Ligand antagonists for treatment of breast cancer

Details Ligand antagonists for treatment of breast cancer Ligand antagonists for treatment of breast cancer

1994-09-19

2002-08-06

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S399000

Reexamination Certificate

active

06429186

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the field of polypeptide ligand and receptor interactions. In particular, it relates to the use of antagonists for treating breast cancer.
2. Description of the Background Art
Ligand induced receptor oligomerization has been proposed as a mechanism of signal transduction for the large family of tyrosine kinase receptors that contain an extracellular ligand binding domain (for reviews see Yarden et al.
Ann. Rev. Biochem.
57:443 (1988); Ullrich et al.
Cell
61:203 (1990)). In these models binding of one hormone molecule (or subunit) (H) per receptor (R) is thought to induce formation of an H
2
R
2
complex. For example, crosslinking and non-dissociating electrophoretic studies suggest that epidermal growth factor (EGF) promotes dimerization of the EGF receptor followed by receptor autophosphorylation and activation of the intracellular tyrosine kinase (Shector et al.
Nature
278:835 (1979); Schreiber et al. J. Biol. Chem. 258: 846 (1983); Yarden et al.
Biochemistry
26:1434 (1987); Yarden et al. Biochemistry 26:1443 (1987)). Studies of other tyrosine kinase receptors including the insulin receptor (Kahn et al.
Proc. Natl. Acad. Sci. U.S.A.
75:4209 (1978); Kubar et al.
Biochemistry
28:1086 (1989); Heffetz et al.
J. Biol. Chem.
261:889 (1986), platelet derived growth factor (PDGF) receptor (Heldin et al.
J. Biol. Chem.
264:8905 (1989); Hammacher et al.
EMBO J.
8:2489 (1989); Seifert et al.
J. Biol. Chem.
264:8771 (1989)) and insulin-like growth factor (IGF-I) receptor (Ikari et al.
Mol. Endocrinol.
2:831), indicate that oligomerization of the receptor is tightly coupled to the biological effect. Other groups have recently crystallized a polypeptide hormone in complex with its extracellular binding domain (Lambert et al.
J. Biol. Chem.
264:12730 (1989); Gunther et al.
J. Biol. Chem.
265:22082 (1990)). However, more detailed analyses of the structural perturbations and requirements for ligand induced changes in these or other receptors have been hampered because of the complexities of these membrane associated systems and the lack of suitable quantities of highly purified natural or recombinant receptors.
When purified receptors were available the assay procedures were often structured so that the nature of the hormone-receptor complex was not recognized. In U.S. Pat. No. 5,057,417, hGH binding assays were conducted using
125
I-hGH competition with cold hGH for binding to the extracellular domain of recombinant hGH receptor (hGHbp), or hGH binding protein; the resulting complex was treated with antibody to the hGHbp, plus polyethylene glycol, to precipitate the complex formed. These immunoprecipitation assays suggested that hGH formed a 1:1 complex with hGHbp. This immunoprecipitation assay correctly detected the amount of
125
I-hGH bound, but it incorrectly indicated a 1:1 molar ratio.
Various solid phase assays for hGH receptor and binding protein have been used. Such assays detected the amount of hGH bound but not the molar ratio of hGH to receptor. Binding assays with solid phase or with membrane fractions containing hGH receptor were not suitable for determining the molar ratio of hGH to receptor due to an inability to detect the total amount of active receptor and/or the amount of endogenous hGH bound. Based upon earlier work, such as with EGF, the art assumed the hGH-receptor complex would be an H
2
R
2
tetramer.
The hGH receptor cloned from human liver (Leung et al.
Nature
330:537 (1987)) has a single extracellular domain (about 28 kD), a transmembrane segment, and an intracellular domain (about 30 kD) that is not homologous to any known tyrosine kinase or other protein. Nonetheless, the extracellular portion of the hGH receptor is structurally related to the extracellular domains of the prolactin receptor (Boutin et al.
Cell
