MXPA98007723A - Erbb3 antibodies - Google Patents

Abstract

Antibodies are disclosed which bind to ErbB3 protein and further possess any one or more of the following properties:an ability to reduce heregulin-induced formation of an ErbB2-ErbB3 protein complex in a cell which expresses ErbB2 and ErbB3;the ability to increase the binding affinity of heregulin for ErbB3 protein;and the characteristic of reducing heregulin-induced ErbB2 activation in a cell which expresses ErbB2 and ErbB3.

Description

ANTIBODIES ErbB3 BACKGROUND OF THE INVENTION Field of the Invention This invention relates generally to antibodies that bind to the ErbB3 receptor. In particular, it relates to ErbB3 antibodies that, surprisingly, increase the binding affinity of heregulin (HRG) of the ErbB3 protein and / or reduce the induced formation of HRG of an ErbB2-ErbB3 protein complex in a cell that expresses the receptors and / or reduces the activation of ErbB2 induced by heregulin in such a cell.

Description of the related art The transduction of signals that regulate cell growth and differentiation is regulated in part by the phosphorylation of several cellular proteins. Proteins tyrosine kinases are enzymes that catalyze this process. The protein receptor tyrosine kinases are thought to direct cell growth via tyrosine phosphorylation stimulated by the ligand substrates REF .: 28128 intracellular. Protein tyrosine kinase receptors of the growth factor of the subfamily of class I include the 170 kDa epidermal growth factor receptor (EGFR) encoded by the erb Bl gene. Erb Bl has been causally implicated in the malignant tumor of human. In particular, increased expression of this gene has been observed in more aggressive carcinomas of the breast, bladder, lung and stomach.

The second member of the subfamily of class I, pl85reu, was originally identified as the product of the gene transformed from neuroblastomas of chemically treated rats. The neu gene (also called er £ &B2; and HER2) encodes a receptor tyrosine kinase protein of 185 kDa.

The amplification and / or overexpression of the human HER2 gene correlates with a poor prognosis in breast and ovarian cancers (Slamon et al., Science, 235: 177-182 (1987); and Slamon et al., Science, 244: 707 -712 (1989)). Overexpression of HER2 has also been correlated with other carcinomas that include carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon and bladder.

In addition, a related gene has also been described, called erjB3 or HER3. See Pat. US Nos. 5,183,884 and 5,480,968; Plowman et al. , Proc. Na ti Acad. Sci. USA, 87: 4905-4909 (1990); Kraus et al., Proc. Nati Acad. Sci. USA, 86: 9193-9197 (1989); EP Pat Appln No. 444,961A1; and Kraus et al., Proc. Nati Acad. Sci. USA, 90: 2900-2904 (1993) Kraus et al., (1989) found that markedly elevated levels of er > B3 mRNAs occur in certain human breast cell lines indicating that er > B3, erbBl similar and er £ > B2, could play a role in some malignant human tumors. These investigations demonstrated that some human breast tumor cell lines exhibit significant elevation of steady state tyrosine phosphorylation of ErbB3, further indicating that this receptor could play a role in malignant tumors in humans. Therefore, diagnostic bioassays using antibodies which bind to ErbB3 are described by Kraus et al., In US Patent Nos. 5,183,884 and 5,480,968.

The role of erL > B3 in cancer has been explored by other researchers. It has been found to be overexpressed in breast cancers (Lemoine et al., Br. J. Cancer, 66: 1116-1121 (1992)), gastrointestinal (Poller et al., J. Pathol., 168: 275-280 ( 1992), Rajkumer et al., J. Pathol., 170: 271-278 (1993) and Sanidas et al., J. Cancer, 54: 935-940 (1993)), and pancreatic (Lemoine et al., J Pathol, 168: 269-273 (1992), and Friess et al., Clinical Cancer Research, 1: 1413-1420 (1995)).

ErbB3 is unique among the family of ErbB receptors in which there is little or no intrinsic tyrosine kinase activity (Guy et al., Proc. Nati, Acad. Sci. USA 91: 8132-8136 (1994) and Kim et al. , J. Biol. Chem. 269: 24747-55 (1994)). When ErbB3 is coexpressed with ErbB2, an active signaling complex is formed and antibodies directed against ErbB2 are capable of breaking this complex (Sliwkowski et al., J. Biol. Chem., 269 (20): 14661-14665 (1994)). ). Additionally, the affinity of ErbB3 for heregulin (HRG) is increased to a higher affinity state when co-expressed with ErbB2. See also, Levi et al. , Journal of Neuroscience 15: 1329-1340 (1995); Morrissey et al. , Proc. Nati Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al. , Cancer Res. , 56: 1457-1465 (1996) with respect to the ErbB2-ErbB3 protein complex.

Raijkumar et al. , Bri tish Journal Cancer, 70 (3): 459-465 (1994), developed a monoclonal antibody against ErbB3 that has an agonistic effect on the anchor-independent growth of cell lines that express this receptor.

The class I subfamily of the protein tyrosine kinase receptors of the growth factor has been further extended to include the HER4 / pl80erbB4 receptor. See Sun. Pat. EP No. 599,274; Plowman et al. , Proc. Nati Acad. Sci. USES, 90: 1746-1750 (1993); and Plowman et al. , Nature, 366: 473-475 (1993). Plowman et al. , found that the increased expression of HER4 closely correlated with certain carcinomas of epithelial origin, including breast adenocarcinomas. Accordingly, diagnostic methods for detecting neoplastic conditions of human (especially breast cancers) that evaluate the expression HER4 are described in Sol Pat EP No. 599,274.

The question of a HER2 oncogene activator has been directed towards the discovery of a family of heregulin polypeptides. These proteins appear to result from the alternative slippage of a single gene that was mapped to the short arm of human chromosome 8 by Lee et al. , Genomi cs, 16: 790-791 (1993); and Orr-Urtreger et al. , Proc. Na ti. Acad. Sci. USA, Vol. 90 pp. 1867-1871 (1993).

Holmes et al. , isolated and cloned a family of polypeptide activators for the HER2 receptor which is called heregulin-a (HRG-a), heregulin-βl (HRG-ßl), heregulin-ß2 (HRG-ß2), similar to heregulin-ß2 ( similar to HRG-ß2), and heregulin-ß3 (HRG-ß3). See Holmes et al. , Science, 256: 1205-1210 (1992); and WO 92/20798. The 45kDa polypeptide, HRG-a, was purified from the conditioned medium of the human breast cancer cell line MDA-MB-231. These investigations demonstrated the ability of the purified heregulin polypeptides to activate tyrosine phosphorylation of the HER2 receptor in MCF-7 breast tumor cells. In addition, the mitogenic activity of the heregulin polypeptides in SK-BR-3 cells (expressing high levels of the HER2 receptor) was illustrated. Like other growth factors belonging to the EGF family, soluble HRG polypeptides appear to be derived from a membrane binding precursor (called pro-HRG) which is processed proteolytically to release the soluble form of 45kDa. These pro-HRGs lack an N-terminal signal peptide.

While heregulins are substantially identical in the first 213 amino acid residues, they are classified into two main types, a and ß, based on two variants as EGF-like domains that differ in their C-terminal portions. However, these EGF-like domains are identical in the spacing of six cysteine residues contained therein. Based on a comparison of the amino acid sequence, Holmes et al. , found that between the first and sixth cysteines in the EGF type domain, the HRGs were 45% similar to the EGF-like growth factor that binds to heparin (HB-EGF), 35% identical to amphiphegulin (AR), 32% identical to TGF-a, and 27% identical to EGF.

The neu-differentiation factor of 44 kDa (NDF), which is the rat equivalent of human HRG, was first described by Peles et al. , Cell, 69: 205-216 (1992); and Wen et al. , Cell, 69: 559-572 (1992). Like the HRG polypeptides, NDF has an immunoglobulin (Ig) homology domain followed by an EGF-like domain and lacks an N-terminal signal peptide. Subsequently, Wen et al. , Mol. Cell Biol. , 14 (3): 1909-1919 (1994) carried out "exhaustive cloning" to extend the family of NDFs. This work revealed six different fibroblastic pro-NDFs. Adopting the nomenclature of Holmes et al. , NDFs were classified as α or β polypeptides based on this sequence of EGF-like domains. Isoforms 1 to 4 are characterized at the base of the variable stretched juxtamembrane (between the EGF type domain and transmembrane domain). Also, isoforms a, b and c are described as having variable length cytoplasmic domains. These investigations conclude that the different NDF isoforms are generated by alternative binding and distinct function of the specific functions of the tissue.

Falls et al. , Cell 72: 801-815 (1993) describes another member of the heregulin family that calls the polypeptide of activity that induces the acetylcholine receptor (ARIA).

Chicken-derived ARIA polypeptide stimulates the synthesis of muscle acetylcholine receptors. See also WO 94/08007. ARIA is a β-type heregulin and lacks the complete "glyco" spacer (rich in glycosylation sites) present between the Ig-type domain and the EGF-like domain of HRGa, and HRGßl-β3.

Marchionni et al., Na ture, 362: 312-318 (1993) identified several bovine derived proteins that call glial growth factors (GGFs). These GGFs share the Ig-like domain and EGF-like domain with the other heregulin proteins described above, but also have an amino terminal binding domain. GGFs in general do not have the complete "glyco" spacer between the Ig type domain and EGF type domain. Only one of the GGFs, GGFII, possesses an N-terminal signal peptide.

The expression of the ErbB2 family of heregulin receptors and polypeptides in breast cancer is reviewed in Bacus et al. , Pa thology Pa tterns, 102 (4) (Supp.1): S13-S24 (1994).

See also, Alimandi et al. , Oncogene, 10: 1813-1821 (nineteen ninety five); Beerli et al. , Molecular and Cellular Biology, 15: 6496-6505 (1995); Karunagaran et al. , EMBO J, 15: 254-264 (1996); Wallasch et al. , EMBO J, 14: 4267-4275 (1995); and Zhang et al. , Journal of Biological Chemistry, 271: 3884-3890 (1996), in relation to the previous recipient's family.

BRIEF DESCRIPTION OF THE INVENTION • This invention provides antibodies that bind to the ErbB3 protein and further possess one or more of the following properties: an ability to reduce the induced formation of heregulin of an ErbB2-ErbB3 protein complex in a cell that expresses ErbB2 and ErbB3. The ability to increase binding affinity of heregulin with the ErbB3 protein, and the characteristic to reduce the activation of ErbB2 induced by heregulin in a cell that expresses ErbB2 and ErbB3.

