Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-07-17
2002-05-14
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S069100, C435S252300, C424S093700, C436S501000, C514S002600, C530S300000, C530S350000, C536S023400, C536S023500
Reexamination Certificate
active
06387638
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to heregulin variants, nucleic acid molecules encoding such variants, and related vectors, host cells, pharmaceutical compositions, and methods. In particular, the invention relates to amino acid substitution variants of human heregulin-&bgr;1 having an enhanced affinity for the ErbB-3 and ErbB-4 receptors.
2. Description of the 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 proteins. Growth factor receptor protein tyrosine kinases of the class I subfamily include the 170 kilodalton (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, p185
neu
(also called the ErbB-2 receptor or p185
HER2
), was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The neu (erbB2 or HER2) gene encodes a 185 kDa receptor protein tyrosine kinase.
Amplification and/or overexpression of the human erbB2 gene correlates with a poor prognosis in breast and ovarian cancers. Slamon et al.,
Science
235:177-82 (1987); Slamon et al.,
Science
244:707-12 (1989). Overexpression of erbB2 has been correlated with other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon and bladder. Accordingly, in U.S. Pat. No. 4,968,603, Slamon et al. describe and claim various diagnostic assays for determining erbB2 gene amplification or expression in tumor cells. Slamon et al. discovered that the presence of multiple copies of the erbB2 oncogene in tumor cells indicates that the disease is more likely to spread beyond the primary tumor site, and that the disease may therefore require more aggressive treatment than might otherwise be indicated by other diagnostic factors. Slamon et al. conclude that the erbB2 gene amplification test, together with the determination of lymph node status, provides greatly improved prognostic utility.
A further related gene, called erbB3 (or HER3), which encodes the ErbB-3 receptor (p180
HER3
) has also been described. See U.S. Pat. No. 5,183,884; Kraus et al.,
PNAS USA
86:9193-97 (1989); EP Patent Application No. 444,961A1; Kraus et al.,
PNAS USA
90:2900-04 (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 human malignancies. Also, Kraus et al. (1993) showed that EGF-dependent activation of the ErbB-3 catalytic domain of a chimeric EGFR/ErbB-3 receptor resulted in a proliferative response in transfected NIH-3T3 cells. Furthermore, these researchers demonstrated that some human mammary tumor cell lines display a significant elevation of steady-state ErbB-3 receptor tyrosine phosphorylation, further implicating this receptor in human malignancies. The role of erbB3 in cancer has been explored by others, and this gene has been found to be overexpressed in breast (Lemoine et al.,
Br. J. Cancer
66:1116-21 [1992]), gastrointestinal (Poller et al.,
J. Pathol
. 168:275-80 [1992]; Rajkumer et al.,
J. Pathol
. 170:271-78 [1993]; Sanidas et al.,
Int. J. Cancer
54:935-40 [1993]), and pancreatic cancers (Lemoine et al.,
J. Pathol
. 168:269-73 [1992], and Friess et al.,
Clinical Cancer Research
1:1413-20 [1995]).
The class I subfamily of growth factor receptor protein tyrosine kinases has been further extended to include the ErbB-4 (HER4) receptor, which is the product of the erbB4 (HER4) gene. See EP Patent Application No. 599,274; Plowman et al.,
PNAS USA
90:1746-50 (1993); and Plowman et al.,
Nature
366:473-75 (1993). Plowman et al. found that increased erbB4 expression closely correlated with certain carcinomas of epithelial origin, including breast adenocarcinomas. Diagnostic methods for detection of human neoplastic conditions (especially breast cancers) that evaluate erbB4 expression are described in EP Patent Application No. 599,274.
The quest for the activator of the erbB2 oncogene has lead to the discovery of a family of heregulin polypeptides. In humans, the heregulin polypeptides characterized thus far are derived from alternate splicing of a single gene which was mapped to the short arm of chromosome 8 by Lee and Wood,
Genomics
16:790-91 (1993).
Holmes et al. isolated and cloned a family of polypeptide activators for the ErbB-2 receptor which they called heregulin-&agr; (HRG-&agr;), heregulin-&bgr;1 (HRG-&bgr;1), heregulin-&bgr;2 (HRG-&bgr;2), and heregulin-&bgr;3 (HRG-&bgr;3). See Holmes et al.,
Science
256:1205-10 (1992); WO 92/20798; and U.S. Pat. No. 5,367,060. These researchers demonstrated the ability of the purified heregulin polypeptides to activate tyrosine phosphorylation of the ErbB-2 receptor in MCF7 breast tumor cells. Furthermore, the mitogenic activity of the heregulin polypeptides on SK-BR-3 cells (which express high levels of the ErbB-2 receptor) was also demonstrated.
Heregulins are large multi-domain proteins that are typically expressed as “pro-heregulins.” Pro-heregulins have been shown to undergo proteolytic processing to a mature soluble form (usually of about 44-45 kDa). Processing has been shown to occur intracellularly or at the cell surface. Domains in the soluble form include (in order from the N- to the C-terminus) an immunoglobulin homology (Ig-like) domain, a spacer region rich in glycosylation sites, and a domain similar to a domain found in EGF that is sufficient for ErbB receptor binding and activation. See Barbacci, et al.,
J. Biol. Chem
. 270:9585-89 (1995).
The heregulin EGF-like domains are characterized by substantial structural similarities to (Jacobsen et al.,
Biochemistry
35:3402-17 [1996]), and limited sequence homology with, EGF residues 1-48 (Holmes, et al., supra). Functional similarities between the heregulin EGF-like domains and EGF have been established by data showing that blocks of EGF sequence substituted into heregulin-&bgr;1 do not impair binding to cells co-expressing ErbB-3 and ErbB-2. Barbacci et al., supra.
While heregulins are substantially identical in the first 213 amino acid residues, they are classified into two major types, &agr; and &bgr;, based on two EGF-like domains that differ in their C-terminal portions. For example, the heregulin-&agr; EGF-like domain differs from that of the &bgr;1-isoform by nine substitutions near the C-terminus. The &bgr;-isoform has been reported to bind ErbB receptors with approximately eight to 10-fold higher affinity than the &agr;-isoform. Wen et al.,
Mol. Cell. Biol
. 14:1909-19 (1994).
The solution structure of the heregulin-&agr; EGF domain has recently been determined at high resolution by NMR. Jacobsen et al., supra; Nagata et al.,
EMBO J
. 13, 3517-3523 (1994). The salient features of this domain include (1) an N-terminal subdomain containing a central three-stranded &bgr;-sheet with an intermittent helix and (2) a smaller C-terminal subdomain that contains a short stretch of &bgr;-sheet. The EGF domain is stabilized by three disulfide bonds, two in the N-terminal subdomain and one in the C-terminal subdomain. The pairing of the six corresponding cysteine residues is conserved in EGF-like domains from all heregulins and from EGF.
The 44 kDa neu differentiation factor (NDF), which is the rat equivalent of human HRG, was first described by Peles et al.,
Cell
, 69:205-16 (1992), and Wen et al.,
Cell
, 69:559-72 (1992). Like the human heregulin polypeptides, NDF has an Ig-like domain followed by an EGF-like
Ballinger Marcus D.
Fairbrother Wayne J.
Jones Jennifer T.
Sliwkowski Mark X.
Wells James A.
Brannock Michael
Eyler Yvonne
Genentech Inc.
Haliday Emily M.
Quine Intellectual Property Law Group P.C.
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