polypeptides encompassed within this term are disclosed, for example, in Holmes et al Science, 256: 1205-1210 (1992); WO 92/20798; wen et al, mol.cell.biol., 14 (3): 1990-1919 (1994); and marchinoni et al, Nature, 362: 312-. The term includes biologically active fragments and/or variants of naturally occurring HRG polypeptides, such as EGF-like domain fragments thereof (e.g., HRG β)_177-244)。

The terms "ErbB 2-ErbB3 protein complex" and "ErbB 2-ErbB4 protein complex" are oligomers of the ErbB2 receptor that are non-covalently associated with the ErbB3 receptor or ErbB4 receptor, respectively. The complex formed when cells expressing both receptors were exposed to HRG, was isolated by immunoprecipitation and was analyzed by SDS-PAGE (as described by Sliwkowski et al, J.biol chem., 269 (20): 14661-14665 (1994)).

"antibody (Ab)" and "immunoglobulin (Ig)" are glycoproteins having the same structural features. Antibodies exhibit binding specificity for a specific antigen, while immunoglobulins include antibodies and other antibody-like molecules that lack antigen specificity. For example, the lymphatic system may produce low levels of the latter class of polypeptides, while myeloma may produce elevated levels of the latter class of polypeptides.

"native antibodies" and "native immunoglobulins" are heterotetrameric proteins of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (V) at one end_H) Followed by a plurality of constant regions. Each light chain has a variable region (V) at one end_L) And the other end has a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. It is believed that particular amino acid residues form the interface between the variable regions of the light and heavy chains.

The term "variable" means that certain portions of the variable regions in an antibody differ in sequence, and apply to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, in a substantially 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 close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol. I, 647, page 669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation of the antibody in antibody-dependent cytotoxicity.

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

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. This region consists of dimers of a heavy and a light chain variable region in close non-covalent association. In this configuration, the three CDRs in each variable region interact, at V_H-V_LThe surface of the dimer defines the antigen binding site. Collectively, the 6 CDRs confer antigen binding specificity to the antibody. However, even a single variable region (or half of an Fv, comprising only three CDRs specific for an antigen) can recognize and bind antigen, albeit with a lower affinity than the entire binding site.

The Fab fragment also contains the light chain constant region and the first constant region of the heavy chain (CH 1). Fab' fragments differ from Fab fragments in that several residues have been added at the carboxy terminus of the CH1 region of the heavy chain, including one or more cysteines from the hinge region. Fab '-SH is designated herein as Fab', in which the cysteine residues of the constant domains carry a free sulfhydryl group. F (ab')₂Antibody fragments were originally produced as Fab' fragment pairs with hinge cysteines in between. Other chemical coupling means of antibody fragments are also known.

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions.

Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the heavy chain constant region. There are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as 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" has its broadest meaning and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments having the desired biological activity.

An "antibody fragment" includes a portion of an intact antibody, preferably the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; antibodies (diabodies); linear antibodies (Zapata et al, Protein Eng.8 (10): 1057-1062 (1995)); a single chain antibody molecule; and antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprised in the population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, unlike 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, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in the present invention can be made by the hybridoma method (first set forth by Kohler et al, Nature, 256: 495 (1975)), or can be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). "monoclonal antibodies" can also be used, for example, in Clackson et al, Nature, 352: 624-: 581-597(1991) from phage antibody library.

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to an antibody sequence derived from that particular species or belonging to a particular antibody class or subclass and the remainder of the chain is homologous to a corresponding sequence derived from an antibody of another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the fragments exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl.Acad.Sci.USA, 81: 6851-6855 (1984)).

A "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab ', F (ab')₂Or other antigen binding sequence of an antibody) that contains minimal sequences of a non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which recipient Complementarity Determining Region (CDR) residues are replaced by CDR residues of an antibody of non-human origin (donor antibody), e.g., mouse, rat or rabbit having the desired specificity, affinity and activity. In some cases, the Fv Framework Region (FR) of the human immunoglobulin is replaced by a corresponding non-human residue. In addition, humanized antibodies may include residues that are not present in either the recipient antibody or the imported CDR or framework sequences. These modifications can be made to further enhance and maximize antibody performance. Typically, a humanized antibody will comprise substantially (at least one, and typically two) all of the variable regions, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody preferably also comprises at least a portion of an immunoglobulin constant region (Fc), typically the Fc portion of a human immunoglobulin. For more details see Jones et al, Nature, 321: 522-525 (1986); reichmann et al, Nature, 332: 323-329 (1988); and PrestaCurr op struct biol, 2: 593-596(1992). The humanized antibody comprises PRIMATIZED^TMAn antibody, wherein the antigen binding region of the antibody is derivable from an antibody made by immunization of cynomolgus monkeys with an antigen of interest.

"Single chain Fv" or "sFv" antibody fragments include the V of an antibody_HAnd V_LRegions, wherein the regions are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises V_HAnd V_LA polypeptide linker between the domains, which linker allows the sFv to form the structure required for binding to an antigen. For an review of sFv it can be found that Pluckthun, Pharmatology for monoclonal antibodies, Vol 113, Rosenburg and Moore eds, Springer-Verlag, New York, p.269-315 (1994).

The term "antibody" refers to a small antibody fragment having two antigen binding sites, which fragment includes a heavy chain variable region (V)_H) And a light chain variable region (V)_L) Connected to the same polypeptide chain (V)_H-V_L) In (1). Two antigen binding sites are formed by using a linker that is too short to allow pairing between two regions on the same strand, forcing the region to pair with a complementary region in the other strand. Antibodies are described, for example, in EP404, 096; WO 93/11161; and Hollinger et al, proc.natl.acad.sci.usa, 90: 6444-.

An "isolated" antibody is one that has been identified and separated and/or recovered from its natural environmental components. Contaminant components of its natural environment are substances that interfere with diagnostic or therapeutic applications of the antibody, which may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody will be purified to: (1) contains more than 95% by weight of antibody (measured by Lowery method), preferably more than 99% by weight; (2) sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence (as determined by a rotor sequencer); or (3) homogeneous as determined by SDS-PAGE under reducing or non-reducing conditions with Coomassie Brilliant blue or better silver staining. Isolated antibodies include antibodies in situ in recombinant cells, as at least one component of the antibody's natural environment is not present. However, isolated antibodies can generally be prepared by at least one purification step.

The term "salvage receptor binding epitope" as used herein refers to an IgG molecule (e.g., IgG)₁，IgG₂，IgG₃Or IgG₄) An epitope of the Fc region, which is responsible for increasing the serum half-life of the IgG molecule in vivo.

"treatment" refers to both therapeutic and prophylactic measures. Those in need of treatment are those already suffering from the disease and those in which the disease is to be prevented.

"mammal" in need of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, zoos, stadium animals, or pets, such as dogs, horses, cats, cows, etc. Preferably the mammal is a human.

A "disease" is any disease that benefits from treatment with an anti-ErbB 2 antibody. This includes chronic and acute diseases or disorders, including those pathological conditions that predispose a mammal to the disease. Non-limiting examples of diseases to be treated herein include benign and malignant tumors; leukemia and lymphoid malignancies; neurological, glial, astrocytic, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocoel diseases; and inflammatory, angiogenic and immunological diseases.

The term "therapeutically effective amount" as used herein refers to any amount that has an anti-proliferative effect. Preferably, the therapeutically effective amount has apoptotic activity, or is capable of inducing cell death, preferably a benign or malignant tumor cell, particularly a cancer cell. The effect can be measured in a conventional manner, depending on the disease to be treated. For cancer treatment, the effect can be determined, for example, by assessing the time to disease progression or determining the Response Rate (RR) (see examples below).

The terms "cancer" and "cancerous" refer to a physiological state in mammals that is typically characterized by unregulated cell growth. Examples of carcinomas include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include 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, breast cancer, colon cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and various head and neck cancers.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or destroys cells. The term includes radioisotopes (e.g., I)¹³¹、I¹²⁵、Y⁹⁰And Re¹⁸⁶) Chemotherapeutic agents and toxins, such as enzymatically active toxins of bacteria, fungi, plants or animals or fragments thereof.