The invention also relates to an antibody that binds to the ErbB3 protein and reduces the binding of heregulin thereto.

Preferred antibodies are monoclonal antibodies that bind to an epitope in the extracellular domain of the ErbB3 receptor. In general, antibodies of interest will bind to the ErbB3 receptor with an affinity of at least about 10nM, more preferably at least about InM, in certain embodiments, the antibody is immobilized in (eg, covalently linked to) a solid phase, p. ex. , by receptor affinity purification or by diagnostic tests.

The antibodies of the preceding paragraphs could be provided in the form of a composition comprising the antibody and a pharmaceutically acceptable carrier or diluent.

The invention also provides: an isolated nucleic acid molecule encoding the antibody of the preceding paragraphs which could also contain a promoter operably linked thereto; an expression vector comprising the nucleic acid molecule operably linked to the control sequences recognized by a host cell transformed with the vector; a cell line containing the nucleic acid (e.g., a hybridoma cell line); and a process for using a nucleic acid molecule encoding the antibody to effect the production of the antibody comprising culturing a cell containing the nucleic acid and, optionally, recovering the antibody from the cell culture and, preferably, the cell culture medium.

The invention also provides a method for treating a mammal, which comprises administering a therapeutically effective amount of the antibody described herein to the mammal, wherein the mammal has a disorder that requires treatment with the antibody.

In a further aspect, the invention provides a method for detecting ErbB3 in vi tro or in vivo comprising contacting the antibody with a cell suspected of containing ErbB3 and detecting whether the link has been presented. Thus, the invention provides a test for detecting a tumor characterized in that the amplified expression of ErbB3 comprises the steps of exposing a cell to the antibody set forth herein and determining the degree of binding of the antibody to the cell. In general, the antibody to be used in such a test will be labeled. The test here could be in an in vi tro test (such as an ELISA test) or an in vivo test. To diagnose the tumor in vivo, the antibody is generally conjugated to a radioactive isotope and administered to a mammal, and the degree of antibody binding to tissues in the mammal is observed by external search for radioactivity.

Brief description of the Ddibos Fig. 1 depicts HRG bound to K562 ErbB3 cells in the presence of various anti-ErbB3 monoclonal antibodies. Purified anti-ErbB3 antibodies were incubated with a suspension of K562 ErbB3 and 125I-HRGßl (17_244) cells. After approximately 18 hours on ice, the beads bound to the cell were measured. The accounts are plotted as a percentage of binding in the absence of the antibody (control). The non-specific binding was determined using an excess of unlabelled HRGßl (177_244) (HRG). Antibodies against the protein ErbB2 (2C4) and HSV (5B6) were used as negative controls.

Fig. 2 shows the effect of antibody concentration on HRG bond. A dose-response experiment was developed in the 3-8D6 antibody that was found to increase the HRG binding. The K562 ErbB3 cells were incubated with a fixed concentration of 125 I-HRG and increased concentrations of the 3-8D6 antibody. The results of the experiment are plotted as counts bound to the cell against the concentration of the antibody.

Fig. 3 illustrates the binding of HRG to K562 ErbB3 cells in the presence and absence of the 3-8D6 antibody or a fragment Fab of it. The competitive ligand binding experiments were carried out in the absence (control) and presence of 3-8D6 or Fab 100 nM. The data was plotted as total / link (B / T) against total HRGßl (177_24).

Detailed Description of the Preferred Modalities I. Definitions Unless otherwise indicated, the term "ErbB3" when used herein refers to the mammalian ErbB3 protein and "er £> B3" refers to the mammalian eri.B3 gene. The preferred ErbB3 protein is the human ErbB3 protein present in the cell membrane of a cell. The gene er > B3 of human is described in US Pat. No. 5,480,968 and Plowman et al. , Na ti. Acad. Sci. USA, 87: 4905-4909 (1990).

The antibody of interest could be one that does not significantly cross-react with other proteins such as those encoded by the erJB1, erbB2 and / or er 'B4 genes. In such embodiments, the degree of antibody binding to these non-ErbB3 proteins (e.g., cell surface binding to the endogenous receptor) will be less than 10% as determined by fluorescence-active cell sorting analysis (FACS). ) or radioimunoprecipitation (RIA). However, sometimes the antibody could be one that cross-reacts with the ErbB4 receptor, and, optionally, for example, does not cross-react with the EGFR receptor and / or ErbB2.

"Heregulin" (HRG) when used herein refers to a polypeptide that activates the ErbB2-ErbB3 protein complex (eg, it induces phosphorylation of tyrosine residues in the ErbB2-ErbB3 complex at the link thereto). Various heregulin polypeptides encompassed by this term have been discussed above. The term includes fragments and / or biologically active variants of a naturally occurring HRG polypeptide, such as an EGF-like domain fragment thereof (eg HRGβ177_244).

The "ErbB2-ErbB3 protein complex" is a non-covalently associated oligomer of the ErbB2 receptor and the ErbB3 receptor. This complex is formed when a cell that expresses both of these receptors is exposed to HRG. The complex is isolated by immunoprecipitation and analyzed by SDS-PAGE as described in the following Example.

The term "reduces the formation induced by heregulin of an ErbB2-ErbB3 protein complex in a cell expressing ErbB2 and ErbB3" refers to the ability of the antibody to statistically significantly reduce the number of ErbB2-ErbB3 protein complexes that are formed in a cell that has been exposed to the antibody and with respect to HRG to an untreated cell (control). The cell expressing ErbB2 and ErbB3 can be a cell or cell line that occurs naturally (eg Caov3 cell) or can be produced recombinantly by introducing the nucleic acid encoding each of these proteins into a host cell. Preferably, the antibody will reduce the formation of this complex by at least 50%, and more preferably at least 70%, as determined by reflectance search densitometry of Western blots of the complex (see the following Example).

The antibody that "reduces the activation of ErbB2 induced by heregulin in a cell that expresses ErbB2 and ErbB3" is one that statistically significantly reduces the tyrosine phosphorylation activity of ErbB2 that occurs when HRG binds to ErbB3 in the protein complex ErbB2-ErbB3 (present on the surface of a cell that expresses the two receptors) with respect to the untreated cell (control). This can be determined based on phosphotyrosine levels in the ErbB2-ErbB3 complex after exposure of the complex to HRG and the antibody of interest. The cell expressing the ErbB2 and ErbB3 protein can be a cell or cell line that occurs naturally (eg Caov3 cell) or can be produced recombinantly. Activation of ErbB2 can be determined by Western blot followed by testing with an anti-phosphotyrosine antibody as described in the following Example. Alternatively, the activation test of the kinase receptor is described in WO 95/14930 and Sadick et al. , Analytical 5 Biochemistry, 235: 207-214 (1996) can be used to quantify ErbB2 activation. Preferably, the antibody will reduce the activation of ErbB2 induced by heregulin by at least 50%, and more preferably by at least 70%, as determined by densitometry by reflectance search of Western blots of the complex tested by a • anti-phosphotyrosine antibody (see the following Example).

The antibody could be one that "increases the binding affinity of heregulin for the ErbB3 protein". This means that, in the presence of the antibody (eg 100 nM antibody), the amount of HRG that binds to ErbB3 (eg, endogenous ErbB3 present in a cell or cell line that occurs naturally or introduced by recombinant techniques, see the following Example), with respect to the control (without antibody), is statistically significantly increased. For example, the amount of HRG that binds to the transfected cell line k562 with erJB3 as described herein could be increased in the presence of the 100 nM antibody by at least 10%, preferably at least 50%, and more preferably at least 100% ( see Fig. 1), with respect to the control.

The antibody that reduces the binding of HRG to the ErbB3 protein (eg ErbB3 present in a cell) is the one that interferes with the binding site of HRG in the ErbB3 protein such that the amount of Heregulin that is allows to link to this site of the molecule. Examples of such antibodies are antibodies 3-2F9, 3-3E9 and 3-6B9 described in the Example herein.

The term "antibody" is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, (eg, bispecific antibodies) formed from at least two intact antibodies, and fragments of antibodies so large that they exhibit the desired biological activity . The antibody could be, for example, an IgM, IgG (eg IgGj, IgG2, IgG3 or IgG4), IgD, IgA or IgE. However, the antibody is preferably not an IgM antibody.

The "antibody fragments" comprise a portion of an intact antibody, generally the antigen binding or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab 'fragments, F (ab ') 2, and Fv; diabodies; single charge antibody molecules; and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g. ex. , the individual antibodies contained in the population are identical except for possible naturally occurring mutations that could occur in smaller amounts. Monoclonal antibodies are highly specific, which are directed against a simple antigenic site. further, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by means of the hybridoma culture, without contaminating other immunoglobulins. The "monoclonal" modifier indicates the character of the antibody as it will be obtained from a substantially homogenous population of antibodies, and will not be constructed as required by the production of the antibody by any particular method, for example, the monoclonal antibodies that they are to be used according to the present invention could be made by the hybridoma method first described by Kohier et al. , Nature, 256: 495 (1975), or could be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). For example, "monoclonal antibodies" could also be isolated from phage antibody libraries using the techniques described in Clackson et al. , Nature, 352: 624-628 (1991) and Marks et al. , J. Mol. Biol. , 222: 581-597 (1991).

The monoclonal antibodies specifically include here "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to the corresponding sequences of the antibodies derived from other species or belonging to another class or subclass of antibody, as well as fragments of such antibodies, of such size that exhibit the desired biological activity (US Patent No. 4,816,567; Morrison et al., Proc. Nati. Acad. Sci. USA, 81: 6851-6855 (1984)) .

The "humanized" forms of non-human antibodies (eg, murine) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other antibody sequences that link antigens) that contain the minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which the residues of a region that determines complementarity (CDR) of the receptor are relocated by residues of a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, the residues of the structural region Fv (FR) of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or in the imported CDR or structural sequences. These modifications are made to refine and further optimize the functioning of the antibody. In general, the humanized antibody will substantially comprise all of at least one, and typically two, variable domains, in which all or substantially all of the 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. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of an immunoglobulin. For additional details, see Jones et al. , Na ture, 321: 522-525 (1986). The humanized antibody includes a Primatized antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing monkeys with the antigen of interest.

The "single chain Fv" or "sFv" fragments comprise the VH and VL domains of the antibody, wherein these domains occur in a single polypeptide chain In general, the Fv polypeptide further comprises a linker polypeptide between the VH domains and VL that allow the sFv to form the desired structure to bind the antigen For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabody" refers to small fragments of antibody with two antigen binding sites, these fragments comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain ( VH-VL). Using a linker that is too short to allow mating between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. The diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. , Proc. Nati Acad. Sci. USA, 90: 6444-6448 (1993).

