1. Field of the Invention
The present invention relates to a growth factor which interacts with the human oncogene erbB-2, and which stimulates as well as inhibits the growth of cells overexpressing this oncogene. A ligand is described which is capable of binding to the expression product of the erbB-2 oncogene. The present invention additionally relates to anti-ligand molecules capable of recognizing and binding to the erbB-2 ligand molecule and to screening assays for such ligands. The present invention further relates to uses for the erbB-2 ligand, the anti-ligand molecules and the screening assay. Furthermore, the invention relates to a cloned gene capable of expressing the erbB-2 ligand of the present invention.
2. Description of the Related Art
Carcinogenesis is believed to be a multi-step process of alteration of genes which are involved in the growth control of cells. A variety of proto-oncogenes and oncogenes have been implicated in the activation of tumor cells as regulating factors. For example, oncogenic protein kinases are believed to induce cellular transformation through either inappropriate or excessive protein phosphorylation, resulting in the uncontrolled growth of malignant neoplasms. See Wrba, F., et al., Histopathology, 15:71-76 (1989).
One group of proto-oncogenes encodes cellular growth factors or their receptors. The c-erbB-1 gene encodes the epidermal growth factor or its receptors. The c-sis gene encodes the B-chain of the platelet-derived growth factor. The c-fms gene encodes a related or identical molecule for the receptor of the granulocyte-macrophage colony stimulating factor. A fourth member of this group of proto-oncogenes, called neu was identified in ethylnitrosourea-induced rat neuroblastomas.
The human counterpart of neu, called HER-2/neu or c-erbB-2, has been sequenced and mapped to the chromosomal locus 17q21. See Schneider, P. M., et al., Cancer Research, 49:4968-4971 (Sep. 15, 1989). The HER-2/neu or c-erbB-2 oncogene belongs to the erbB-like oncogene group, and is related to, but distinct from the epidermal growth factor receptor (EGFR). The c-erbB-2 oncogene is known to express a 185 kDa transmembrane glycoprotein (p185.sup.erbB-2). The expressed protein has been suggested to be a growth factor receptor due to its structural homology with EGFR. However, known EGFR ligands, such as EGF or TGF.alpha., do not bind to p185.sup.erbB-2.
The oncogene has been demonstrated to be implicated in a number of human adenocarcinomas leading to elevated levels of expression of the p185 protein product. For example, the oncogene has been found to be amplified in breast, ovarian, gastric and even lung adenocarcinomas. Furthermore, the amplification of the c-erbB-2 oncogene has been found in many cases to be a significant, if not the most significant, predictor of both overall survival time and time to relapse in patients suffering from such forms of cancer. Carcinoma of the breast and ovary account for approximately one-third of all cancers occurring in women and together are responsible for approximately one-fourth of cancer-related deaths in females. Significantly, the c-erbB-2 oncogene has been found to be amplified in 25 to 30% of human primary breast cancers. See Slamon, D., et al., Science, 244, 707-712 (May 12, 1989).
Although ligands for EGFR are known, namely EGF and TGF.alpha., few ligands for the oncogene-encoding transmembrane proteins such as erbB-2, ros, etc., have been characterized. Transforming growth factor ligands belong to a family of heat and acid-stable polypeptides which allow cells to assume a transformed morphology and form progressively growing colonies in anchorage-independent growth assays (DeLarco, et al., Proc. Natl. Acad. Sci. USA, 75:4001-4005 (1978); Moses, et al., Cancer Res., 41:2842-2848 (1981); Ozanne, et al., J. Cell. Physiol., 105:163-180 (1980); Roberts, et al., Proc. Natl. Acad. Sci. USA, 77:3494-3498 (1980)). The epidermal growth factor receptor (EGFR) and its physiologic ligands, epidermal growth factor (EGF) and transforming growth factor .alpha. (TGF.alpha.), play a prominent role in the growth regulation of many normal and malignant cell types (Carpenter, G., Annu. Rev., Biochem., 56:881-914 (1987)).