A "chemotherapeutic agent" is a chemical substance used to treat cancer. Examples of chemotherapeutic agents include doxorubicin (adriamycin) (doxorubicin), epirubicin (epirubicin), 5-fluorouracil (5-FU), cytarabine (cytidine arabinoside) (Ara-C), cyclophosphamide (cyclophosphamide) (CYTOXAN)^TM) Thiotepa (thiotepa), busulfan (busufan), tacrolids (taxoids) (e.g. paclitaxel (paclitaxel) (r))Bristol-Myers Squibbonology, Princeton, NJ) and docetaxel (doxetaxel) (Taxotere, Rhone-Poulenc Rorer, Anthony, France)), methotrexate (methotrexate), cisplatin (cissplatin), vinblastine (vinblastine), bleomycin (bleomycin), epipodophyllotoxin (etoposide), ifosfamide (ifosfamide), mitomycin C (mitomycin C), Mitoxantrone hydrochloride (Mitoxantrone), vincristine (vincristine), Vinorelbine (Vinorelbine), Carboplatin (Carboplatin), epipodophyllotoxin thiophene glycoside (teniposide), daunomycin (daunomycin), carminomycin (carminomycin, carminomycin (4,675,187), carminomycin (carminomycin, carminomycin (carminomycin, carminomycin(s) and other related patents (see, U.S.S.S. Also included in this definition are hormonal agents which act to regulate or inhibit hormones acting on tumours, such as tamoxifen (tamoxifen) and onapristone (onapristone).

Other examples of chemotherapeutic agents include alkyl sulfonates such as improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodopa), carboquone (carboquone), metotepipa (meturedpa) and uredepa (uredpa); aziridines and melamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and trimethylmelamine; nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chloramphazine), chlorophosphamide (chlorophosphamide), estramustine (estramustine), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxydichloride), norubicin (novembichin), cholesteryl-p-phenylacetic acid mustard (phenylesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard (uramustard); nitrosoureas (nitrosureas), such as nitrosourea mustard (carmustine), chlorzotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), ramustine (ranimustine); antibiotics such as aclacinomycin (aclacinomycin), actinomycin (actinomycin), amicynin (aurramycin), azaserine (azaserine), actinomycin C (cactinomycin), calicheamicin (calicheamicin), karacin (carabicin), carzinophilin (carzinophilin), chromomycin (chromomycin), 6-diazo-5-oxo-L-norleucine (6-diazo-5-oxo-L-norleucin), daunomycin (daunorubicin), mycophenolic acid (mycophenolic acid), nogacin (nogalamycin), olivomycin (olivomycin), pelomomycin (polyplomycin), podoficin (potomycins), puromycin (puromycin), streptomycin (streptozocin), gentamycin (gentamycin), tuberculin (zostatin), and gentamycin (zostatin); folic acid analogues, e.g. denopterin (denopterin), serolo(iv) pteropterin (pteropterin), trimetrexate (trimetrexate); purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (6-mercaptopurine), thiamiroproline (thiamiriprine), thioguanine (thioguanine); pyrimidine analogs such as cyclocytidine (ancitabine), azacitidine (azacitidine), 6-azauridine (6-azauridine), carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), and floxuridine (floxuridine); androgens such as dimethyltestosterone (calusterone), drostanolonepropionate, epitioandrostanol (epitiostanol), mepiquane (mepiquantene), testolactone (testolactone); anti-adrenals such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements, such as frilic acid; acetoglucuronide (acegulenone), phosphoramide aldoside (aldophosphamide glycoside); aminolevulinic acid (aminolevulinic acid); amsacrine (amsacrine); bessburyl (beslabucil); bisantrene; ideback (edatraxate); desphosphamide (defofamine); dimecorsine (demecolcine); diazaquinone (diaziqutone); fornixing (elfornitine); ammonium etitanium acetate; etodolu (etoglucid), gallium nitrate (galliumnitrate), hydroxyurea (hydroxyurea), lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); nitramines (nitracrine); gustducin (pentostatin); benzene nanotide (phenamett); pirarubicin (pirarubicin); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide (2-ethyl hydrazide); toluhydrazide (procarbazine);(ii) a Razoxane (rizoxane); cilostan (sizofiran); germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2, 2 ', 2' -trichlorotriethylamine (2, 2 ', 2' -trichlorotriethylamine); ethyl carbamate (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromo mannitol (mitobronit)ol); dibromodulcitol (mitolactol); pipobromane (pipobroman); gatifloxacin (gacytosine); chlorambucil (chlorambucil); gaicetobine (gemcitabine); 6-thioguanine (6-thioguanine); mercaptopurine (mercaptoprine); platinum (platinum); neomycin amide (navelbine); novatrone (novantrone); tiodol (xeloda); ibandronate (ibandronate); CPT-11; topoisomerase (topoisomerase) inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid (retinic acid); capecitabine, and pharmaceutically acceptable salts, acids or derivatives thereof.

Other examples of hormonal agents that modulate or inhibit hormones acting on tumors include other antiestrogens such as raloxifene (Evista), aromatase inhibiting4(5) -imidazole (aromatase inhibiting4(5) -imidazoles), 4-hydroxytryptamine (4-hydroxytryptafene), trioxifene (trioxifene), kexiphenol (keoxifene), and LY 117018; and anti-androgens such as flutamide (flutamide) and nilutamide (nilutamide); and pharmaceutically acceptable salts, acids or derivatives thereof.

The term "growth inhibitory agent" as used herein refers to a compound or composition that inhibits the growth of cells, particularly cancer cells that overexpress ErbB2, in vitro or in vivo. Thus the growth inhibitory agent significantly reduced the percentage of cells overexpressing ErbB2 in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (beyond S phase), such as agents that induce G1 arrest and M phase arrest. Typical M-phase blockers include the vinca alkaloids (vincristine) and vinblastine (vinblastine)),and topo type II inhibitors such as doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide), and bleomycin (bleomycin). Those agents that arrest G1 will also spill over (spill over) to S phase arrest, for example DNA alkylating agents such as tamoxifen, prednisone,Dacarbazine (dacarbazine), mechlorethamine (mechlorethamine), cisplatin (cispain), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil) and cytarabine (ara-C). Further information can be found in molecular basis for cancer, edited by Mendelsohn and Israel, Chapter1 entitled "cell cycle Regulation, oncogenes and antitumor drugs", Murakami et al, (WB Saunders: Philadelphia, 1995), especially page 13. The 4D5 antibody (and functional equivalents thereof) may also be used for this purpose.

"Adriamycin" is an anthracycline antibiotic. The chemical name of adriamycin is (8S-cis) -10- [ (3-amino-2, 3, 6-trideoxy-alpha-L-lyxose-hexopyranosyl) oxy ] -7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5, 12-naphthalenedione.

The term "cytokine" is a genetic term for a protein released by one type of cell that acts on another cell as an intercellular medium. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; (ii) prorelaxin; glycoprotein hormones (such as Follicle Stimulating Hormone (FSH); Thyrotropin (TSH) and Luteinizing Hormone (LH)); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and beta; a Miller inhibitor; mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factor (such as NGF-beta); platelet growth factor; transforming Growth Factors (TGF) (e.g., TGF-alpha and TGF-beta); insulin-like growth factors I and II; erythropoietin (EPO); osteoinductive factor (osteoinductive factor); interferons (e.g., interferon alpha; beta and gamma); colony Stimulating Factors (CSFs) (e.g., macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF)); interleukins (IL) (e.g., 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); tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and cassette ligands (KL, kit ligand). The term cytokine as used herein includes proteins from natural or recombinant cell culture as well as biologically active equivalents of the native sequence cytokines.