An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of their natural environment are materials that would interfere with the diagnosis of therapeutic uses for the antibody, and could include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the antibody will be purified (I) to greater than 95% by weight of the antibody as determined by the Lowry method., and more preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by the use of a rotary cup sequencer, or (3) for homogeneity by SDS-PAGE under reduced or non-reduced conditions using Coomassie blue or, preferably, silver tincture. The isolated antibody includes the antibody in itself within recombinant cells since at least one component of the antibody's natural environment will not occur. Ordinarily, however, the isolated antibody will be prepared by at least one purification step.

As used herein, the term "epitope that binds the wild-type receptor" refers to an epitope of the Fc region of an IgG molecule (eg, IgGlf IgG2, IgG3, or IgG4) that is responsible for increasing the half-life of in vivo serum of the IgG molecule.

"Treatment" refers to therapeutic treatment and prophylactic or preventive measures. Those who need treatment include those who already have the disorder • as those who are going to avoid disorder.

"Mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human. • 10 A "disorder" is any condition that would benefit from treatment with the anti-ErbB3 antibody. This includes chronic and acute disorders or diseases that include the pathological conditions that predispose the mammal to the disorder in question. In general, the disorder will be one whose excessive activation of the ErbB2-ErbB3 protein complex occurs by means of heregulin. Non-limiting examples of disorders to be treated include benign and malignant tumors; leukemias and malignant lymphoid tumors; neuronal, glial, astrocytal, hypothalamic and other disorders glandular, macrophagal, epithelial, stromal and blastocoelic; and inflammatory, angiogenic and immunological disorders.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, cancer. colon, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes (eg I, Y, Pr), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

A "chemotherapeutic agent" is a compound, chemical useful in the treatment of cancer. Examples of chemotherapeutic agents include Adriamycin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Tiotepa, Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carmiomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamycins (see US Pat. No. 4,675,187), Melphalan and other nitrogen-related mustards.

The term "cytosine" is a generic term for proteins released by a cell population that act in another cell as intracellular mediators. Examples of such cytosines are lymphokines, monocins, and traditional polypeptide hormones. Included among the cytosines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; Prorrelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and lutenization hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; factor a and ß of tumor necrosis; substance that inhibits mullerian; peptide associated with mouse gonadotropin; activin; Vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon a, β, and y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulacy-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (lys) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α and TNF-β; and other polypeptide factors including LIF and the ligand kit (KL). As used herein, the term cytosine includes proteins from natural or recombinant cell culture sources and biologically active equivalents of the native sequence cytosines.

The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the original drug and is capable of being enzymatically activated or converted to the most active original form . See, p. ex. , Wilman, "Produgs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al. , "produgs: A Chemical Approach to Targeter Drug Delivery", Directed Drug Delivery, Borchardt et al. , 8ed. ), pp. 247-267, Humana Press (1985). Prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, prodrugs containing peptides, modified D-amino acid prodrugs, glycosylated prodrugs, prodrugs containing β-lactam, prodrugs containing optionally substituted phenoxyacetamide or prodrugs containing optionally substituted phenylacetamide, 5-fluorocytosine and other prodrugs of 5-fluorouridine which can be converted to the most active cytotoxic free drug. Examples of cytotoxic prodrugs that can be derived in a prodrug form for use in this invention include, but are not limited to, the chemotherapeutic agents described above.

The word "label" when used herein refers to a detectable compound or composition that is conjugated directly or indirectly with antibody. The label could be detected by itself (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, could catalyze the chemical alteration of a compound substrate or composition that is detectable.

By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or completely of glass (eg, controlled pore glass), polysaccharides (eg, agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase may comprise the well of a test plate; in others it is a purification column (eg, an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.

A "liposome" is a small vesicle composed of various types of lipids, phospholipids and / or surfactants that are useful for releasing a drug (such as the anti-ErbB3 antibodies set forth herein and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of the biological membranes.

An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is different in the form or environment in which it is found in nature. Therefore the isolated nucleic acid molecules are distinguished from the nucleic acid molecule since they exist naturally in the cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

The term "control sequences" refers to DNA sequences necessary for the expression of a coding sequence operably linked in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

The nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, the DNA of a presequence or secretory guide is operably linked to the DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. In general, "operably linked" means that the DNA sequences that are linked are contiguous, and, in the case of a secretory guide, contiguous and in reading phase. However, the augmentators do not have to be contiguous. The binding is carried out by binding at convenient restriction sites. If such sites do not exist, the adapters of the synthetic oligonucleotide are used in accordance with conventional practice.

As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably and all designations include progeny. Thus, the words "transformants" and "transformed cells" include the subject elementary cell and the cultures derived therefrom without estimating the number of transfers. It is also understood that all progeny could not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity are included since they were screened from the originally transformed cell. Where different designations are intended, it will be clear in the context.

II. Ways to Carry Out the Invention ? Preparation of the Antibody A description follows as examples of techniques for the production of the claimed antibodies. (i) Polyclonal antibodies Polyclonal antibodies are generally cultured in animals by means of multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen with a protein that is immunogenic in the species to be immunized, e.g. ex. , keyhole hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivative agent, eg, maleimidobenzoyl ester sulfosuccinimide (conjugation through the cysteine residues), N-hydroxysuccinimide (by the lysine residues), glutaraldehyde, succinic anhydride, S0C12, or R1N = C = NR, where R and R1 are different alkyl groups.

The animals are immunized against the antigen, immunogenic conjugates, or derivatives by combination, e.g. ex. , 100 μg or 5 μg of the protein or conjugate (from rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally in multiple sites. One month later the animals were raised with 1/5 to 1/10 of the original amount of the peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals were bled and the serum was tested for antibody titre. The animals are raised to the titration plate. Preferably, the animal is raised with the conjugate of the same antigen, but with a different protein and / or a different cross-linking reagent. The pools can also be made in recombinant cell culture as protein fusions. Also, aggregation agents such as alumina are used appropriately to increase the immune response. (ii) Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, e.g. ex. , the individual antibodies contained in the population are identical except for possible naturally occurring mutations that could occur in smaller amounts. Thus, the "monoclonal" modifier indicates the character of the a-antibody since it is not a mixture of discrete antibodies.

For example, monoclonal antibodies could be made using the hybridoma method first described by Kholes et al. , Nature, 256: 496 (1975), or it could be done by recombinant DNA methods (U.S. Patent No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, the lymphocytes could be immunized in vi tro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parenteral myeloma cells lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), whose substances prevent cell growth. HGRPT-deficient.

Preferred myeloma cells are those that efficiently fuse, stable support for high level production of the antibody by the selected cells that produce the antibody, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63 cells -Ag8-653 available at the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbos, J. I munol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The culture medium in which the hybridoma cells are growing is tested for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding test, such as radioimmunoassay (RIA) or linked enzyme-linked immunosorbent assay (ELISA).

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

After the hybridoma cells were identified that produce antibodies of the specificity, affinity, and / or desired activity, the clones could be subcloned by limiting the dilution and growth procedures by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, p. 59-103 (Academic Press, 1986)). The culture medium suitable for this purpose includes, for example, D-MEM medium or RPMI-1640. In addition, the hybridoma cells could grow in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional methods of immunoglobulin purification such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional methods (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA could be placed in expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that will not otherwise produce the immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al. , Curr. Opinion in Immunol. , 5: 256-262 (1993) and Plückthun, Immunol. Revs. , 130: 151-188 (1992).

In a further embodiment, antibodies or antibody fragments can be isolated from an antibody phage library generated using the techniques described in McCafferty et al. , Na ture, 348: 552-554 (1990). Clackson et al. , Na ture, 352: 624-628 (1991) and Marks et al. , J. Mol. Biol. , 22: 581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity human antibodies (nM range) by means of chain rearrangement (Marks et al., Bio / Technology, 10: 779-783 (1992)), as well as combinatorial infection and in vivo recombination. I live as a strategy to build very large phage libraries (Waterhouse et al., Nuc.Acids.Res., 21: 2265-2266 (1993)). Thus, these techniques are viable techniques for traditional antibody hybridoma techniques for the isolation of monoclonal antibodies.

The DNA could also be modified, for example, by substituting the sequence coding for light and heavy chain constant domains in place of the homologous murine sequences (US Patent No. 4,816,567; Morrison et al., Proc. Nati. Acad. Sci. USA, 81: 6851 (1984)), or by covalently joining the sequence encoding immunoglobulin all or part of the sequence encoding a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or are substituted for the variable domains of a site that combines antigen having specificity for one antigen and another site that combines antigen having specificity for a different antigen. (iii) Anti humanized and human bodies Methods for immunizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced therein from a source that is non-human. These non-human amino acid residues are often referred to as "imported" residues, which are typically taken from an "imported" variable domain. Humanization can be developed essentially following the method of Winter and co-authors (Jones et al., Na ture, 321: 522-525 (1986), Riechmann et al., Na ture, 331: 323-327 (1988), Verhoeyen. et al., Science, 239: 1534-1536 (1988)), substituting rodent sequences CDRs or CDRs for the corresponding sequences of a human antibody. Therefore, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues of analogous sites in rodent antibodies.

The choice of human variable domains, light and heavy, which are going to be used to make the humanized antibodies are very important to reduce the antigenicity. According to the method also called "best arrangement", the variable domain sequence of a rodent antibody is screened against the entire library of known sequences of the human variable domain. The human sequence that is closest to that of the rodent is then accepted as the human structure (FR) for the humanized antibody (Sims et al., J. Im unol., 151: 2296 (1993); Chothia et al., J Mol. Biol., 196: 901 (1987)). Another method uses a derivative of particular structure from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same structure could be used for several different humanized antibodies (Cárter et al., Proc.Nat.Acid.Sci.USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993). ).

