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Isolated nucleic acids, vectors and host cells encoding erbb3 antibodies

Isolated nucleic acids, vectors and host cells encoding erbb3 antibodies description/claims

This is a non-provisional application filed under 37 CFR 1.53(b)(1), claiming priority under USC Section 119(e) to provisional application Ser. No. ______ to be assigned) filed on Mar. 27, 1996.

1. Field of the Invention

This invention relates generally to antibodies which bind the ErbB3 receptor. In particular, it relates to anti-ErbB3 antibodies which, surprisingly, increase the binding affinity of heregulin (HRG) for ErbB3 protein and/or reduce HRG-induced formation of an ErbB2-ErbB3 protein complex in a cell which expresses both these receptors and/or reduce heregulin-induced ErbB2 activation in such a cell.

2. Description of Related Art

Transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases are enzymes that catalyze this process. Receptor protein tyrosine kinases are believed to direct cellular growth via ligand-stimulated tyrosine phosphorylation of intracellular substrates. Growth factor receptor protein tyrosine kinases of the class I subfamily include the 170 kDa epidermal growth factor receptor (EGFR) encoded by the erbB1 gene. erbB1 has been causally implicated in human malignancy. 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 class I subfamily, p185neu, was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The neu gene (also called erbB2 and HER2) encodes a 185 kDa receptor protein tyrosine kinase. 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 including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon and bladder.

A further related gene, called erbB3 or HER3, has also been described. See U.S. Pat. Nos. 6,183,884 and 6,480,968; Plowman et al., Proc. Natl. Acad. Sci. USA, 87:4906-4909 (1990); Kraus et al., Proc. Nat. Acad. Sci. USA, 86:9193-9197 (1989); EP Pat Appln No 444,961A1; and Kraus et al., Proc. Natl. Acad. Sci. USA, 90:2900-2904 (1993). Kraus et al.

(1989) discovered that markedly elevated levels of erbB3 mRNA were present in certain human mammary tumor cell lines indicating that erbB3, like erbB1 and erbB2, may play a role in some human malignancies. These researchers demonstrated that some human mammary tumor cell lines display significant elevation of steady-state ErbB3 tyrosine phosphorylation, further indicating that this receptor may play a role in human malignancies. Accordingly, diagnostic bioassays utilizing antibodies which bind to ErbB3 are described by Kraus et al. in U.S. Pat. Nos. 5,183,884 and 5,480,968.

The role of erbB3 in cancer has been explored by others. It has been found to be overexpressed in breast (Lemoine et al., Br. J. Cancer, 66:1116-1121 (1992)), gastrointestinal (Poller et al., J. Pathol., 168:276280 (1992), Rajkumer et al, J. Pathol., 170:271-278 (1993), and Sanidas et al., Int. J. Cancer, 54:935-940 (1993)), and pancreatic cancers (Lemoine et al., J. Pathol., 168:269-273 (1992), and Friess et al., Clinical Cancer Research, 1:1413-1420 (1996)).

ErbB3 is unique among the ErbB receptor family in that it possesses little or no intrinsic tyrosine kinase activity (Guy et al., Proc. Natl. Acad. Sci. USA 91:8132-8136 (1994) and Kim et al. J. Biol. Chem. 269:24747-55 (1994)). When ErbB3 is co-expressed with ErbB2, an active signaling complex is formed and antibodies directed against ErbB2 are capable of disrupting 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. Natl. Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al., Cancer Res., 66:1467-1465 (1996) with respect to the ErbB2-ErbB3 protein complex.

Rajkumar et al., British Journal Cancer, 70(3):469-465 (1994), developed a monoclonal antibody against ErbB3 which had an agonistic effect on the anchorage-independent growth of cell lines expressing this receptor.

The class I subfamily of growth factor receptor protein tyrosine kinases has been, further extended to include the HER4/p180erbB4 receptor. See EP Pat Appln No 699,274; Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al., Nature, 366:473-476 (1993). Plowman et al. found that increased HER4 expression closely correlated with certain carcinomas of epithelial origin, including breast adenocarcinomas. Accordingly, diagnostic methods for detection of human neoplastic conditions (especially breast cancers) which evaluate HER4 expression are described in EP Pat Appln No. 599,274.

