Cystine depleted glycoprotein hormones

Details Cystine depleted glycoprotein hormones Cystine depleted glycoprotein hormones

1997-09-19

2004-02-17

Borin, Michael (Department: 1631)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S397000, C530S398000, C530S399000

Reexamination Certificate

active

06693074

ABSTRACT:

TECHNICAL FIELD
The invention relates to modified forms of single chain glycoprotein hormones wherein one or more cystine bonds in the &agr; and/or &bgr; chain is disrupted. The cystine depleted forms are agonists or antagonists of the receptors for the relevant glycoprotein hormones.
BACKGROUND ART
The glycoprotein hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (CG) are heterodimers composed of an &agr; subunit, which is identical for all four hormones in a particular species, and &bgr; subunits which differ within a species according to the identity of the hormone. These hormones are found in all mammals although chorionic gonadotropin has been determined as present only in primates and equine species. Each of the &agr; and &bgr; subunits contain multiple cystine bridges which appear necessary to effect the proper conformation for dimerization and secretion.
Recently, single chain forms of these glycoprotein hormones have been disclosed. PCT Application at WO 96/05224 published Feb. 22, 1996, and PCT Application WO 95/22304 published Aug. 24, 1995, describe various single chain forms of these hormones and recombinant methods for their production. See also Sugahara, T. et al.
Proc Natl Acad Sci USA
(1995) 92:201-2045; Narayan, P. et al.,
Mol Endocrinol
(1995) 9:172-1726. In these single chain forms, the &agr; and &bgr; portions are provided as a single fusion protein, optionally containing a linker, wherein either the &agr; or &bgr; is at the N-terminus.
Cysteine depleted mutants of the &agr; and &bgr; subunits and of the heterodimers of some of the human glycoprotein hormones have been reported previously. It has been shown that chemical reduction or site specific mutations of individual cysteine residues in either the common &agr; or &bgr; subunit alters dimer assembly and secretion rate. Suganuma, N. et al,
J Biol Chem
(1989) 264:19302-19307 and Furuhashi, M. et al,
J Biol Chem
(1994) 269:25543-25548. In the present work, it is shown that mutants lacking either the 7-31 or 59-87 bonds of the a subunit were secreted and that heterodimers containing the &agr; 7-31 mutation bound to the LH/hCG receptor with comparable affinity to wild type hCG, whereas the &agr;59-87 mutant interacted with lower binding affinity. Mutants comprising the cystine knot (10-60, 28-82 and 32-84) were not secreted in sufficient quantities to permit biological activity to be determined. The &agr;7-31 and &agr;59-87 forms had previously been prepared by Furuhashi, M., et a., supra.
Recently, the present applicant has reported the construction of &bgr;&agr; single-chain forms of chorionic gonadotropin wherein, in each such form, one of the six cystine bridges normally present in the &bgr; subunit has been deleted. These cystine depleted forms are still able to bind to the appropriate receptor and stimulate the production of cyclic AMP. (Ben-Menahem D. etal,
J Biol Chem
(1997)272: 6827-6830). Similarly, the present applicant as shown that deletion of single cystine bridges in the &agr; chain portion of this &bgr;&agr; single chain hCG results in compounds with similar activity (Sato, A. et at.,
J Biol Chem
(1997) 272:18098-18103).
The modified single-chain forms of the present invention provide additional members for the repertoire of agonists of the various heterodimeric glycoprotein hormones in various mammalian species and are useful as immunogens. Some members of this group may also act as antagonists.
DISCLOSURE OF THE INVENTION
Cystine bridge depleted single chain glycoprotein hormones derived from various species provide a new class of agonists (and antagonists) useful in enhancing (or inhibiting) fertility, modulating conditions associated with the reproductive system, and in the treatment of thyroid disorders. Enrichment of the resource of available agonists and antagonists provides added possibilities for treatment of individuals according to their unique metabolic and physiological profiles.
Thus, in one aspect, the invention is directed to a glycoprotein hormone agonist or antagonist which is a modified form of a glycoprotein hormone selected from the group consisting of follicle stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (CG) and thyroid stimulating hormone (TSH) of the formula:
&agr;-(linker)
n
−&bgr;  (1)
or
(&bgr;)−(linker)
n
−&agr;  (2);
wherein &bgr; represents the &bgr; subunit of said FSH, LH, CG or TSH or a variant thereof; &agr; represents the a subunit common to said glycoprotein hormones or a variant thereof; “linker” represents an amino acid sequence providing a fusion protein between the C-terminus of a and the N-terminus of &bgr; in formula (1) or the C-terminus of &bgr; and the N-terminus of &agr; in formula (2); and n is 0 or 1; and wherein one or more cystine bridges contained in said &agr; and/or &bgr; subunit is deleted.
In other aspects, the invention is directed to recombinant materials and methods for the production of the agonists and antagonists of the invention, to pharmaceutical compositions containing these compounds, and to methods to modify the metabolism or physiology of a subject using the modified hormones of the invention.


REFERENCES:
patent: WO 95/22304 (1995-08-01), None
patent: 96/05224 (1996-02-01), None
Sugahara et al. Proceedings of the National Academy of Sciences of the United States of America, vol. 92, No. 6, pp. 2041-2045, Mar. 1995.*
Suganuma et al. J. Biol. Chem, 264:19302-19307, Oct. 1989.*
Sato et al. J. Biol. Chem, 272:18098-18103, Jul. 1997.*
Cystine Knot of the Gonadotropin &agr; Subunit is Critical for Intracellular Behavior but Not for In Vitro Biological Activity, Asomi Sato, et al.,The Journal of Biological Chemistry, 1997, vol. 272, No. 29 Issue of Jul. 18, pp 18098-18103 (1997).
The Biologic Action of Single-chain Choriogonadotropin Is Not Dependent on the Individual Disulfide Bonds of the &bgr; Subunit, David Ben-Menahem, et al.,The Journal of Biological Chemistry, vol. 272, No. 11, Issue of Mar. 14, pp. 6827-6830 (1997).
Elimination of Di-Sulfide Bonds Affects Assembly and Secretion of the Human Chorionic Gonadotropin &bgr;Subunit, Nobuhiko Suganuma et al.,Journal of Biological Chemistry, vol. 264, No. 32, Issue of Nov. 15, pp. 19302-19307 (1989).
Mutagenesis of cysteine Residues in the Human Gonadotropin &agr;Subunit, Madoka Furuhashi, et al.,Journal of Biological Chemistry, vol. 269, No. 41, Issue of Oct. 14, pp. 25543-25548 (1994).
Functional Expression of Yoked Human Chorionic Gonadotropin in Baculovirus-Infected Insect Cells, Prema Narayan, et al.,Molecular Endocrinology, vol. 9, No. 12, pp. 1720-1726 (1995).

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