The term "prodrug" as used herein refers to a precursor or derivative form of a pharmaceutically active agent which is less toxic to tumor cells than the parent drug and which is capable of being enzymatically activated into or converted into the more active parent form. See, for example, Wilman, "prodrugs in cancer chemotherapy" biochemical society Transactions, 14, pp.375-382, 615th Meeting Belfast (1987) and Stella et al, "prodrugs: a chemical method for the Directed Delivery of drugs, "Directed Drug Delivery, Borchardt et al, (ed.), pp.247-267, Humana Press (1985). Prodrugs of the invention include, but are not limited to, phosphate-containing prodrugs, phosphorothioate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β -lactam-containing prodrugs, prodrugs containing optionally substituted phenoxyacetamide or prodrugs containing optionally substituted phenylacetamide, 5-fluorocytosine and other 5-fluorouracil prodrugs capable of being converted to the more cytotoxic free drug. Examples of cytotoxic drugs that may be derivatized into prodrug forms for use in the present invention include, but are not limited to, those chemotherapeutic agents described above.

"solid phase" refers to a non-aqueous matrix to which an antibody of the invention can adhere. Examples of solid phases herein include those made partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrenes, polyvinyl alcohols, and silicones. In certain examples, depending on the context, the solid phase may comprise wells of a test plate; in other examples, it may be a purification column (e.g., an affinity chromatography column). The term also includes a discontinuous solid phase of dispersed particles as disclosed in U.S. Pat. No.4,275,149.

"liposomes" are vesicles composed of various classes of lipids, phospholipids and/or surfactants used to deliver drugs (such as the anti-ErbB 2 antibodies disclosed herein, and optionally chemotherapeutic agents) to mammals. The components of liposomes are typically arranged in a bilayer format, 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, containing information about the indication, use, dosage, mode of administration, contraindications and/or warnings concerning the use of such therapeutic products.

Production of anti-ErbB 2 antibodies

A typical protocol for producing the antibodies of the invention will now be described. The ErbB2 antigen used to produce antibodies can be, for example, a soluble form of the extracellular domain of ErbB2 or a portion thereof containing the desired epitope. Alternatively, antibodies can be produced using cells expressing ErbB2 on the cell surface (e.g., NIH-3T3 cells transformed to overexpress ErbB 2; or cancer cell lines such as SKBR3 cells, see Stancovski et al, PNAS (USA) 88: 8691-8695 (1991)). Other forms of ErbB2 that can be used to produce antibodies will be apparent to those skilled in the art.

(i) Polyclonal antibodies

Preferably, polyclonal antibodies are produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Using bifunctional or derivatizing reagents (e.g. sulphosuccinimidyl maleimidobenzyloxy ester (coupled via a cysteine residue), N-hydroxysuccinimide (coupled via a lysine residue), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N ═ C ═ NR (where R and R are¹Different alkyl groups)) may be coupled to a protein that is immunogenic in the animal to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor).

Animals are immunized with antigen, immunogenic conjugate or derivative by mixing, for example, 100. mu.g or 5. mu.g of protein or conjugate (to rabbit or mouse, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution into multiple sites of the intradermal space. After 1 month, injections into multiple subcutaneous sites were boosted with a prior amount of 1/5 to 1/10 of peptide or conjugate in Freund's complete adjuvant. After 7 to 14 days, the animals were bled and the serum was assayed for antibody titer. The booster animals were continued until the titer rose to a plateau. Preferably, the animals are boosted with conjugates of the same antigen, but conjugated to different proteins and/or via different cross-linking agents. The conjugates can also be made into fusion proteins in recombinant cell culture. In addition, aggregating agents (e.g., alum) are also useful for enhancing immune responses.

(ii) Monoclonal antibodies

Monoclonal antibodies can be obtained from a substantially homogeneous class of antibodies, i.e., each antibody contained in the population is identical. Except for the possible presence of very few naturally occurring mutations. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of unrelated antibodies.

For example, monoclonal antibodies can be made by the hybridoma method (first developed by Kohler et al, Nature, 256: 495 (1975)), or by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other suitable host animal (e.g., hamster) is immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes and myeloma cells are then fused with a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, monoclonal antibodies: Principles and Practice, pp.59-103(Academic Press, 1986)).

The hybridoma cells thus produced are then seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), hypoxanthine, aminopterin, and thymidine (HAT medium) are typically added to the hybridoma culture medium, which substances inhibit the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stable and high-level production of antibodies by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Preferred myeloma Cell lines among these are mouse myeloma Cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, Calif. USA) and SP-2 or X63-Ag8-653 cells (available from American Type clinical Collection, Rockville, Maryland USA). In addition, human myeloma and mouse-human hybrid myeloma cell lines for producing human Monoclonal antibodies have also been described (Kozbor, J.Immunol., 133: 3001 (1984); Brodeur et al, Monoclonal antibody Production Techniques and applications, pp51-63(Marcel Dekker, Inc., New York, 1987)).

The presence or absence of monoclonal antibody production against the antigen in the culture medium in which the hybridoma grows is determined. The binding specificity of monoclonal antibodies produced by hybridoma cells is preferably determined by immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).

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

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

Monoclonal antibodies secreted by the subcloned cells are suitably isolated from the culture medium, ascites fluid or serum using conventional immunoglobulin purification procedures (e.g., protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography).

DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of a mouse antibody). Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells (e.g., E.coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein) and monoclonal antibodies synthesized in the recombinant host cells. For review of recombinant expression of DNA encoding antibodies in bacteria, literature includes Skerra et al, curr. 256-: 151-188(1992).

In another embodiment, the method may be selected from the group consisting of methods employing McCafferty et al, Nature, 348: 552 (1990) and isolating the antibody or antibody fragment from the antibody phage library. Clackson et al, Nature, 352: 624-: 581-597(1991) describes the isolation of murine and human antibodies, respectively, using phage libraries. Later publications describe the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology, 10: 779-. Thus, these methods are viable alternatives to the isolation of monoclonal antibodies by traditional monoclonal antibody hybridoma techniques.

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

Typically, the substitution of these non-immunoglobulin polypeptides for the constant regions of an antibody, or their substitution for the variable regions of one antigen-binding site in an antibody, results in a chimeric bivalent antibody comprising an antigen-binding site specific for one antigen and another antigen-binding site specific for a different antigen.

(iii) Humanized antibody and human antibody

Methods for humanizing non-human antibodies are well known in the art. Preferably, the humanized antibody has one or more amino acid residues of non-human origin introduced therein. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable region. Humanization can be based essentially on Winter and its 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 replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, these "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which substantially less than an entire human variable region is replaced by the corresponding sequence of a non-human animal. 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 selection of human light and heavy chain variable regions to make humanized antibodies is important for reducing antigenicity. The entire library of known human variable region sequences is used to screen rodent antibody variable region sequences according to the so-called "best fit" method. The human sequence closest to the rodent sequence is then used as the human Framework Region (FR) of the humanized antibody (Sims et al, J.Immunol., 151: 2296 (1993); Chothia et al, J.mol.biol., 196: 901 (1987)). Another approach employs specific framework regions derived from consensus sequences of a specific subset of all human antibody light or heavy chains. The same framework region can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al, J.Immunol., 151: 2623 (1993)).

More importantly, humanization of antibodies should retain high affinity for the antigen as well as other favorable biological properties. To achieve this, a preferred method of preparing humanized antibodies is to analyze the parental sequences and various conceptual (concept) humanized antibodies using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are generally available and well known to those skilled in the art. Computer programs are available that describe and display the likely three-dimensional conformational structures of candidate immunoglobulin sequences. Observing these indications, one can analyze the likely role residues play in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. Thus, FR residues can be selected from the donor antibody and the introduced sequence and combined to obtain the desired antibody properties (e.g., increased affinity for the target antigen). Generally, CDR residues directly and most substantially affect antigen binding.