The quest for an activator of the HER2 onoogene has lead to the discovery of a family of heregulin polypeptides. These proteins appear to result from alternative splicing of a single gene which was mapped to the short arm of human chromosome 8 by Lee et al., Genomics, 16:790-791 (1993); and Orr-Urtreger et al., Proc Natl. Acad. Sci. USA, 1962:1746-1750 (1993).

Holmes et al. isolated and cloned a family of polypeptide activators for the HER2 receptor which they termed heregulin-α (HRG-α), heregulin-β1 (HRG-β1), heregulin-β2 (HRG-β2), heregulin-p2-like (HRG-β2-like), and heregulin-β3 (HRG-β3). See Holmes et al., Science, 256:1206-1210 (1992); and WO 92/20798. The 45 kDa polypeptide, HRG-α, was purified from the conditioned medium of the MDA-MB-231 human breast cancer cell line. These researchers demonstrated the ability of the purified heregulin polypeptides to activate tyrosine phosphorylation of the HER2 receptor in MCF-7 breast tumor cells. Furthermore, the mitogenic activity of the heregulin polypeptides on SK-BR-3 cells (which express high levels of the HER2 receptor) was illustrated. Like other growth factors which belong to the EGF family, soluble HRG polypeptides appear to be derived from a membrane bound precursor (called pro-HRG) which is proteolytically processed to release the 45 kDa soluble form. These pro-HRGs lack a N-terminal signal peptide.

While heregulins are substantially identical in the first 213 amino acid residues, they are classified into two major types, α and β, based on two variant EGF-like domains which differ in their C-terminal portions. Nevertheless, these EGF-like domains are identical in the spacing of six cysteine residues contained therein. Based on an amino acid sequence comparison, Holmes et al. found that between the first and sixth cysteines in the EGF-like domain, HRGs were 46% similar to heparin-binding EGF-like growth factor (HB-EGF), 35% identical to amphiregulin (AR), 32% identical to TGF-α, and 27% identical to EGF.

The 44 kDa neu differentiation factor (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, 69:659-672 (1992). Like the HRG polypeptides, NDF has an immunoglobuin (Ig) homology domain followed by an EGF-like domain and lacks a 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 distinct fibroblastic pro-NDFs. Adopting the nomenclature of Holmes et al., the NDFs are classified as either a or P polypeptides based on the sequences of the EGF-like domains. Isoforms 1 to 4 are characterized on the basis of the variable juxtamembrane stretch (between the EGF-like domain and transmembrane domain). Also, isoforms a, b and c are described which have variable length cytoplasmic domains. These researchers conclude that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions.

Falls et al., Cell, 72:801-816 (1993) describe another member of the heregulin family which they call acetylcholine receptor inducing activity (ARIA) polypeptide. The chicken-derived ARIA polypeptide stimulates synthesis of muscle acetylcholine receptors. See also WO 94/08007. ARIA is a β-type heregulin and lacks the entire “glyco” spacer (rich in glycosylation sites) present between the Ig-like domain and EGF-like domain of HR a, and HRGβ1-β3.

Marchionni et al., Nature, 362:312-318 (1993) identified several bovine-derived proteins which they 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 kringle domain. GGFs generally do not have the complete “glyco” spacer between the Ig-like domain and EGF-like domain. Only one of the GGFs, GGFII, possessed a N-terminal signal peptide.

Expression of the ErbB2 family of receptors and heregulin polypeptides in breast cancer is reviewed in Bacus et al., Pathology Patterns, 102(4)(Supp. 1):S13-S24 (1994).

See also, Alimandi et al., Oncogene, 10: 1813-1821 (1995); Beerli et al., Molecular and Cellular Biology, 16:6496-6505 (1995); Karunagaran et al., EMBO J. 16:254-264 (1996); Wallasch et al., EMBO J, 14:4267-4276 (1995); and Zhang et al., Journal of Biological Chemistry, 271:3884-3890 (1996), in relation to the above receptor family.

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