One role the EGF receptor system may play in the oncogenic growth of cells is through autocrine-stimulated growth. If cells express the EGFR and secrete EGF and/or TGF.alpha., then such cells could stimulate their own growth. Since some human breast cancer cell lines and tumors express EGFR (Osborne, et al., J. Clin. Endo. Metab., 55:86-93 (1982); Fitzpatrick, et al., Cancer Res., 44:3442-3447 (1984); Filmus, et al., Biochem. Biophys. Res. Commun., 128:898-905 (1985); Davidson, et al., Mol. Endocrinol, 1:216-223 (1987); Sainsbury, et al., Lancet, 1:1398-1402 (1987); Perez, et al., Cancer Res. Treat., 4:189-193 (1984)) and secrete TGF.alpha. (Bates, et al., Cancer Res., 46:1707-1713 (1986); Bates, et al., Mol. Endocrinol, 2:543-555 (1988)), an autocrine growth stimulatory pathway has been proposed in breast cancer (Lippman, et al., Breast Cancer Res. Treat., 7:59-70 (1986)).
The erbB-2 proto-oncogene amplification has been found in breast, ovarian, gastric, salivary gland, and in non-small cell carcinomas of the lung (King, et al., Science, 229:974 (1985); Slamon, et al., Science, 244:707 (1989); Yokota, et al., Lancet, 1:765 (1986); Fukushige, et al., Mol. Cell. Biol., 6:955 (1986); Semba, et al., Proc. Natl. Acad. Sci. USA, 82:6497 (1985); Weiner, et al., Cancer Res., 50:421 (1990)). Amplification and/or overexpression of the erbB-2 proto-oncogene has been found to correlate with poor prognosis in breast, ovarian and non-small cell lung carcinomas (Slamon, et al., Science, 235:177 (1986); Slamon, et al., Science, 244:707 (1989); Guerin, et al., Oncogene Research, 3:21 (1988); Wright, et al., Cancer Res., 49:2087 (1989); Kern, et al., Cancer Res., 50:5184 (1990); DiFiore, et al., Science, 237:178 (1987)). In addition to these clinical studies, in vitro studies strongly suggest that overexpression of the erbB-2 transmembrane receptor (p185.sup.erbB-2) may have an important role in tumor progression (DiFiore, et al., Science, 237:178 (1987); Hudziak, et al., Proc. Natl. Acad. Sci. USA, 84:7159 (1987)).
An autocrine growth stimulatory pathway analogous with that proposed for epidermal growth factor receptor and its ligands may also be employed by a growing list of oncogene encoded transmembrane proteins that have structure reminiscent of growth factor receptors. This list includes the protooncogenes neu and its human equivalent erbB-2 or HER2 (Bargmann, et al., Nature, 319:226-229 (1986); Coussens, et al., Science, 230:1131-1139 (1985); Yamamoto, et al., Nature, 319:230-234 (1986); c-kit (Yarden, et al., EMBO, 6:341-3351 (1987); ros (Neckameyer, et al., Mol. Cell. Biol. 6:1478-1486 (1986); met (Park, et al., PNAS, 84:6379-6383 (1987); trk (Martin-Zanca, et al., Nature, 319:743-748 (1986); and ret (Takahashi, et al., Mol. Cell. Biol., 7:1378-1385 (1987)). The erbB-2 and c-kit protooncogenes encode factors that display remarkable structural homology with EGFR (Yarden, et al., Annu. Rev. Biochem., 57:443-478 (1988). Although erbB-2 and its related oncogene neu are related to EGFR, these proteins are distinct. For example, known EGFR ligands such as EGF and TGF.alpha. do not bind to erbB-2 receptor. (King, et al., EMBO, 7:1647 (1988); and Stern, et al., EMBO, 7:995 (1988).
If, according to the autocrine growth stimulatory pathway, malignant cells are capable of secreting a potent tumor growth factor in vivo, it is plausible that the growth factor ligand might be detected in body fluids, much like human chorionic gonadotropin or .alpha.-fetoprotein, and could be used as a tumor marker and a prognostic variable. Studies suggest that TGF.alpha. activity can be detected in body fluids of cancer patients and that its presence may provide important information concerning the biology of a patient's tumor (Stromberg, et al., J. Cell. Biochem., 32:247-259 (1986); Twardzick, et al., J. Natl. Cancer Inst., 69:793-798 (1982); Sherwin, et al., Cancer Res., 43:403-407 (1983)).
Prior to the present invention, no ligand was known which binds to p185.sup.erbB- 2 protein. Thus, a need continues to exist for a ligand for p185.sup.erbB-2. Such a ligand might be used to counteract the effects of c-erbB-2 oncogene overexpression in facilitating carcinogenesis.