Alternatively, transgenic animals (e.g., mice) can now be prepared that can be immunized to produce the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, antibody heavy chain joining regions (J) have been described in chimeric germline mutant mice_H) Homozygous deletion of the gene completely suppresses endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into these germline mutant mice allows them to produce 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, Yeast in immunity, 7: 33(1993). Human antibodies can also be obtained 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 for preparing antibody fragments have been developed. Traditionally, these fragments have been obtained by protein digestion of intact antibodies (see, e.g., Morimoto et al, Journal of biochemical and Biophysical Methods 24: 107-nan et al, Science, 229: 81(1985)). However, these fragments can now be produced directly by recombinant host cells. For example, as described above, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically crosslinked to form F (ab')₂Fragments (Carter et al, Bio/Technology 10: 163-. According to another approach, the F (ab') 2 fragment can be isolated directly from the recombinant host cell culture. Other methods of making antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185.

(v) Bispecific antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. A typical bispecific antibody can bind to two different epitopes of ErbB2 protein. For example, one arm can bind to an epitope in domain 1 of ErbB2 (e.g., the 7C2/7F3 epitope) and the other can bind to a different ErbB2 epitope (e.g., the 4D5 epitope). Other such antibodies may combine the ErbB2 binding site with the EGFR, ErbB3, and/or ErbB4 binding site. Alternatively, the anti-ErbB 2 arm may be combined with an arm that binds to a trigger molecule on a leukocyte (e.g., T cell receptor molecule CD2 or CD3) or Fc receptor (fcyr) of IgG (e.g., fcyri (CD64), fcyrii (CD32), and fcyriii (CD16) to focus cellular defense mechanisms on cells expressing ErbB 2. bispecific antibodies may also be used to localize cytotoxic agents to cells expressing ErbB 2. these antibodies have a2 binding arm and a cytotoxic agent (e.g., saponin, antiinterferon alpha, vinca alkaloid, ricin a chain, methotrexate, or radioactive isotope hapten) binding arm₂Bispecific antibodies).

Methods for making bispecific antibodies are known in the art. The traditional approach to making full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs with different specificities (Millstein et al, Nature, 305: 537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadracy) may produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule (usually by affinity chromatography) is quite laborious and yields are low. WO93/08829 and Traunecker et al, EMBO J.10: 3655-3659(1991) disclose a similar process.

According to different methods, the variable region of an antibody with the desired binding specificity (antibody-antibody binding site) is fused to an immunoglobulin constant region sequence. Preferably to the constant region of an immunoglobulin heavy chain, and includes at least a portion of the hinge, CH2 and CH3 regions. Preferably, at least one of the fusions contains a first heavy chain constant region (CH1) that contains the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains are used in the construction to provide optimal yields, this provides greater flexibility in adjusting the ratios of the three polypeptide fragments to each other. However, when expression of at least two polypeptide chains in equal ratios results in high yields, or the ratio is inconsequential, the coding sequences for two or all three polypeptide chains can be inserted into one expression vector.

In a preferred embodiment of the method, the bispecific antibody consists 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. This asymmetric structure has been found to be advantageous for isolating the desired bispecific antibody from unwanted immunoglobulin chain compositions, since the immunoglobulin light chain is present in only half of the bispecific molecule and is therefore easily isolated. This method is disclosed in WO 94/04690. More details of the preparation of bispecific antibodies can be found, for example, in Suresh et al, Methods in Enzymology, 121: 210(1986). According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered such thatThe heterodimers obtained from the recombinant cell culture were recovered to a maximum percentage. Preferred interfaces include at least antibody constant region C_H3 domain. In this method, one or more of the small side chains of amino acids at the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). Compensatory "holes" of the same or similar size to the large side chains (in the primary antibody) are formed at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller side chains (such as alanine or threonine). This provides a mechanism to allow higher yields of heterodimers than other undesired end products (e.g., homodimers).

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

Methods for making bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared by chemical linkage. Brennan et al, Science, 229: 81(1985) describes a method of proteolytic cleavage of intact antibodies to F (ab')₂And (3) fragment. These fragments are reduced in the presence of the dimercapto complexing reagent sodium arsenite to stabilize vicinal dimercapto groups and thereby prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One Fab '-TNB derivative is then converted back to the Fab' -sulfhydryl group by reduction with mercaptoethylamine. And then mixed with an equimolar amount of another Fab' -TNB derivative to form the bispecific antibody. The bispecific antibody prepared can be used as a reagent for selectively immobilizing enzymes.

More recentProgress has been made to recover and obtain directly from E.coli Fab' -SH fragments that can be chemically cross-linked to form bispecific antibodies. Shalaby et al, j.exp.med., 175: 217-225(1992) describes fully human bispecific antibodies F (ab')₂The preparation process of the molecule. The respective Fab' fragments were secreted separately from E.coli and then subjected to directed chemical cross-linking in vitro to form bispecific antibodies. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells and trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various methods have been described for the preparation and isolation of bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies can be made using leucine zippers. Kostelny et al, j.immunol., 148 (5): 1547-1553(1992). The leucine zipper peptides of the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The hinge region of the antibody homodimer is reduced to form a monomer, and then re-oxidized to form an antibody heterodimer. This method can also be used to prepare antibody homodimers. Hollinger et al proc.natl.acad.sci.usa, 90: the "antibody" technique described in 6444- > 6448(1993) provides an alternative mechanism for the preparation of bispecific antibody fragments. These fragments comprise the heavy chain variable region (V) linked via a linker_H) Linked to the variable region of the light chain (V)_L) The linker is so short that no pairing can occur between two functional domains on the same chain. Thus, V on a segment_HAnd V_LThe functional domain is forced to complement V on another fragment_LAnd V_HThe functional domains pair, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments using single chain fv (scFv) dimers has also been reported. See Grubber et al, j.immunol., 152: 5368(1994).

Antibodies with more than a bivalent antibody are also contemplated. For example, trispecific antibodies can be made. Tutt et al, j.immunol.147: 60(1991).

(vi) Screening for antibodies of desired Properties

Methods of making antibodies are described above. Those antibodies having the properties described herein are now selected.

To select antibodies that induce cell death, loss of membrane integrity is indicated, for example, by assessment of PI, trypan blue, or 7AAD uptake relative to controls. The preferred test method is the "PI uptake assay using BT474 cells". According to this assay, BT474 cells (available from the American type culture Collection (Rochville, Md)) were cultured in Dulbecco's modified Eagle Medium (D-MEM): ham's F-12 (50: 50) (with the addition of 10% heat-inactivated FBS (Hyclone) and 2mM L-glutamine). (thus, the assay is performed in the absence of complement and immune effector cells). BT474 cells were plated at 3X 10 cells per dish⁶Individual densities were inoculated into 100X 20mm dishes and allowed to adhere overnight. The medium is then removed and replaced with fresh medium alone or with medium containing 10. mu.g/ml of the appropriate monoclonal antibody. Cells were incubated for 3 days. After each treatment, the monolayers were washed with PBS and detackified with trypsin digestion. Then, the cells were centrifuged at 1200rpm for 5 minutes at 4 ℃ and the pellet was resuspended in 3ml of ice-cold Ca²⁺Binding buffer (10mM Hepes, pH7.4, 140mM NaCl, 2.5mM CaCl₂) In (b), the cells were aliquoted into 35mm 12X 75 test tubes (1 ml per tube, 3 tubes as a treatment group) covered with a filter to remove clumps of cells. PI (10. mu.g/ml) was then added to the tube. Using FACSCAN^TMFlow cytometer and FACSCOPERT^TMThe samples were analyzed by CellQuest software (Becton Dickinson). Those antibodies were selected that induced a statistically significant level of cell death (as determined by PI uptake).

To select antibodies that induce apoptosis, an "annexin binding assay using BT474 cells" may be used. BT474 cells were cultured and plated into dishes as described in the previous paragraph. The medium was then removed and replaced with fresh medium alone or with medium containing 10. mu.g/ml monoclonal antibody. After 3 days of incubation, the monolayers were washed with PBS and detackified with trypsin digestion. The cells were then centrifuged and resuspended in Ca²⁺Combined in buffer and aliquoted into tubes as described above for cell death assays. Then theLabelled annexin (e.g.annexin V-FTIC) (1. mu.g/ml) was added to the tube. Using FACSCAN^TMFlow cytometer and FACSCOPERT^TMThe samples were analyzed by CellQuest software (Becton Dickinson). Those antibodies that induce a statistically significant level of annexin binding (compared to control) were selected as apoptosis-inducing antibodies.