53:69 (1988)) and broadly to at least eight other cytokine and related receptors. hGHbp expressed in
Escherichia coli
has been secreted in tens of milligrams per liter (Fuh et al.
J. Biol. Chem.
265:3111 (1990)). The highly purified hGHbp retains the same specificity and high affinity for hGH (K
D
about 0.4 nM) as compared to the natural hGHbp found in serum.
hGH is a member of a homologous hormone family that includes placental lactogens, prolactins, and other genetic and species variants of growth hormone (Nicoll et al.
Endocrine Reviews
7:169 (1986)). hGH is unusual among these in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung et al.
Nature
330: 537 (1987)) or prolactin receptor (Boutin et al.
Cell
53:69 (1988)). The cloned gene for hGH has been expressed in a secreted form in
Eschericha coli
(Chang et al.
Gene
55:189 (1987)) and its DNA and amino acid sequence has been reported (Goeddel et al. Nature 281:544 (1979); Gray et al.
Gene
39:247 (1985)). The three-dimensional structure of hGH has not previously been available. However, the three-dimensional folding pattern for porcine growth hormone (pGH) has been reported at moderate resolution and refinement (Abdel-Meguid et al.
Proc. Natl. Acad. Sci. U.S.A.
84:6434 (1987)). hGH receptor and antibody binding sites have been identified by homologue-scanning mutagenesis (Cunningham et al.
Science
243:1330 (1989)). Growth hormones with N-terminal amino acids deleted or varied are known. See Gertler et al.
Endocrinology
118:720 (1986); Ashkenazi et al.
Endocrinology
121:414 (1987), Binder,
Mol. Endo.
7:1060 (1990), and WO 90/05185. Antagonist variants of hGH are described by Chen et al.
Mol. Endo.
5:1845 (1991) and literature set forth in the bibliography thereof; and WO 91/05853. hGH variants are disclosed by Cunningham et al.
Science
244:1081 (1989) and
Science
243:1330 (1989).
Since the mode of interaction of many polypeptide ligands with their receptors has remained uncertain it has been difficult to engineer amino acid sequence variants of such ligands to achieve desired properties. Essentially, the art has introduced variation at random, perhaps in some cases with guidance from homology analyses to similarly-acting ligands or animal analogs, or from analysis of fragments, e.g., trypsin digest fragments. Then the art has screened the candidates for the desired activity, e.g., agonist or antagonist activity. The screening methods have been tedious and expensive, e.g., the use of transgenic animals (WO 91/05853). Methods are needed for improving the efficiency of selection of candidates. In particular, methods are needed for focusing on candidates likely to be either antagonists or agonists. Antagonists are substances that suppress, inhibit or interfere with the biological activity of a native ligand, while agonists exhibit greater activity per se than the native ligand.
That prolactin (PRL) and growth hormone have a role in the development and progression of breast cancer has been well established in the experimental animal (Tornell et al.
Int. J. Cancer
49:114 (1991)). For example, a high serum level of growth hormone was found to induce the formation of breast cancer (Tornell, id.), while reduction of the circulating level of growth hormone correlated with the regression of breast cancer (Phares et al.
Anticancer Res.
6:845 (1986)). Higher serum level of lactogenic hormones have been found in breast cancer patients in some studies (Maddox et al.
Brit. J. Cancer
65:456 (1992)) but not in others (Love et al.
Cancer
68:1401 (1991)). 40-70% of breast cancer biopsies were positive for the presence of prolactin receptor (Bonneterre et al.
Cancer Res.
47:4724 (1987); Murphy et al.
Cancer Res.
44:1963 (1984)).
Most human breast cancer cells in culture contain prolactin receptors. In fact, the majority of breast cancer cell lines overexpressed prolactin receptor 2-10 fold (Shiu, “Prolactin, Pituitary Hormones, and Breast Cancer,” in
Hormones and Breast Cancer,
Pike et al., eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1981)). Lactogenic hormones have been found to induce the growth of the huma

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