It is also important that the antibodies are humanized with high affinity retention for the antigen and other important biological properties. To achieve this goal, according to a preferred method, the humanized antibodies are prepared by a process of analysis of the parental sequences and several conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. The computer programs are arranged in such a way that they illustrate and display probable three-dimensional conformational structures of immunoglobulin candidate sequences. The inspection of these presentations allows analysis of the probable role of the residues in the functioning of the immunoglobulin candidate sequence, p. ex. , the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, the FR residues can be selected and combined from the recipient and importer sequences for the characteristics of the desired antibody, such that increased affinity is achieved for the target antigen. In general, CDR residues are directly and more substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g., mice) that are capable, in immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the removal of homozygote from the heavy-chain antibody (JH) binding region gene in chimeric mutant mice and germ line results in complete inhibition of endogenous antibody production. The transfer of the arrangement of the human germ line immunoglobulin gene in such a germ line mutant mouse will result in the production of human antibodies upon passage of the antigens. See, p. ex. , Jakobovits et al. , Proc. Na ti. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al. , Na ture, 362: 255-258 (1993); Bruggermann et al. , Year in Immuno. , 7:33 (1993). Human antibodies can also be derived from libraries that represent phage (Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581-597 (1991 )). (iv) Antibody fragments Several techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, eg, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229 : 81 (1985)). However, these fragments can now be produced directly by means of recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be recovered directly from E. coli and chemically coupled to form F (ab ') 2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, the F (ab ') 2 fragments can be isolated directly from the culture of recombinant host cells. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. (v) Bispecific antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Example of bispecific antibodies could bind to two different epitopes of the ErbB3 protein. Other antibodies could combine an ErbB3 binding site with EGFR, ErbB2 and / or ErbB4 binding sites. Alternatively, an anti-ErbB3 arm could be combined with an arm that binds to a molecule that activates a leukocyte such as a T cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG (FcyR) , such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) to focus the cell defense mechanism towards the cell expressing ErbB3. Bispecific antibodies could also be used to localize cytotoxic agents to cells expressing ErbB3. These antibodies possess an arm that binds ErbB3 and an arm that binds the cytotoxic agent (eg, saporin, anti-interferon-a, vinca alkaloid, castor A chain, methotrexate or hapten radioactive isotope). Bispecific antibodies can be prepared as full-length antibodies with antibody fragments (e.g., F (ab ') 2 specific antibodies).

Methods for making bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where these two chains have different specificities (Millstein et al., Nature, 305: 537-539 1983)). Due to the random variety of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule that is usually given by affinity chromatography steps, is rather uncomfortable, and the product yield is low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al. , EMBO J., 10: 3655-3659 (1991).

According to a different approach, the variable domains of antibody with the desired binding specificities (antibody-antigen combining sites) are fused for immunoglobulin constant domain sequences). The fusion is preferably with a heavy chain immunoglobulin constant domain, comprising at least part of the binding, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site necessary to bind the light chain present in at least one of the fusions. The DNAs encoding the heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and cotransfected into a suitable host organism. This provides great flexibility to adjust the mutual proportions of the three polypeptide fragments in the modalities when the unequal ratios of the three polypeptide chains are used in the construct that provides the optimum performance. However, it is possible to insert the sequences coding for two or three polypeptide chains into an expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of non-particular significance.

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

According to another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from the culture of recombinant cells. The preferred interface comprises at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more side chains of small amino acids at the interface of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chains are created at the interface of the second antibody molecule by replacing side chains of large with smaller amino acids (eg alanine or threonine). This provides a mechanism to increase the performance of the heterodimer over other undesired end products such as homodimers.

Bispecific antibodies include cross-linked antibodies or "heteroconjugate" for example, one of the antibodies in the heteroconjugate can be coupled to avidin, the others to biotin. Such antibodies, for example, have been proposed to direct cells of the immune system to unwanted cells (US Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies could be made using any convenient cross-linking method. Suitable crosslinking agents are well known in the art, and are disclosed in US Patent No. 4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describes a method wherein the intact antibodies are proteolytically cut to generate F (ab ') 2 fragments. These fragments are reduced in the presence of sodium arsenite dithiol complexing agent to stabilize neighboring dithiols and prevent formation of intermolecular disulfide. The generated Fab 'fragments are then converted to thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted to Fab '-thiol by the reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al. , J. Exp. Med., 175: 217-225 (1992) describes the production of a bispecific antibody molecule fully immunized F (ab ') 2. Each Fab 'fragment was secreted separately from E. coli and subjected to directed chemical coupling in vi tro to form the bispecific antibody. The bispecific antibody thus formed allowed to bind cells that overexpress the HER2 receptor and normal human T cells, as well as activate the lytic activity of human cytotoxic lymphocytes against human breast receptor tumors.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zipper. Kostelny et al. , J. Immunol. , 148 (5): 1547-1553 (1992). The leucine zipper peptides of the Fos and Jun proteins were bound to the Fab 'portions of two different antibodies by fusion of the gene. The antibody homodimers are reduced to the binding region to form monomers and then re-oxidized to form the antibody heterodimers. This -method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al. , Proc. Na ti. Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by means of a linker that is too short to allow pairing between the two domains on the same chain. Therefore, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment thereby forming two antigen binding sites. Another strategy has also been reported for making bispecific antibody fragments by the use of single chain dimers Fv (sFv). See Gruber et al. , J. Immunol. , 152: 5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. , J. Immunol. 147: 60 (1991). (vi) Screening for antibodies with the desired properties The techniques for generating antibodies have been described before. Antibodies having the characteristics described herein are selected.

To select antibodies that reduce the induced formation of HRG of the ErbB2-ErbB3 complex protein, cells expressing both receptors (eg Caov3 cells) can be pre-incubated with a buffer (control or antibody, then treated with HRG or control buffer. The cells are then lysed and the crude lysates can be centrifuged to remove the insoluble material.The supernatants could be incubated with an antibody specific for ErbB2 covalently coupled to their solid phase.After washing, the immunoprecipitates could be separated by means of SDS-PAGE. Western blots of the gels are then tested with anti-ErbB2 antibody.After visualization, the spots could be cut into strips and retested with an anti-ErbB2 antibody.The densitometry by reflectance search of the gel can be performed to quantify the effect of the antibody in question on the induced formation of HRG of the complex. Antibodies that reduce the formation of the ErbB2-ErbB3 complex relative to the control (untreated cells). See the following Example.

To select the antibodies that reduce the activation of ErbB2 induced by HRG in a cell that expresses the ErbB2 and ErbB3 receptor, the cells can be pre-incubated with buffer (control) or antibody, then treated with HRG or control buffer. The cells are then lysed and the crude lysate can be centrifuged to remove the insoluble material. Activation of ErbB2 can be determined by means of Western blot followed by testing with an anti-phosphototein antibody as described in the following Example. For example, ErbB2 activation can be quantified by means of gel reflectance search densitometry. Alternatively, the kinase receptor activation test is described in WO 95/14930,? 51 and Sadick et al. , Analytical Biochemistry, 235: 207-214 (1996) can be used to determine the activation of ErbB2.

The effect of the antibody on HRG binding to ErbB3 can be determined by incubating the cells expressing this receptor 5 (eg, 4E9H3 cells transfected to express ErbB3) with radiolabeled HRG (eg the EGF-like domain thereof), absence (control) or presence of the ErbB3 antibody, for example, as described in the following Example. Antibodies that increase the binding affinity of HRG for the ErbB3 receiver can be selected for further development. Antibodies that increase the affinity binding of HRG to the ErbB3 receptor can be selected for further development. Where the antibody of choice is one that blocks the binding of HRG to ErbB3, they can identify the antibodies that make this test.

To screen for antibody binding to the epitope on linked ErbB3 by means of an antibody of interest (eg, those that block the binding of antibody 3-8B8 to ErbB3), a routine cross-blockade test such as described in Antibodies, A Labora tory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). (vii) Engineering of the effector function It may be desirable to modify the antibody of the invention with respect to effector function, for example, to increase the effectiveness of the antibody in the treatment of cancer. For example, cysteine residues could be introduced into the Fc region, thus allowing the formation of the interchain disulfide bond in this region. The homodimeric antibody so generated could have improved internalization capacity and / or increased cell death by complement and antibody-dependent cellular cytotoxicity (ADCC). See Carón et al. , J. Exp. Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 81992). Homodimeric antibodies with enhanced anti-tumor activity could also be prepared using cross-linked heterobifunctional linkers as described in Wolff et al. , Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and could thus have complement lysis and enhanced ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3: 219-230 (1989). (viii) Immunocon played The invention also pertains to immunoconjugates comprising the antibody described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) , or a radioactive isotope (eg, a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. The enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, unbound, active fragments of diphtheria toxin, exotoxin chain A (from Pseudomonas aeruginosa), castor A chain, abrin A chain, A chain, modecina, alfa-sarcina, proteins of Aleuri tes fordii, diantin proteins, proteins of Phytolaca americana (PAPI, PAPII, and PAP-S), inhibitor of morrantine charantia, curcinia, crotina, inhibitor of sapaonaria officinalis, gelonin, mitogeline, restrictocin , fenomycin, enomycin and trichothecenes. A variety of radionuclides are available for the production of radioconjugated anti-ErbB3 antibodies. Examples include 212Bi, 131I, 131In, 90Y and 186Re.

The conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as adipose). dimethyl HCl idato), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azido benzoyl) hexandiamine), bis-diazonium derivatives (such as bis- ( p-diazonium benzoyl) -ethylenediamine), diisocyanates (such as 2,6-tolienium diisocyanate), and bis-active fluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a castorium immunotoxin can be prepared as described in Vitetta et al. , Science 238: 1098 (1987). The labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminpentaacetic acid at carbon 14 (MX-DTPA) is an example of a chelating agent for conjugation of the radionucleotide to the antibody. See WO 94/11026.

In another embodiment, the antibody could be conjugated to a "receptor" (such as streptavidin) for use in prereceptor tumors wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clarifying agent. and then the administration of a "ligand" (eg, avidin) that is conjugated with a cytotoxic agent (eg, a radionuclide). (ix) Immunoliposomes The anti-ErbB3 antibodies set forth herein could also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al. , Proc. Na ti. Acad. Sci. USA, 82: 3688 (1985); Hwuang et al. , Proc. Na ti. Acad. Sci. USA, 77: 4030 (1980); and Pat. U.S. 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 generated by the reverse phase evaporation method with a lipid composition containing phosphatidylcholine, cholesterol and phosphatidylethanolamine derived from PEG (PEG-PE). The liposomes are extruded through filters of defined pore size to obtain liposomes with the desired diameter. The Fab 'fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) by way of a disulfide exchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al. J. Na tional Cancer Inst. 81 (19) 1948 (1989). (x) Dependent Antibody-Mediated Prodrug Therapy (ADEPT) The antibody of the present invention could also be used in ADEPT by conjugating the antibody with an enzyme that activates the prodrug which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81/01145) to an active anti-cancer drug. . See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includes an enzyme capable of acting on a prodrug in such a way that it converts it into its most active cytotoxic form.

Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine to the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), which are useful for converting prodrugs containing peptides into free drugs; D-alanylcarboxypeptidase, useful for converting prodrugs containing D-amino acid substituents; enzymes that break carbohydrates such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derived with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase useful for the conversion of drug derivatives into their amino nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, eg, Massey, Na ture 328: 457-458 (1987 )). The antibody-abzyme conjugates can be prepared as described herein to release the abzyme to a tumor cell population.

Enzymes of this invention can be covalently linked to anti-ErbB3 antibodies by techniques well known in the art such as the use of heterobifunctional cross-linking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see , eg, Neuberger et al., Nature, 312: 604-608 (1984)). (xi) Epitope fusions that bind the wild-type antibody receptor.

In certain embodiments of the invention, it may be desirable to use an antibody fragment, rather than an intact antibody, for example, to increase tumor penetration. In this case, it may be desirable to modify the antibody fragment to increase the half-life of its serum. This could be achieved, for example, by the incorporation of an epitope that binds the wild-type receptor to the antibody fragment (eg, by mutation of the appropriate region in the antibody fragment or by incorporating the epitope on a tip of the peptide that is then fuses the antibody fragment at the end or in the middle, eg, by means of DNA or peptide synthesis).

A systematic method for preparing such an antibody variant having an increased half-life in vivo comprises several steps. The first involves identifying the sequence and conformation of an epitope that binds to the wild type receptor of an Fc region of an IgG molecule. Once this epitope is identified, the antibody sequence of interest is modified to include the sequence and conformation of the identified binding epitope. After the sequence is mutated, the antibody variant is tested to see if it has a longer half-life in vivo than that of the original antibody. If the antibody variant does not have a longer half-life in vivo in the test, its sequence is further altered to include the sequence and conformation of the identified binding epitope. The altered antibody is tested for longer half-life in vivo, and this process is continued until a molecule having a longer half-life in vivo is obtained.

The epitope that binds to the wild-type receptor that is thus incorporated into the antibody of interest is any suitable epitope as defined above, and its nature will depend, e.g. ex. , of the type of antibody that is going to be modified. The transfer is made such that the antibody of interest still possesses the biological activities described herein.

The epitope is generally a region in which one or more amino acid residues of one or two cycles of an Fc domain are transferred to an analogous position of the antibody fragment. Even more preferably, three or more residues of one or two cycles of the Fc domain are transferred. Even more preferred the epitope is taken from the CH2 domain of the Fc region (e.g., from an IgG) and transferred to the CH1, CH3, or VH region, or more than one region, of the antibody. Alternatively, the antibody is taken from the CH2 domain of the Fc region and transferred to the CL region or VL region, or both, of the antibody fragment.

In a more preferred embodiment, the epitope that binds to the wild-type receptor comprises the sequence (5 'to 3'): PKNSSMISNTP (SEQ ID NO: 1), and further optionally comprises a sequence selected from the group consisting of HQSLGTQ (SEQ ID NO. : 2), HQNLSDGK (SEQ ID NO: 3), HQNISDGK (SEQ ID NO: 4), or VISSHLGQ (SEQ ID NO: 5), particularly where the antibody fragment is Fab or F (ab ') 2. In another more preferred embodiment the epitope that binds the wild type receptor is a polypeptide containing the sequences (5 'to 3'): HQNLSDGK (SEQ ID NO: 3), HQNISDGK (SEQ ID NO: 4), or VISSHLGQ (SEQ ID NO: 4) NO: 5) and the sequence PKNSSMISNTP (SEQ ID NO: 1).

B. Vectors, Host Cells and Recombinant Methods The invention also provides isolated nucleic acids encoding an antibody as set forth herein as vectors and host cells containing the nucleic acid, and recombinant techniques for the production of the antibody.

For the recombinant production of the antibody, the encoding nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. The DNA encoding the monoclonal antibody is easily isolated and sequenced using conventional methods (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components include in general, but not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence . (i) Component signal sequence The anti-ErbB3 antibody of this invention could be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the protein mature or polypeptide. The selected heterologous signal sequence is preferably one that is recognized and processed (e.g., cut by means of a peptidase signal) by the host cell. For prokaryotic host cells that do not recognize or process the native signal sequences of the anti-ErbB3 antibody, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, lpp, or thermostable enterotoxin guides. . For yeast secretion the native signal sequence could be replaced by, eg. ex. , the guide of the yeast invertase, the a-factor guide (including the a-factor guides of Saccharomyces and Kluyveromyces), or the guide of the acid phosphates, the glucoamylase guide of C. albi cans, or the signal described in WO 90/13646. In mammalian cell expression, the mammalian signal sequences are available as are the viral secretion guides, for example, the herpes simplex gD signal.

The DNA of such a region of the precursor is linked in the reading frame to the DNA encoding the anti-ErbB3 antibody. (ii) Origin of replication component The expression and cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. In general, in cloning vectors this sequence is what allows the vector to replicate independently of chromosomal DNA, and includes origins of replication or sequences that replicate autonomously. Such sequences are well known for a variety of bacteria, yeasts, and viruses. The origin of replication of plasmid pBR322 is suitable for most Gram-negative bacteria, the origin of the 2μ plasmid is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. In general, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin could typically be used because it contains the initial promoter). (iii) Selection gene component The expression and cloning vectors could contain a selection gene, also called a selective marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g. ex. , ampicillin, neomycin, methotrexate, or tetracycline, (b) auxotrophic supplemental deficiencies, or (c) critical nutrient supplementation not available in the complex medium, p. ex. , the gene encoding Bacilli D-alanine racemase.

An example of a selection scheme uses a drug to stop the growth of a host cell. Cells that successfully transform with a heterologic gene produce a protein that confers resistance to the drug and thus survives the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selective markers for mammalian cells are those that allow the identification of competent cells to take the anti-ErbB3 antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes. , adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are first identified by growing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, the transformed host cells (particularly wild-type host containing endogenous DHFR) or co-transformed with DNA sequences encoding the anti-ErbB3 antibody, the wild-type DHFR protein, and another selective marker such as aminoglycoside 3'-phosphotransferase ( APH) can be selected by means of cell growth in medium containing a selection agent for the selective marker such as an aminoglycoside antibiotic, e.g. eg, kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

A suitable selection gene for use in yeast is the trpl gene present in yeast plasmid YRp7 (Stinchcomb et al., Na ture, 282: 39 (1979)). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 4407 ^ 6 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trpl lesion in the genome of the yeast host cell then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, eu2 deficient yeast strains (ATCC 20,622 or 38,626) are supplemented with known plasmids having the Leu2 gene.

In addition, vectors derived from the circular plasmid pKDl of 1.6 μm can be used for the transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale production of calf chemokine was reported for K. lactis. Van den Berg, Bio / Technology, 8: 135 (1990). Stable multicopy expression vectors have also been exposed for secretion of mature recombinant human serum albumin by means of industrial strains of Kl. Uveromyces. Fleer et al. , Bio / Technology, 9: 968-975 (1991). (iv) Promoter component The expression and cloning vectors usually contain a promoter which is recognized by the host organism and is operably linked to the nucleic acid of the anti-ErbB3 antibody. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgarno sequence (S.D.) operably linked to the DNA encoding the anti-ErbB3 antibody.

Promoter sequences are known from eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the site where transcription starts. Another sequence found 70 to 80 bases upstream of the start of transcription of many genes is a CNCAAT region where N could be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence that could be the signal for the addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- Phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters that have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degenerative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for the use of maltose and galactose. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.

Transcription of the anti-ErbB3 antibody from the vectors in mammalian host cells is controlled, for example, by promoters obtained from virus genomes such as polyoma virus, chicken pox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, bird sarcoma virus, cytomegalovirus, retrovirus a, hepatitis B virus and more preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g. ex. , the actin promoter or an immunoglobulin promoter, from heat shock promoters, are provided such promoters that are compatible with the host cell systems.

The initial and final promoters of the SV40 virus are conveniently obtained as a restriction fragment SV40 that also contains the viral origin of replication SV40. The initial promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is set forth in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al. , Nature, 297: 598-601 (1982) on expression of human β-interferon cDNA in mouse cells under the control of a herpes simplex virus thymidine kinase promoter. Alternatively, the repeated terminal length rous sarcoma virus can be used as a promoter. (v) Component element enhancer or enhancer The transcription of DNA encoding the anti-ErbB3 antibody of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, an eukaryotic cell virus enhancer will be used. Examples include the SV40 enhancer on the final side of the replication origin (bp 100-270), the cytomegalovirus initial promoter enhancer, the polyoma enhancer on the final side of the replication origin, and the adenovirus enhancers. See also Yaniv, Na ture, 297: 17-18 (1982) in enhancing elements for the activation of eukaryotic promoters. The enhancer could be spliced into the vector at a position 5 'or 3 * to the sequence encoding the anti-ErbB3 antibody, but is preferably located at a 5' site of the promoter. (vi) Component termination of the transcript Expression vectors used in eukaryotic host cells (yeast, fungus, insect, plant, animal, human, or nucleated cells of other multicellular organisms) will also contain the sequences necessary for the termination of transcription and to stabilize the mRNA. Such sequences are commonly available in 5 'and occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA that encodes the anti-ErbB3 antibody. A useful transcription termination component is the polyadenylation region of bovine growth hormone. See WO 94/11026 and the expression vector set forth herein. (vii) Selection and transformation of host cells Suitable host cells for cloning or expressing the DNA in the vectors here are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteria such as Escherichia, - p. ex. , E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, p. ex. , Salmonella typhimurium, Serratia, p. ex. , Serra tia marcescans, and Shigella, as well as Bacilli such as B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published April 12, 1989) Pseudomonas such as P. aeruginosa, and Streptomyces. A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

In addition to prokaryotic, eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding the anti-ErbB3 antibody. Saccharomyces cerevisiae, or common bread yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are available and are commonly useful here, such as Schizosaccharomyces pombe; Guests Kluyveromyces such as, p. ex. , K. lactis, K. fragilis (ATCC 12,424), K. b? lgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. wal tii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. termotolerans and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Western Scwanniomyces i s; and filamentous fungi such as, p. ex. , hosts Neurospora, Penicillium, Tolypocladium, and Aspergillus such as A. nidulans and A, niger.

Suitable host cells for the expression of glycosylated anti-ErbB3 antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous strains of baculoviruses and variants and host cells of corresponding permissive insects have been identified such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombix mori. A variety of virus strains for transfection are patented, e.g. ex. , the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombix mori NPV, and such viruses could be used as the viruses according to the present invention, particularly by transfection of Spodoptera frugiperda cells.