In addition to the annexin binding assay, the "DNA staining assay with BT474 cells" can also be used. To perform this assay, BT474 cells treated with the antibody of interest as described in the previous two paragraphs were incubated at 9. mu.g/ml HOECHST 33342^TMIncubated at 37 ℃ for 2 hours, then MODFIT LT was added^TMSoftware (Verity Software House) at EPICS ELITE^TMAnalyzed on a flow cytometer (Coulter corporation). Antibodies that induce a change in the percentage of apoptotic cells (2-fold or greater (preferably 3-fold or greater) over untreated cells (up to 100% apoptosis)) may be selected as pro-apoptotic antibodies using this assay.

To screen for antibodies that bind to the epitope of ErbB2 that binds to the antibody of interest, a conventional cross-blocking assay can be used, as described in the handbook of antibodies, Cold Spring Harbor Laboratory, Ed Harbor and David Lane (1988). Alternatively, epitope mapping can be performed using methods known in the art.

To identify anti-ErbB 2 antibodies that inhibit the growth of 50-100% SKBR3 cells in cell culture, the SKBR3 assay described in WO89/06692 was performed. According to this experiment, SKBR3 cells were grown in F12 and DMEM medium mixture (1: 1) supplemented with 10% fetal bovine serum, glutamine and penicillin streptomycin. 20,000 SKBR3 cells (2ml/35mm dish) were seeded in 35mm cell culture dishes. anti-ErbB 2 antibody was added at 2.5. mu.g/ml to each dish. After 6 days, COULTER was used^TMThe electronic cell counter counts the number of cells compared to untreated cells. Those antibodies that inhibited growth of 50-100% SKBR3 cells were selected and used in combination with apoptotic antibodies as needed.

(vii) Engineering of effector functions

It may be desirable to modify the antibodies of the invention in effector function to enhance the effectiveness of the antibodies in treating, for example, cancer. For example, cysteine residues may be introduced in the Fc region, thereby forming interchain disulfide bonds in this region. The homodimeric antibody thus produced may have improved internalization capacity and/or increased complement-mediated cell killing capacity and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, j.exp med.176: 1191-1195(1992) and shop, B.J.Immunol.148: 2918-2922(1992). Also, for example, Wolff et al Cancer Research 53: 2560 2565(1993), heterodimeric antibodies with enhanced antitumor activity were prepared using heterobifunctional cross-linkers. Alternatively, antibodies can be engineered to have a dual Fc region, potentially enhancing complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design 3: 219-230(1989).

(viii) Immunoconjugates

The invention also relates to immunoconjugates comprising an antibody as described herein conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents used to prepare such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof which may be used include diphtheria toxin a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin a chain, abrin a chain, α -sarcina, Aleurites fordii protein, diaphin protein, pokeweed protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, maple toxin, croton toxin, saposin inhibitor, erulonin (gelonin), mitogellin source (mitogellin), restrictocin, phenomycin, enomycin and tribasic western (tricothecenes). Various radionuclides can be used to prepare radioconjugated anti-ErbB 2 antibodies. Examples include²¹²Bi，¹³¹I，¹³¹In，⁹⁰Y and¹⁸⁶Re。

conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3- (2-pyridyldithio) -propionate (SPDP), Iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipate, HCL), active esters (e.g., succinimidyl suberate), aldehydes (e.g., glutaraldehyde), diazides (e.g., bis (p-azidobenzoyl) hexanediamine), diazide derivatives (e.g., bis (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (e.g., toluene 2, 6-diisocyanate), and bis-active fluorine compounds (e.g., 1, 5-difluoro-2, 4-dinitrobenzene). For example, the ratio can be determined according to Vitetta et al Science 238: 1098(1987) the immunotoxin for the preparation of ricin. C¹⁴Labeled 1-isothiocyanatophenyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is a typical chelator used to couple radionucleotides to antibodies. See WO 94/11026.

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

(ix) Immunoliposomes

The anti-ErbB 2 antibodies disclosed herein can also be made into immunoliposomes. Methods for preparing liposomes containing such antibodies are known in the art, such as Epstein et al, proc.natl.acad.sci.usa, 82: 3688 (1985); hwang et al, proc.natl.acad.sci.usa, 77: 4030 (1980); and U.S. patent nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be prepared by reverse evaporation from lipid compositions comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of defined pore size to obtain liposomes of the desired diameter. The antibody Fab' fragment of the invention can be represented by Martin et al, J.biol.chem.257: 286-288(1982) were coupled to the liposomes by a disulfide interchange reaction. The liposomes may optionally contain a chemotherapeutic agent. See Gabizon et al, J.national Cancer Inst.81(19)1484 (1989).

(x) Antibody-dependent enzyme-mediated prodrug therapy (ADEPT)

The antibodies of the invention may also be used in ADEPT by coupling the antibody to an enzyme which activates a prodrug, which is capable of converting a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) into an active anticancer agent. See, for example, WO88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate used for ADEPT includes any enzyme that acts on the prodrug to convert it to a more active and cytotoxic form.

Enzymes useful in the methods of the invention include, but are not limited to: alkaline phosphatase for converting a phosphate-containing prodrug into a free drug; arylsulfatase for converting a sulfate-containing prodrug into a free drug; cytosine deamidase for converting non-toxic 5-fluorocytosine into an anticancer drug (5-fluorouracil); proteases for converting peptide-containing prodrugs into free drugs, such as serratia (serata) protease, thermolysin, subtilisin, carboxypeptidase, and cathepsin (e.g., cathepsins B and L); a D-alanylcarboxypeptidase for converting a prodrug containing a D-amino acid substituent; carbohydrate cleaving enzymes such as β -galactosidase and neuraminidase for converting glycosylated prodrugs into free drugs; a beta-lactamase for converting a beta-lactam derived drug into a free drug; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, for converting phenoxyacetyl or phenylacetyl-derived amino nitrogen, respectively, into free drug. Alternatively, a prodrug of the invention may be converted to a free, active drug using an enzymatically active antibody (also known as abzyme) (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein to deliver abzymes to a tumor cell population.

The enzyme of the invention is covalently bound to anti-ErbB 2 antibodies using techniques known in the art (e.g., using the above-described heterobifunctional cross-linking agents). Alternatively, using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al, Nature, 312: 604-608(1984)), fusion proteins can be constructed which comprise at least the antigen binding region of an antibody of the invention and associated therewith at least one functionally active portion of an enzyme of the invention.

(xi) Fusion of antibody-salvage receptor binding epitopes

In certain embodiments of the invention, it is desirable to employ antibody fragments rather than whole antibodies, for example, to improve tumor penetration. In such cases, it is desirable to modify the antibody fragment to increase its serum half-life. This can be accomplished, for example, by incorporating a salvage receptor binding epitope in the antibody fragment (e.g., by mutation in an appropriate region in the antibody fragment, or by incorporating the epitope in the peptide tail end, which is then fused into the end or middle of the antibody fragment by DNA or peptide synthesis).

The systematic method for preparing such antibody variants with increased in vivo half-life comprises the following steps. First, the sequence and configuration of the salvage receptor binding epitope of the Fc region of the IgG molecule was identified. Once the epitope is determined, the sequence of the antibody of interest can be modified to include the sequence and configuration of the identified binding epitope. After mutation of this sequence, antibody variants were tested to see if their half-life in vivo was longer than that of the original antibody. If the antibody variant does not have a longer in vivo half-life when tested, its sequence is again altered to include the sequence and configuration of the identified binding epitope. The altered antibody is tested for a longer in vivo half-life and the process is continued until a molecule exhibiting a longer in vivo half-life is obtained.

The salvage receptor binding epitope thus incorporated into the antibody of interest is any suitable epitope described above, the characteristics of which depend, for example, on the type of antibody being modified. This transfer should allow the antibody of interest to still have the biological activity described herein.

Preferably, the epitope constitutes a region in which any one or more amino acid residues from one or both loops of the Fc region are transferred to a similar position on the antibody fragment. More preferably, three or more residues of one or two loops in the Fc region are transferred. It is also preferred that the epitope is taken from the CH2 region in the Fc region (of IgG, for example) and is transferred to the antibodies CH1, CH3 or V_HA region, or more than one such region. Alternatively, the epitope is taken from the CH2 region of the Fc region and transferred to the C of the antibody fragment_LRegion or V_LIn a zone or in both.

In a most preferred embodiment, the salvage receptor binding epitope comprises the sequence (5 'to 3'): PKNSSMISNTP (SEQ ID NO: 3), and optionally further comprising a sequence selected from HQSTGTG (SEQ ID NO: 4), HQNLSDGK (SEQ ID NO: 5), HQNISDGK (SEQ ID NO: 6) or VISSLGQ (SEQ ID NO: 7), especially when the antibody fragment is a Fab or (Fab')₂Then (c) is performed. In another preferred embodiment, the salvage receptor binding epitope is a polypeptide comprising the following sequence (5 'to 3'): HQNLSDGK (SEQ ID NO: 5), HQNISDGK (SEQ ID NO: 6) or VISSHLGGQ (SEQ ID NO: 7) and sequence PKNSSMISNTP (SEQ ID NO: 8).

(xii) Purification of anti-ErbB 2 antibodies

When recombinant techniques are employed, the antibody may be produced intracellularly (in the periplasmic space) or secreted directly into the culture medium. If the antibody is produced intracellularly, the first step is to remove particulate debris of the host cell or lysed fragment, e.g., by centrifugation or ultracentrifugation. Carter et al, Bio/Technology 10: 163-167(1992) describes a method for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell slurry was thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris was removed by centrifugation. If the antibody is secreted into the culture medium, it is preferred to first concentrate the supernatant of the expression system using a commercially available protein concentration filter (e.g., an ultrafiltration device from Amicon or Millipore Pellicon). Protease inhibitors (e.g., PMSF) which inhibit proteolysis may be added at any of the preceding steps, and antibiotics may also be added to prevent the growth of adventitious contaminants.

For example, antibody compositions prepared from cells can be purified using hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification method. The degree to which protein a is suitable as an affinity ligand depends on the animal species and isotype of any immunoglobulin Fc region in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, J.Immunol. meth.62: 1-13 (1983)). For all mouse isotypes and human gamma 3, protein G is proposed (Guss et al, EMBO J.5: 1567-1575 (1986)). The matrix used for affinity ligand attachment is typically agarose, but other matrices may be used. Mechanically stable matrices such as pore size controlled glass or poly (styrene divinyl) benzene can achieve faster flow rates and shorter run times than agarose. When the antibody comprises C_HWhen the 3-zone is available, Bakerbond ABX can be used^TMPurification was performed on resin (j.t. baker, phillips burg, NJ). Depending on the antibody to be recovered, other protein purification methods may be used, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin Sepharose on anion or cation exchange resins (e.g., polyaspartic acid columns)^TMChromatography, focusing chromatography, SDS-PAGE and ammonium sulfate precipitation.

After any initial purification, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., about 0-0.24M salt).

C. Pharmaceutical preparation

The antibody therapeutic agent for use in the present invention is prepared and deposited in the form of a lyophilized preparation or an aqueous solution by mixing the antibody having a desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical sciences, 16 th edition, Osol, editors (1980)). Acceptable carriers, excipients or stabilizers at the dosages and concentrations employed should not be deleterious to the recipient thereofToxic, they include: buffers such as phosphate, citrate and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (e.g. octadecyl dimethyl benzyl ammonium chloride, hexa methyl quaternary amine 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, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, e.g. TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

The formulations herein may also contain one or more active compounds as required for the particular indication being treated, especially those compounds which have complementary activity and which do not adversely affect each other. For example, in one formulation, it would also be desirable to provide antibodies that bind to EGFR, ErbB2 (e.g., antibodies that bind to different epitopes on ErbB2), ErbB3, ErbB4, or Vascular Endothelial Growth Factor (VEGF). Alternatively, the composition may additionally comprise a cytotoxic agent, cytokine or growth inhibitory agent, provided that the cytotoxic agent is not an anthracycline derivative (e.g., doxorubicin or epirubicin). These molecules should be combined in effective doses to achieve the desired purpose.

The active ingredient may also be encapsulated in microcapsules prepared, for example, by agglomeration techniques or interfacial polymerization methods, such as hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microbeads, microemulsions (micro-emulsions), nano-sized particles and nano-sized capsules) or macroemulsions (macroemulsions). These techniques are disclosed in Remington's Pharmaceutical Sciences 16 edition, eds Osol, A. (1980).

Formulations for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

Can be made into sustained release preparation. Suitable examples of sustained release formulations include solid hydrophobic polymeric semipermeable matrices containing the antibody, wherein the matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and γ -ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(injectable microbeads consisting of lactic-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid release the molecule for over 100 days, some hydrogels release proteins for shorter periods of time. When encapsulated antibodies stay in the body for a long time, they are denatured or aggregated by exposure to moisture at 37 ℃, resulting in loss of biological activity, and immunogenicity may be changed. Rational strategies for stabilization can be designed according to the mechanism involved. For example, if the mechanism of aggregation is found to be intermolecular S — S bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling the degree of humidity, employing appropriate additives, and developing specific polymer matrix compositions.

Treatment of anti-ErbB 2 antibodies

The present invention contemplates that anti-ErbB 2 antibodies may be used to treat a variety of disorders characterized by overexpression and/or activation of the ErbB2 receptor. Examples of typical conditions or diseases include benign or malignant tumors (e.g., kidney, liver, kidney, bladder, breast, stomach, ovary, colorectal, prostate, pancreas, lung, vulva, thyroid, hepatocellular carcinoma; sarcomas; glioblastoma and various head and neck tumors); leukemia and lymphoid malignancies; other diseases such as neurological, glial, astrocytic, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocoel diseases; as well as inflammatory, angiogenic and immune disorders.

The antibody of the present invention can be administered to a human patient according to known methods, such as intravenous administration as a bolus, or continuous infusion for a period of time via the intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred.

The therapeutic methods of the present invention involve the administration of an anti-ErbB 2 antibody in combination with a chemotherapeutic agent other than an anthracycline derivative. Co-administration includes co-administration, use of separate formulations or one pharmaceutical formulation, and sequential administration in any order, preferably for a period of time when both (or all) active agents exert their biological activity simultaneously. The formulation and dosage regimen of these chemotherapeutic agents can be used according to the manufacturer's instructions or can be determined according to the practical experience of a skilled physician. Formulations and dosage regimens for these chemotherapeutics are also disclosed in Chemotherapy Service ed, m.c. perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may be administered before, after, or simultaneously with the administration of the antibody. The antibodies may be combined with anti-estrogen compounds (e.g., tamoxifen) or anti-progestin compounds (e.g., onapristone) (see EP616812), and dosages of these molecules are known in the art.

It may also be desirable to administer antibodies against other tumor associated antigens, such as antibodies that bind to EGFR, ErbB3, ErbB4, or Vascular Endothelial Growth Factor (VEGF). Alternatively, two or more anti-ErbB 2 antibodies can be administered to the patient. Sometimes, it may be beneficial to also administer one or more cytokines to the patient. In a preferred embodiment, the anti-ErbB 2 antibody is administered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by administration of the ErbB2 antibody. However, simultaneous administration, or administration of the ErbB2 antibody first, is also contemplated. Suitable dosages of growth inhibitory agents are those presently employed, and may also be reduced due to the combined effect (synergy) of the growth inhibitory agent and the anti-ErbB 2 antibody.