Plant cultures of cotton, corn, potato, soy, petunia, tomato, and tobacco could also be used as hosts.

However, interest has increased in vertebrate cells, and the spread of vertebrate cells in culture (tissue culture) has been a routine procedure. Examples of useful mammalian host cell lines are the monkey kidney CVL line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc.Nat.Acid.Sci.USA, 77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRl cells (Mather et al., Annals N. Y. Acad. Sci., 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed with the expression or cloning vectors described above for production and culture of anti-ErbB3 antibodies in modified conventional nutrient medium as appropriate to induce promoters, selection of transformants, or amplification of genes encoding the desired sequences. (viii) Culture of host cells The host cells used to produce the anti-ErbB3 antibody of this invention could be cultured in a variety of media. Commercially available media such as Ham's FIO (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culture of In addition, any of the media described in Ham et al., Meth. Enz., 58:44 (1979), Barnes et al., Anal. Biochem., 102: 255 (1980), Pat. US Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985 could be used as a culture medium for the host cells. Any of these means could be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN *), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent source of energy. Any other necessary supplements could also be included in appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cells selected for expression, and will be apparent to those skilled in the art. (ix) Purification of anti-ErbB3 antibody When recombinant techniques are used, the antibody can be produced intracellularly, in the periplasmic space, or secreted directly into the medium. If the antibody is produced intracellularly, as a first step, the particulate residues, of host cells or lysate fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al. , Bio / Technology 10: 163-167 (1992) describes a method for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, the cell paste is melted in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 min. The cellular debris can be removed by centrifugation. Where the antibody is secreted into the medium, the supernatants of such expression systems are generally concentrated first using an available protein concentration filter, for example, an Amicon or a Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF could be included in any of the aforementioned steps to inhibit proteolysis and antibiotics could be included to prevent the growth of upstart contaminants.

The prepared antibody composition of the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography which is the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any Fc domain immunoglobulin that occurs in the antibody. Protein A can be used to purify antibodies that are based on human?,? 2, or? 4 heavy chains (Lindmark et al., J. Immunol., Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human? 3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity ligand binds is most often agarose, but other matrices are arranged. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, Bakerbond ABX ^ resin (J.T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation in an ion exchange column, ethanol precipitation, Reverse Phase HPLC, silica chromatography, heparin chromatography Sepharose "1 * chromatography on anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

After any additional purification step, the mixture containing the antibody of interest and contaminants could be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably developed at low salt concentrations (p. eg salt of approximately 0-0.25 M).

C. Pharmaceutical Formulations Therapeutic formulations of the antibody are prepared from the pool by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients, and stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), form of lyophilized formulations or aqueous solutions. Acceptable vehicles, excipients, or stabilizers are non-toxic to the recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; organic acids including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzothonium chloride, phenolic, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m- cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt that forms ion accounts such as sodium; metal complexes (eg, Zn-protein complexes); and / or nonionic surfactants such as Tween *, Pluronics1111 or polyethylene glycol (PEG).

The formulation herein could also contain more than one active compound if necessary for the particular indication to be treated, preferably those with complementary activities that do not adversely affect one another. For example, it may be desirable to further provide antibodies that bind to EGFR, ErbB2, ErbB4, or vascular endothelial factor (VEGF) in the formulation. Alternatively, or in addition, the composition could comprise a chemotherapeutic agent or a cytosine. Such molecules are suitably presented in combination in amounts that are effective for the intended purpose.

The active ingredients could also be entrapped in microcapsules prepared, for example, by coacervation or interfacial polymerization techniques, for example, hydroxymethyl cellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (eg example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are set forth in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile. This is easily done by filtration through sterile filtration membranes.

Sustained-release preparations could be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of molded articles, e.g. ex. films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L -glutamate, non-degradable vinyl ethylene acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot1 ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leprolide acetate), and poly-D- (- -3-hydroxybutyric. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow to release molecules for 100 days, certain hydrogels release proteins for shorter periods of time. When the encapsulated antibodies remain in the body for a long time, they could become denatured or aggregated as a result of exposure to humidity at 37 ° C, resulting in a loss of biological activity and possible changes in immunogenicity. Additional strategies can be projected for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be SS intermolecular bond formation by thio-disulfide exchange, stabilization could be achieved by modifying sulfhydryl residues, lyophilizing in acid solutions, controlling moisture content, using appropriate additives, and developing the compositions of the specific polymer matrix.

D. Non-therapeutic Uses of the Antibody The antibodies of the invention could be used as affinity purification agents. In this process, the antibodies are immobilized on a solid phase such as Sefadex resin or filter paper, using methods well known in the art. The immobilized antibody is contacted with a sample containing the ErbB3 protein (or fragment thereof) to be purified, and subsequently the support is washed with a suitable solvent that will remove substantially all the material in the sample except the protein ErbB3, which binds to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, which will release the ErbB3 protein from the antibody.

Anti-ErbB3 antibodies could also be useful in diagnostic tests for the ErbB3 protein, p. ex. , detecting its expression in specific cells, tissues or serum. Thus, the antibodies could be used in the diagnosis of human malignant tumors (see, for example, US Patent 5,183,884).

For diagnostic applications, the antibody will typically be labeled with a detectable radical. Numerous brands are available that can be grouped in general in the following categories: (a) Radioisotopes, such as 35S, C, 125I, 3H, and 131I. For example, the antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al. , Ed., Wiley-Interscience, New York, Pubs., (1991) and radioactivity can be measured using scintillation counting. (b) Fluorescent labels are available such as rare earth chelates (europium chelates) or fluorescein and its derivatives, radamine and its derivatives, dansyl, Lisamine, phycoerythrin and Texas Red. For example, fluorescent labels can be conjugated to the antibody using the technique set forth in Current Protocols in Immunology, supra. Fluorescence can be quantified using a fluorimeter. (c) Various enzyme-substrate labels and U.S. Pat. No. 4, 275,149 provides a review of some of these. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme could catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme could alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and could then emit light that can be measured (for example, using a chemiluminometer) or donate energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacteria luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydronaphthalazindione, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidases, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes with antibodies are described in O'Sullivan et al. , Methods for Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (Ed J. Langone &H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

Examples of substrate-enzyme combinations include, for example: (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (eg, orthophenylene diamine (OPD) or 3, 3 ', 5, 5' -tetramethyl hydrochloride benzidine (TMB)); (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as a chromogenic substrate; Y (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (eg p-nitrophenyl-β-D-galactosidase) or the fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

Other numerous enzyme-substrate combinations are available to those skilled in the art. For a general review of this, see U.S. Patents. Nos. 4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. The art expert will be aware of several techniques to achieve this. For example, the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vi ce versa. Biotin binds selectively to avidin and therefore, the label can be conjugated with the antibody in this direct manner. Alternatively, to achieve indirect conjugation of the label, the antibody is conjugated with a small hapten (eg dioxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (eg antibody). anti-dioxin). Thus, indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the anti-ErbB3 antibody does not need to be labeled, and the presence thereof can be detected using a labeled antibody that binds to the anti-ErbB3 antibody.

The antibodies of the present invention could be employed in any known test method, such as competitive binding tests, placement tests between two direct and indirect parts, and immunoprecipitation tests. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding tests are based on the ability of a labeled standard to compete with the analyte in the test sample to bind with a limited amount of antibody. The amount of ErbB3 protein in the test sample is inversely proportional to the amount of standard that is bound to bind. To facilitate the determination of the amount of standard that is bound, the antibodies are generally insolubilized before or after the competition, so that the standard and analyte that bind to the antibodies can be conveniently separated from the standard and analyte. remains unlinked Placement tests between two parties involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a test of placing between two parts, the analyte in the test sample is bound by a first antibody that is immobilized on a solid support, and subsequently a second antibody is bound to the analyte, thus forming an insoluble complex of three parts . See, p. ex. , Pat US No. 4,376,110. The second antibody could be labeled by itself with detectable radical (direct tests of placing between two parts) or could be measured using an anti-immunoglobulin antibody that is labeled with a detectable radical (indirect test of placing between two parts). For example, one type of test to place between two parts is an ELISA test, in which case the detectable radical is an enzyme.

For immunohistochemistry, the tumor sample could be fresh or frozen or it could be embedded in paraffin and fixed with a condom, for example, such as formalin.

The antibodies could also be used for in vivo diagnostic tests. In general, the antibody is labeled with a radionucleus (such as? NIn, "Te, 1C, 131I, 125I, 3H, 32P or 35S) so that the tumor can be localized using immunocytography.

E. Diagnostic Cases As a matter of convenience, the antibody of the present invention can be provided in a kit, e.g. ex. , a combination of reagent packaging in predetermined quantities with instructions to develop the diagnostic test. Where the antibody is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a precursor substrate that provides the detectable chromophore or fluorophore). In addition, other additives such as stabilizers, buffers (e.g., a blocking buffer or lysis buffer) and the like may be included. The relative amounts of various reagents could be varied widely to provide solution concentrations of reagents that substantially optimize the sensitivity of the test. Particularly, the reagents could be provided as dry powders, usually lyophilized, including excipients which in solution will provide a reagent solution having the appropriate concentration.

F. Therapeutic Uses of the Antibody It is contemplated that the anti-ErbB3 antibody of the present invention could be used to treat conditions in which excessive activation of the ErbB2-ErbB3 complex is occurring, particularly activation is mediated by a heregulin polypeptide. Example of conditions of disorders to be treated with the ErbB3 antibody include benign or malignant tumors (eg renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic carcinomas) , lung, vulval, thyroid, liver, sarcomas, glioblastomas, and various head and neck tumors); leukemias and malignant lymphoid tumors; other disorders such as neuronal, glial, astrocital, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunological disorders.

The antibodies of the invention are administered to a mammal, preferably a human, according to known methods, such as intravenous administration such as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebro-spinal, subcutaneous, intra-administration. -articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred.

Other therapeutic regimens could be combined with the administration of the anti-ErbB3 antibodies of the present invention. For example, the patient to be treated with the antibodies exposed here could also receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent could be administered to the patient. The preparation and dosing schedules for such chemotherapeutic agents could be used according to the manufacturer's instructions or as determined empirically by the skilled physician. The preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent could precede, or follow the administration of the antibody or could occur simultaneously with the same.

It may also be desirable to administer antibodies against other tumor-associated antigens, such as antibodies that bind to EGFR, ErbB2, ErbB4, or vascular endothelial factor (VEGF). Two or more anti-ErbB3 antibodies could be co-administered to the patient.

For the prevention or treatment of disease, the appropriate dosage of the antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, prior therapy , the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or a series of treatments.

Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (eg 0.1-20 mg / kg) of antibody is an initial candidate dosage for administration to the patient, either, for example, by half of one or more separate administrations, or by continuous infusion. A typical daily dosage could be in the range of about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations for several days or longer, depending on the condition, treatment is maintained until a desired suppression of the symptoms of the disease occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and tests. 6. Processing Articles In another embodiment of the invention, there is provided a processing article containing materials useful for the treatment of the disorders described above. The processing article comprises a container and a mark. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers could be formed from a variety of materials such as glass or plastic. The container maintains a composition that is effective in treating the condition and could have a sterile access door (e.g. the container could be an intravenous solution bag or vial having a cap penetrable by a hypodermic injection needle). The active agent in the composition is the anti-ErbB3 antibody. The mark on, or associated with, the container indicates that the composition is used to treat the chosen condition. The processing article could further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution and dextrose solution. It could also include other desirable materials from a commercial and use point of view, including other shock absorbers, diluents, filters, needles, syringes, and packaging booklets with instructions for use.

H. Deposit of Materials The following hybridoma cell line has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA (ATCC): ATCC Designation No. Deposit Date Bibridoma / Antibody 8B8 HB-1270 March 22, 1996 This deposit was made under the conditions of the Budapest treaty in the International Recognition of the Deposit of Microorganisms for the End of Procedure and the Patent Regulations under it (Budapest Treaty). This ensures the maintenance of a viable crop for 30 years from the date of deposit. The cell line will be available at the ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which ensures (a) access to the culture will be available for the duration of the patent application to someone determined by the Commissioner to authorize the same according to 37 CFR §1.14 and 35 USC §122, and (b) that all restrictions on the availability to the public of the crop thus deposited shall be irrevocably withdrawn as a guarantee of the patent.

The allocation of the present application has agreed that if the crop in deposit could die or be lost or destroyed when cultivated under suitable conditions, it will be promptly replaced in notification with a viable specimen of the same crop. The availability of the deposited cell line will not be built as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The aforementioned written specification is considered sufficient to enable an expert in the art to practice the invention. The present invention is not limited in scope by the deposited culture, since the deposited mode is intended as a single illustration of one aspect of the invention and any culture that is functionally equivalent is within the scope of this invention. The deposit of the material here does not constitute an admission that the written description contained herein is inadequate to allow the practice of any aspect of the invention, including the best form thereof, nor is it constructed as limiting the scope of the claims for illustration specific that it represents. Actually, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the aforementioned description and fall within the scope of the appended claims.

Regarding the designations in which the European patent is requested, a sample of the deposited microorganism will become available until the publication of the European patent granting mention or until the date on which the application has been discarded or withdrawn or consider to withdraw, only by the succession of such sample to an expert nominated by the person requesting the sample (Rule 28 (4) EPC) The following examples are provided by way of illustration and not by way of limitation. Exhibits of all citations in the specification are expressly incorporated herein by reference.

EXAMPLE PRODUCTION OF ANTIBODIES ANTI-ErbB3 This example describes the production of antibodies that have the characteristics described herein.

Materials and methods Cell lines The human myeloid leukemia cell line K562 (which lacks the receptor class I subfamily protein tyrosine kinase determined by Western blot) and the human ovarian carcinoma cell line Caov3 were obtained from the American Type Culture Collection (Rockville, MD). They were cultured in RPMI 1640 medium supplemented with 10% bovine fetal serum, 2 mM glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10 mM HEPES ("growth medium").

Stable Transfection of K562 Cells. The cell line K562 was transfected and the clones expressing ErbB3 were selected. Briefly, er £ > B3 cDNA was subcloned into the expression vector of mammalian cells pcDNA-3 (Invitrogen) and introduced into K652 cells by electroporation (1180 mF, 350 V). The transfected cells were cultured in growth medium containing 0.8 mg / mL of G418. Resistant clones were obtained by limiting dilution and were tested for ErbB3 expression by Western blot and heregulin binding assays (HRG). The 4E9H3 clone expressing ErbB3 was used in the experiments described in this report. The phorbol ester stimulation was found to significantly increase the expression of ErbB3 in the K562 transfectants. Therefore, 4E9H3 cells were placed in growth medium containing 10 ng / mL phorbol-12-myristate acetate (PMA) overnight before use in several tests described below.

Antibodies Monoclonal antibodies specific for the ErbB3 protein were generated against a recombinant fragment of the receptor corresponding to the extracellular domain (ECD) thereof fused at its amino terminus for herpes simplex virus type 1 (HSV I) glycoprotein D (gD) epitope antibody monoclonal 5B6. The sequence encoding the ErbB3 signal sequence was replaced with a sequence encoding amino acids 1-53 of the gD polypeptide. Amino acids 1-25 encode the gD signal sequence while amino acids 26-53 contain an epitope for monoclonal antibody 5B6. See WO 95/14776. The resulting construct, gD.ErB3.ECD, was purified using an affinity column of anti-gD antibodies. Immunizations were made as follows. Female Balb / c mice (Charles River) were initially injected via the base of the paw with 5 μg of gD.ErbB3.ECD in 100 μl of 'RIBI'sMR adjuvant (Ribi Immunochem Research, Inc., Hamilton, MT) . The animals were raised 2 times with 5 μg of gD.ErbB3.ECD in their paw every two weeks followed by a final injection of 5 μg of gD.ErbB3.ECD. Three days after the last immunization, the popliteal lymph nodes were removed and a single cell suspension was prepared by PEG fusion.

The monoclonal antibodies were purified and cloned by immobilization ELISA and phase solution for cross-reactivity with ErbB2 and ErbB4. For immobilized ELISA, 1 μg / ml of ErbB2.ECD, gD.ErbB3.ECD or gD.ErbB4.ECD was used to cover a 96-well microtiter plate overnight. Anti-ErbB3 Mab at 1 μg / ml was added and incubated for 1 hour at room temperature (RT), washed and by goat anti-mouse IgG (gam) conjugated with HRPO. The ELISA was developed and read at 490 nm. For the phase solution ELISA, 1 μg / ml gamma IgG (specific Fc) was used to cover a 96-well microtiter plate overnight. Anti-ErbB3 Mab at 1 μg / ml was added and incubated for 1 hour at RT, washed and by ErbB2.ECD, gD.ErbB3.ECD or gD.ErbB4.ECD. This reaction was incubated for 1 hour at RT, washed and followed with HRPO estrepavidin. The ELISA was developed and read at 490 nm. In this test, none of the anti-ErbB3 antibodies reacted cross-over with ErbB2 or ErbB4.

Fab fragments of the 3-8D6 antibody were generated by digestion of papain. The undigested IgG and Fc fragments were removed by protein A affinity chromatography followed by gel filtration chromatography. No IgG was detected in the Fab pool by SDS-PAGE and by Western blot tested with a specific antibody Fc.

HRG Link Testing All experiments were carried out using the similar EGF domain of the ß1 isoform, p. ex. HGR1717_244 (Sliwkowski et al., J. Biol. Chem. 269: 14661-5 (1994)). The ErbB3 antibody panel was screened for an HRG binding effect by incubating 5.0 x 104 cells of 4E9H3 with 100 pM 125 I-HRG overnight at 0 ° C, absent (control) or presence of 100 nM anti-ErbB3 antibody. Irrelevant IgGs were used as negative controls. The cells were harvested and washed rapidly with ice-cold test buffer (RPMI medium containing 10 mM HEPES, pH = 7.2) in a 96-well filtration device. (Millipore). The filters were then removed and counted.

For antibody dose-response experiments, 4E9H3 cells were incubated with 100 pM 125I-HRG in the presence of increased antibody concentrations. The affinity measurements of HRG were determined in the absence (control) or presence of antibody fragment or 100 nM Fab. These experiments were carried out in a competitive inhibition format with increasing amounts of unlabeled HRG and a fixed concentration (35 pM) of 125 I-HRG. For the control experiment (without antibody), 1 x 105 4E9H3 cells were used for each sample. Due to limitations in the dynamic range of the test, the number of 4E9H3 cells used to bind in the presence of the antibody or Fab was reduced to 2.5 x 104 cells per sample.

Reduction of phosphorylation antibody stimulated by HRG. Caov3 cells, which naturally express ErbB2 and ErbB3, were preincubated with 250 nM anti-ErbB3 anti-ErbB3 antibody, Fab fragments of this antibody, or buffer (control), for 60 minutes at room temperature. The anti-ErbB2 antibody, 2C4 (Fendly et al., Cancer Res., 50: 1550-1558 (1990)), which was previously shown to block the HRG stimulated phosphorylation of ErbB2 was included as a positive control. The cells were then stimulated with HRG to a final concentration of 10 nM for 8 minutes at room temperature, or left unstimulated. The reaction was stopped by removing the supernatants and dissolving the cells in SDS sample buffer. The lysates were then run on SDS-PAGE. The Western blots of the gels were then probed with anti-phosphotyrosine conjugated with horseradish peroxidase (Transduction Labs), and the spots were visualized using a chemiluminescent substrate (Amersham). The spots were searched with a reflectance search densitometer as described in Holmes et al. , Science, 256: 1205-1210 (1992).

Reduction of the antibody for the formation of the ErbB2-ErbB3 protein complex. Caov3 cells were pre-incubated with buffer (control), anti-ErbB3 250 nM anti-ErbB3 antibody, or Fab fragments of this antibody, or anti-ErbB2 antibody (2C4) for 60 minutes at room temperature, then treated with HRG 10 nM or control buffer for 10 minutes. The cells were lysed in 25 mM Tris, pH = 7.5, 150 mM NaCl, 1 mM EDTA, 1.0% Triton X-100%, CHAPS 1.0%, 10% v / v glycerol, containing 0.2 mM PMSF, 50 mTU / mL of aprotinin, and 10 mM leupeptin ("lysis buffer"), and the crude lysates were centrifuged briefly to remove the insoluble material.The supernatants were incubated with 3E8, a monoclonal antibody specific for ErbB2 (Fendly et al., Cancer Res., 50: 1550-1558 (1990)), were covalently coupled to an insoluble support (Affi Prep-10, BioRad.) The incubation was carried out overnight at 4 ° C. The immunoprecipitates were washed twice. times with ice-cooled lysis buffer, resuspended in a minimum volume of SDS sample buffer, and run on SDS-PAGE.The Western blots of the gels were then probed with a polyclonal anti-ErbB3 (Santa Cruz Biotech). spots were searched with a reflectance search densitometer as described in Holmes et al., Sicenc e, 256: 1205-1210 (1992). After visualization with the ECL chemiluminescence substrate, the spots were cut into strips and failed with a polyclonal anti-ErbB2 (Santa Cruz Biotech). A duplicate test stained with anti-ErbB2 showed that equal amounts of ErbB2 were immunoprecipitated in each sample.