For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease to be treated, the severity and course of the disease (whether the antibody is administered for prophylactic or therapeutic purposes), previous therapy, patient history and response to the antibody, and the judgment of the attending physician. The antibody may suitably be administered to the patient at one time or over a series of treatments.

Depending on the severity and type of disease, the initial candidate antibody dose administered to the patient is about 1. mu.g/kg to about 15mg/kg (e.g., 0.1-20mg/kg), whether by one or more administrations or continuous infusion. Typical daily dosages will be in the range of about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administration for several days or longer, depending on the condition, the treatment is continued until the condition is desirably suppressed. However, other dosage regimens may be employed. The progress of such treatment is readily monitored by routine techniques and experimentation.

Further information on suitable dosages is provided in the examples below.

G. Production of products

In another embodiment of the invention, a product containing a substance for treating the above-mentioned diseases is provided. The product includes a container, a label, and a package insert. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be made of various substances such as glass or plastic. The container contains a composition effective for treating the condition and may also have a sterile access port (e.g., the container may be a bag containing an intravenous solution 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 the container or attached thereto indicates that the composition can be used to treat the conditions listed. The product may also comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution and dextrose solution. It may also include other materials that are commercially and practically desirable, including other buffers, diluents, filters, needles and syringes. In addition, the product comprises a package insert with instructions for use, the insert including a warning that the composition cannot be used in combination with an anthracycline chemotherapeutic agent (e.g., doxorubicin or epirubicin).

H. Preservation of materials

The following hybridoma cell lines have been deposited with the American type culture Collection (12301Parklawn Drive, Rockville, Md., USA (ATCC)):

antibody nomenclature ATCC accession number deposit date

7C2 ATCC HB-122151996 year 10 months 17 days

7F3 ATCC HB-122161996 year 10 month 17 day

4D5 ATCC CRL 104631990 24/5

The following non-limiting examples further describe the details of the invention.

Examples

Materials and methods

An anti-ErbB 2 monoclonal antibody. According to Fendly et al Cancer Res.50: 1550 and 1558(1990) and WO89/06692, generate anti-ErbB 2 IgG specific for the extracellular domain of ErbB2₁The kappa murine monoclonal antibody 4D 5. Briefly, as in Hudziak et al, proc.natl.acad.sci. (USA) 84: 7159(1987) the production of NIH 3T3/HER2-3₄₀₀Cells (each cell expresses about 1X 10)⁵ErbB2 molecules), cells were harvested with Phosphate Buffered Saline (PBS) containing 25mM EDTA and used to immunize BALB/c mice. Mice were injected intraperitoneally with 10 injections at weeks 0, 2, 5, and 7⁷0.5 ml of PBS to the cells. At weeks 9 and 13, antisera were immunoprecipitated³²P-labeled ErbB2 mice were injected intraperitoneally with maltolectin-Sepharose (TM) ((TM))WGA) purified ErbB2 membrane extract. Then, 0.1ml of the ErbB2 preparation was injected once intravenously and splenocytes were fused with the mouse myeloma cell line X63-Ag8.653. Hybridoma supernatants were screened for ErbB2 binding capacity by ELISA and radioimmunoprecipitation. MOPC-21(IgGl) (Cappell, Durham, NC) was used as an allotypic matching control.

Humanized version of the murine 4D5 antibody for treatment () The process is carried out. Engineering the humanized antibody by inserting the complementarity determining region of murine 4D5 antibody into human immunoglobulin IgG₁Framework regions of consensus sequences (IgG)₁) Medium (Carter et al, proc.natl.acad.sci.usa 89: 4285-4289[1992]). The resulting humanized anti-ErbB 2 monoclonal antibody p185^HER2Has very high affinity (Dillomethylation constant K)_d]0.1nmol/L), significant inhibition of p185 at high levels in vitro and in human allografts^HER2Induces antibody-dependent cellular cytotoxicity (ADCC) and is found to be clinically active as a monotherapy in patients with metastatic breast cancer overexpressing ErbB2 who have undergone extensive prior therapy.Produced by a genetically engineered Chinese Hamster Ovary (CHO) cell line that grows on a large scale and secretes the antibody into the culture medium. The antibody was purified from CHO media using standard chromatography and filtration methods. Each batch of antibody used in this study was tested to confirm its properties, purity and potency and to meet the sterility and safety requirements of the "food and drug administration".

Eligibility criteria patients must meet the following study-approved eligibility criteria:

metastatic breast cancer

The ErbB2(HER2) oncogene is overexpressed (2 + to 3+ as determined by immunohistochemistry or Fluorescence In Situ Hybridization (FISH)).Tumor expression of [ ErbB2 can be as described previously (Slamon et al, 1987]And [ 1989)]) As such, it is determined by immunohistochemical analysis of a series of thin sections made from paraffin-preserved tumor masses of patients. The primary detection antibody used was the 4D5 monoclonal antibody, which has the same CDRs as the humanized antibody used for treatment. If at least 25% of the tumor cells exhibit characteristic p185^HER2Membrane staining, the tumor was considered to overexpress ErbB2]。

Two-dimensionally detectable disease (including soluble bone lesions) by radiographic means, physical examination or photographs

Measurable disease is defined as mass that can be reproducibly measured in two perpendicular diameters by physical examination, X-ray (plain film), Computerized Tomography (CT), Magnetic Resonance Imaging (MRI), ultrasound or photographs.

Osteoblastic metastases, pleural effusion or ascites are not considered determinable. The largest measurable lesion size is at least 1 cm. The statement of the evaluable sites of metastatic disease and the number of lesions in the evaluable sites (e.g., lung) must be recorded in a suitable Case Report Form (CRF). If there are a large number of lung or liver lesions, a maximum of 6 lesions per site are followed.

-able to understand and willing to sign a form with written indication of consent

Women should be 18 years old

The suitability of a candidate for receiving concomitant cytotoxic chemotherapy is demonstrated by screening laboratory evaluations of blood, kidney, liver and metabolic function.

Criteria for exclusion patients with either of the following conditions should be excluded from the study:

cytotoxic chemotherapy of previously metastatic breast cancer

The patient may have previously been treated with a hormone for metastatic disease (e.g., tamoxifen) or cytotoxic treatment in an adjuvant setting.

Associated with malignant tumors that are not effectively cured

-Karnofsky score Performance status lower than 60%

-pregnant or nursing women; women likely to give birth unless the investigator determines that effective contraception has been achieved

Bilateral breast cancer (primary tumors must have 2+ to 3+ over-expression of HER2, or metastatic sites must have 2+ to 3+ over-expression of HER2)

Investigational or unlicensed formulations were used within 30 days before the study

Clinically unstable or refractory metastases to the brain (e.g. requiring radiotherapy)

469 patients were selected for the study based on the criteria described above. Randomly additional receiving half of the patients (stratified by chemotherapy)Antibodies (see below).

Administration and dosage

anti-ErbB 2 antibodies

4 mg/kg humanized anti-ErbB 2 antibody administered intravenously over a 90 minute period on day 0: (H). From day 7 onwards, patients received 2 mg/kg of antibody intravenously every week over a 90 minute period.

Chemotherapy

Patients received a minimum of 6 cycles of either of the following two chemotherapy regimens, as long as the patient's disease has not progressed: a) cyclophosphamide and doxorubicin or epirubicin (AC) if the patient has not received anthracycline treatment in an adjuvant setting, or b) paclitaxel: (A)) If the patient has been treated with an anthracycline in an adjuvant setting.The initial administration of the antibody was 24 hours prior to the first cycle of any chemotherapy regimen. The antibody is then administered immediately prior to the start of the chemotherapy administration if the initial administration of the antibody is well tolerated. If the first administration of antibody is not well tolerated, the subsequent infusion is continued 24 hours prior to chemotherapy administration. The patient is allowed to continue chemotherapy for more than 6 cycles if it appears to the treating physician that the patient is advantageous to continue treatment.

Administration of cyclophosphamide (600 mg/m) can be either intravenously pushed over a minimum of 3 minutes or infused by infusion over a maximum of 2 hours.

Doxorubicin (60 mg/m) or epirubicin (75 mg/m) can be slowly pushed intravenously over a minimum of 3-5 minutes or infused by infusion over a maximum of 2 hours according to institutional procedures.

Paclitaxel (A)) The dose administered was 175 mg/cm intravenously over 3 hours. All patients receiving paclitaxel had previously administered 20mg x 2 doses of oral dexamethasone (or equivalent) 12 to 6 hours prior to paclitaxel administration; intravenous administration of 50 mg diphenhydramine (or its equivalent) 30 minutes prior to paclitaxel and administration of dimetridine (or another H) 30 minutes prior to paclitaxel₂A blocking agent).

Reaction standard

Progressive disease: there was objective evidence of an increase of 25% or more in any measurable lesion. Progressive disease responses also include those in which new lesions appear. For bone lesions, progression was defined as a 25% increase in plain, CT, MRI objective measurements; symptomatic new lesions not caused by fractures; or require mild radiation therapy.

And (3) complete reaction: all radiographs and/or macroscopic tumors disappeared in the shortest 4 weeks. Complete skin and chest wall reactions must be confirmed by biopsy.

Partial reaction: the sum of the vertical diameters of all measurable lesions was reduced by at least 50% in a minimum of 4 weeks. No new lesions appeared and no progress was made in the size of all lesions.

Minimum reaction: the sum of the vertical diameters of all measurable lesions was reduced by 25% to 49%. No new lesions appeared and no progress was made in the size of all lesions.

Stable diseases: the size of the lesion can be determined to vary by no more than 25%. No lesions appeared.

The time To Tumor Progression (TTP) was calculated from the start of treatment to progression. The confidence limits for the reaction rates were calculated using the exact method for the single fractions. (Fleiss, JL, Statistical Methods for Rates and Proportions (2 nd edition), New York, NY, Wiley, 1981, pages 13-17).

Results

The results of the evaluation of the time to disease progression (TTP, month unit) and the Rate of Response (RR) at mid-term follow-up of 10.5 months showed that,the results of the chemotherapeutic agents were significantly increased, and the overall severe side effects (AE) were not increased:

participant TTP (month) 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 of logarithmic rank test is less than 0.001;^**X²checking that p is less than 0.01; CRx: chemotherapy; AC: anthracycline/cyclophosphamide treatments; h:

combination treatment with AC + H (18% grade 3/4) was reported to be more frequent than with AC (3%), T (0%) or T + H (2%) alone, with a syndrome of myocardial dysfunction similar to that observed with anthracyclines.

These data indicate that treatment with anti-ErbB 2 antibody in combination with chemotherapeutic agents can increase clinical efficacy (as determined from the rate of response and the outcome of the assessment of disease progression). However, the combined use of anthracyclines with anti-ErbB 2 antibody therapy is contraindicated because doxorubicin or epirubicin increases myocardial side effects. From the risk and benefit considerations, the results showAnd paclitaxelThe combination therapy of (1) is advantageous.

All references cited in the specification are expressly incorporated herein by reference.

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Claims (22)

1. An article of manufacture comprising (1) a container, (2) a composition comprising an anti-ErbB 2 antibody that binds to epitope 4D5 of the extracellular domain sequence of ErbB2 in the container, (3) a label on or associated with the container that indicates that the composition can be used to treat breast cancer characterized by overexpression of the ErbB2 receptor, and (4) a package insert with instructions for avoiding the use of an anthracycline chemotherapeutic agent in combination with the composition.

2. The article of claim 1, wherein the label indicates that the composition can be used to treat metastatic breast cancer.

3. The article of manufacture of claim 1, wherein the antibody is a humanized 4D5 anti-ErbB 2 antibody.

4. The article of manufacture of claim 1 or 2, wherein the antibody binds to any one or more residues of SEQ ID No. 9.

5. The article of manufacture of claim 1 or 2, wherein said antibody is a humanized 4D5 anti-ErbB 2 antibody, said 4D5 anti-ErbB 2 antibody being obtainable from deposit No. ATCC CRL 10463.

6. The article of manufacture of claim 1 or 2, wherein the antibody is an antibody fragment.

7. The article of manufacture of claim 1 or 2, wherein the antibody is an antibody conjugate.

8. The article of manufacture of claim 1 or 2, wherein the antibody is an intact antibody.

9. Use of an anti-ErbB 2 antibody that binds to epitope 4D5 in the ErbB2 ectodomain sequence in combination with tacrolide in the manufacture of a medicament for the treatment of breast cancer characterized by overexpression of ErbB2 in a human patient.

10. The use of claim 9, wherein the medicament comprises a single formulation of the anti-ErbB 2 antibody in combination with tacrolide.

11. The use of claim 9, wherein said medicament comprises (1) said antibody, and (2) a separate formulation made of said tacrolide.

12. The use of claim 11, wherein the separate formulations are formulations that are administered sequentially in any order.

13. The use of claim 9, wherein said antibody is contained within a container of an article of manufacture, the article of manufacture further comprising a package insert with instructions for avoiding the use of an anthracycline chemotherapeutic agent in combination with said antibody.

14. The use according to claim 9, wherein the cancer is metastatic breast cancer.

15. Use according to claim 9 or 14, wherein the antibody binds to any one or more residues of SEQ ID No. 9.

16. The use according to claim 9 or 14, wherein the antibody is a humanized 4D5 anti-ErbB 2 antibody, said 4D5 anti-ErbB 2 antibody being obtainable from deposit No. ATCC CRL 10463.

17. Use according to claim 9 or 14, wherein the antibody is an antibody fragment.

18. Use according to claim 9 or 14, wherein the antibody is an antibody conjugate.

19. Use according to claim 9 or 14, wherein the antibody is a whole antibody.

20. Use according to claim 9 or 14, wherein the medicament is administered together with a further chemotherapeutic agent selected from the group consisting of: 5-fluorouracil (5-FU), cytarabine (Ara-C), cyclophosphamide, thiotepa, leucinolone, methotrexate, vinblastine, bleomycin, epipodophyllotoxin glucopyranoside, ifosfamide, mitomycin C, mitoxantrone hydrochloride, vincristine, vinorelbine, carboplatin, epipodophyllotoxin thiopheneglycoside, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, epothilones, melphalan and other related nitrogen mustards, hormonal agents that modulate or inhibit the hormonal effects on tumors, alkyl sulfonates, aziridines or methylpropanes, melamines, nitrosureas, antibiotics, folic acid analogs, purine analogs, pyrimidine analogs, androgens, anti-adrenal agents, folic acid supplements, acetoglucuronolactone, phosphoramidalditoside; aminolevulinic acid, amsacrine, besiflozin, bisantrene, idazoxanide, dimecorsin, diazaquinone, fornicin, etiracetam, etoglut, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidanol, nitramine, pentostatin, pheniramate, pirarubicin, podophyllotonic acid, 2-ethyl hydrazide, toluhydrazide, Krestin, Razoxan, pyrazox, gerospinamine, altenonic acid, triimine, 2 ', 2' -trichlorotriethylamine, urethane, vindesine, dacarbazine, mannitol mustard, dibromomannitol, dibromodulcitol, pipobroman, calico-ciosine, chlorambucil, calicheamicin, 6-thioguanine, mercaptopurine, platinum, neomycin amide, novitone, tiaprofenic, elodean, etodolac, CPT-11 CPT, Topoisomerase inhibitor RFS2000, Difluoromethylornithine (DMFO), retinoic acid or carboplatin, and pharmaceutically acceptable salts, acids or functionally equivalent derivatives of any of the foregoing.

21. Use according to claim 9 or 14, wherein the tacrolide is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid or functionally equivalent derivative thereof.

22. Use according to claim 9 or 14, wherein the amount of said antibody in combination with tacrolide in said medicament is lower than the sum of the amounts of said antibody and said chemotherapeutic agent when said antibody and tacrolide are administered separately.