Results A panel of monoclonal antibodies directed against the extracellular domain of ErbB3 were evaluated for their ability to affect the binding of HRG with ErbB3. Initial screening was carried out by incubating each of the purified antibodies in a final concentration of 100 nM with 4E9H3 cells in the presence of 125 I-HRG. 4E9H3 cells are ErbB3 transfectants of the human myeloid leukemia cell line K562. The K562 cell line does not express endogenous ErbB receptors or HRG. Therefore, heregulin binding to 4E9H3 cells occurs exclusively through ErbB3. After incubation the samples were chilled on ice overnight, cell-associated beads were measured. As shown in Fig. 1, two of the anti-ErbB3 monoclonal antibodies (2F9 and 3E9) reduced the amount of 125I-HRG bound to 4E9H3 cells relative to control (without antibody). However, there were several linked ligands increased significantly. These results suggested that these anti-ErbB3 antibodies were capable of increasing the affinity for binding HRG and / or increasing the availability of HRG binding sites. To further characterize the influence of these antibodies on the HRG link to ErbB3, dose-response experiments were performed using the 3-8D6 antibody that increased the HRG linkage. 4E9H3 cells were incubated with 100 pM 125 I-HRG in the presence of increasing concentrations of the 3-8D6 antibody. The beads associated with cells were then measured after one night incubation on ice. The results are shown in Fig. 2 as graphs of counts associated with cells against antibody concentrations. There is a correlation between the increase in HRG binding and the increase in antibody concentration. The heregulin binding reached saturation between 10 and 100 nM IgG. The EC50 value for antibody 3-8D6 was 722 pM. No dose-response curves decreased at high antibody concentrations were observed for any antibody.

The Scatchard analysis of HRG binding was determined in the presence of these antibodies and the results are shown in Table 1.

Table 1 In the absence of the antibody, a Kd of 1200 pM was measured for HGR binding to ErbB3, which is in agreement with a previously measured affinity measurement of binding of HRG to ErbB3. The number of binding sites per cell was determined to be 36,000. In the presence of the antibody, 3-8D6, the binding constant measured for the HRG binding is significantly increased up to 210 pM. However, the number of HRG binding sites does not increase in the presence of 3-8D6.

To determine whether the increase in binding affinity of the ErbB3 ligand was dependent on the antibody that is divalent, the HRG binding experiments were performed in the presence of 100 nM of a Fab fragment prepared by papain digestion of the 3-8D6 antibody. The Fab fragments used for these experiments were purified by protein A affinity chromatography and by gel filtration chromatography. Non-intact IgG was detected in this purified preparation by SDS-PAGE. As shown in Fig. 3, the HRG binding in the presence of the intact antibody or the resulting Fab is almost identical. The Scatchard analysis of these results provides a dissociation constant for the HRG binding in the presence of 280 pM Fab and the number of receptors per cell determined from this experiment was also essentially the same as that of the control. These results are consistent with those presented in Fig. 2, where the dose response curves with the intact antibodies show a plateau more than a bell-shaped curve at a higher concentration of the antibody, where univalent antibody binding could occur. Without theorizing, these results suggest that the alteration in the HRG bond observed in the presence of these antibodies does not require a divalent antibody.

The effect of the 3-8D6 antibody on a tyrosine receptor phosphorylation test, using the Caov3 ovarian tumor cell line that co-expresses ErbB2 and ErbB3 was then examined. The cells were stimulated with 10 nM HRG after a 60-minute pre-incubation with the antibody 3-8D6 (at 250 nM) or buffer (control). The whole cell lysates were analyzed in a Western blot with anti-phosphotyrosine. The HRG treatment did not stimulate phosphorylation in 4E9H3 cells. Treatment of 4E9H3 cells with antibody 3-8D6 did not induce phosphorylation of ErbB3 on its own or had any effect on tyrosine phosphorylation in Caov3 cells. A marked tyrosine phosphorylation signal was detected in a protein with a molecular size ~ 180 kDa after stimulation of HRG. The treatment of Caov3 cells with 2C4, an antibody specific for ErbB2, allowed to block the tyrosine phosphorylation signal mediated by HRG. When the cells were treated with the anti-ErbB3 antibody, 3-8D6, before stimulation of HRG, tyrosine phosphorylation also decreased. By means of search densitometry of the anti-phosphotyrosine stains of the whole cell lysates, it was observed that 3-8D6 inhibits the phosphotyrosine signal at 180-185 kDa to approximately 80% (range 76-84%). This signal is contributed by the tyrosine phosphate residues in ErbB3 and ErbB2. The treatment of Caov3 cells with the Fab fragments prepared from the 3-8D6 antibody also reduced the stimulated phosphorylation of HRG from the 180 kDa band relative to the control. However, the inhibitory activity of Fab was slightly less potent than the intact antibody.

The increase mediated by the 3-8D6 antibody in the receptor affinity of cells expressing ErbB3 alone is analogous to the increase in affinity associated with the coexpression of ErbB2 with ErbB3. In addition, this antibody blocks the ErbB2 kinase activity stimulated by HRG in cells expressing both receptors. To determine whether the anti-ErbB3 antibody competes directly with ErbB2 to bind to ErbB3, a series of co-immunoprecipitation experiments were performed using Caov3 cells. The cells were pre-incubated with antibody, or buffer (control) and then treated with 10 nM HRG for 10 minutes. The cell lysates were then immunoprecipitated with a monoclonal antibody against ErbB2. The immunoprecipitates were then analyzed by Western blot for the presence of ErbB3. The results of these experiments indicate that ErbB3 was present in the ErbB2 immunoprecipitate of the cell lysate stimulated by HRG, but not in the immunoprecipitate of the unstimulated lysate. These results suggest that HRG directs the formation of an ErbB2-ErbB3 complex in Caov3 cells. ErbB3 was not detected in the immunoprecipitate of the sample treated with the anti-ErbB2 monoclonal antibody, 2C4. A significant decrease in the ErbB3 signal was observed when the cells were pre-incubated with the 3-8D6 antibody or its resulting Fab before HRG stimulation. These results indicate that the 3-8D6 antibody inhibits the formation of an ErbB2-ErbB3 complex following the HRG treatment. The search densitometry of the anti-ErbB3 Western blots of the anti-ErbB2 immunoprecipitates revealed that the anti-ErbB3 signal (indicating the number of ErbB2-ErbB3 complexes present) also decreases by 3-8D6 by approximately 80% (range 71- 90%). When duplicate spots were tested with anti-ErbB2, equivalent amounts of ErbB2 were present on all routes.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Genentech, Inc. (ii) TITLE OF THE INVENTION: ErbB3 Antibodies (iii) SEQUENCE NUMBER: 5 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Genentech, Inc. (B) STREET: 460 Point San bruno Blvd (C) CITY: South, San Francisco (D) STATE: California (E) COUNTRY: USA (F) ZIP CODE (ZIP): 94080 (v) COMPUTER READING FORM: (A) TYPE OF MEDIUM: 3.5 inches, 1.44 Mb floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PACKAGE: WinPatin (Genentech) (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) PUBLICATION DATE: (C) CLASSIFICATION: (viii) ATTORNEY / MANDATORY INFORMATION: (A) NAME: Lee, Wendy M. (B) REGISTRATION NUMBER: 40,378 (C) REFERENCE NUMBER / LIST: P1003PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415 / 225-1994 (B) TELEFAX: 415 / 952-9881 (C) TELEX: 910 / 371-7168 (2) INFORMATION FOR SEC ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 Pro Lys Asn Ser Ser Met Lie Ser Asn Thr Pro 1 5 10 11 (2) INFORMATION FOR SEC ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: is Gln Ser Leu Gly Thr Gln 1 5 7 (2) INFORMATION FOR SEC ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: s Gln Asn Leu Ser Asp Gly Lys 5 8 (2) INFORMATION FOR SEC ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: s Gln Asn lie Ser Asp Gly Lys 5 8 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Val lie Ser Ser His Leu Gly Gln 1 5 8 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Having described the invention as above, the content of the following is claimed as property.

Claims (21)

1. An antibody that binds to the ErbB3 protein and reduces the formation of induced by heregulin of an ErbB2-ErbB3 protein complex in a cell, characterized in that it expresses ErbB2 and ErbB3.

2. The antibody of claim 1, characterized in that it increases the binding affinity of heregulin for the ErbB3 protein.

3. The antibody of claim 1, characterized in that it further reduces the activation of ErbB2 induced by heregulin in the cell.

4. The antibody of claim 1, characterized in that it is a monoclonal antibody.

5. The antibody of claim 1, characterized in that it is humanized.

6. The antibody of claim 1, characterized in that it is human.

7. The antibody of claim 1, characterized in that it is an antibody fragment.

8. The antibody fragment of claim 8, characterized in that it is a Fab.

9. The antibody of claim 1, characterized in that it is labeled.

10. The antibody of claim 1, characterized in that it is immobilized on a solid phase.

11. An antibody, characterized in that it binds to the ErbB3 protein and increases the binding affinity of heregulin for the ErbB3 protein.

12. An antibody, characterized in that it binds to the ErbB3 protein and reduces the activation of ErbB2 induced by heregulin in a cell that expresses ErbB2 and ErbB3.

13. An antibody, characterized in that it binds to the ErbB3 protein and reduces the binding of heregulin thereto.

14. The antibody of claim 13, characterized in that it further reduces the activation of ErbB2 induced by heregulin in a cell that expresses ErbB2 and ErbB3.

15. The antibody of claim 1, characterized in that it binds to the epitope bound by the antibody 8B8.

16. The antibody of claim 1, characterized in that it has the regions that determine complementarity of the 8B8 antibody.

17. A composition, characterized in that it comprises the antibody of claim 1 and a pharmaceutically acceptable carrier.

18. A cell line, characterized in that it produces the antibody of claim 1.

19. The cell line of claim 18, characterized in that it is a hybridoma cell line that produces the 8B8 antibody.

20. A method for determining the presence of the ErbB3 protein, characterized in that it comprises exposing a cell suspected of containing the ErbB3 protein with the antibody of claim 1 and determining the binding of said antibody to the cell.

21. A kit, characterized in that it comprises the antibody of claim 1 and instructions for using the antibody to detect the ErbB3 protein.