Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Reexamination Certificate
1998-10-19
2003-10-21
Spector, Lorraine (Department: 1646)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
C424S198100, C530S397000, C530S398000
Reexamination Certificate
active
06635256
ABSTRACT:
TECHNICAL FIELD
The invention relates to the field of protein engineering, specifically to modified forms of certain glycoprotein hormones which native forms occur normally as heterodimers. The invention concerns multiple domain complexes of chorionic gonadotropin (CG), thyroid stimulating hormone (TSH), luteinizing hormone (LH), and follicle stimulating hormone (FSH), wherein an a subunit covalently linked to a &bgr; subunit may associate with an additional &bgr; subunit or may be covalently linked to two &bgr; subunits. These multiple domain glycoprotein hormones can provide two or more effects or functions, or can behave like agonists and/or antagonists of the native hormones.
BACKGROUND ART
In humans, four important glycoprotein hormones (LH, FSH, TSH and CG) are heterodimers that have identical &agr; subunits and differing &bgr; subunits. Three of these hormones are present in virtually all other vertebrate species as well; CG has so far been found only in primates and in the placenta and urine of pregnant mares.
PCT application WO90/09800, published Sep. 7, 1990, and incorporated herein by reference, describes a number of modified forms of these hormones. One important modification is C-terminal extension of the &bgr; subunit by the carboxy terminal peptide (CTP) of human chorionic gonadotropin or a variant thereof. Other muteins of these hormones are also described. CTP is the sequence of amino acids extending from any one of positions 112-118 to position 145 of the &bgr; subunit of human chorionic gonadotropin. The PCT application describes variants of the CTP extension obtained by conservative amino acid substitutions such that the capacity of the CTP to alter the clearance characteristics of the hormone is not destroyed. In addition, PCT application WO94/24148 published Oct. 27, 1994, incorporated herein by reference, describes modifying these hormones by extension or insertion of the CTP at locations other than the C-terminus and CTP fragments shorter than the sequence extending from positions 112-118 to 145.
The CTP-extended &bgr; subunit of FSH is also described in two papers by applicants herein: LaPolt, P. S. et al.;
Endocrinology
(1992) 131:2514-2520 and Fares, F. A. et al.;
Proc Natl Acad Sci USA
(1992) 89:4304-4308. Both of these papers are incorporated herein by reference.
The crystal structure of human chorionic gonadotropin has been published in more or less contemporaneous articles; one by Lapthorn, A. J. et al.
Nature
(1994) 369:455-461 and the other by Wu, H. et al.
Structure
(1994) 2:545-558. The results of these articles are summarized by Patel, D. J.
Nature
(1994) 369:438-439.
PCT application WO91/16922 published Nov. 14, 1991 describes a multiplicity of chimeric and otherwise modified forms of the glycoprotein hormones. In general, the disclosure is focused on chimeras of &agr; subunits or &bgr; subunits involving portions of various &agr; or &bgr; chains respectively. One construct simply listed in this application, and not otherwise described, fuses substantially all of the &bgr; chain of human chorionic gonadotropin to the &agr; subunit preprotein, i.e., including the secretory signal sequence for this subunit.
Two additional published PCT applications describe single-chain forms of these glycoprotein hormones wherein the &agr; and &bgr; subunits are covalently linked to result in a compound of the general formula:
&bgr;(linker)
n
&agr;; or
&agr;(linker)
n
&bgr;
wherein n is 0 or 1 and &agr; and &bgr; represent the respective subunits of these hormones: Moyle, W. R., PCT application WO95/22340 published Aug. 24, 1995 and the application of the inventor herein, WO96/05224 published Feb. 22, 1996. The disclosure of these documents is also incorporated herein by reference.
Forms of the above-described single-chain forms of these hormones in which the number of cystine bridges has been depleted are disclosed in U.S. Ser. No. 08/933,693 filed Sep. 19, 1997, and incorporated herein by reference.
The &agr; subunit of a single-chain form of a glycoprotein hormone, CG&bgr;-&agr;, was found to bind noncovalently to an FSH&bgr; subunit as disclosed by the applicants in Society for the Study of Reproduction, Abstract 193, 1996.
Recently, the &agr; subunit of the single-chain glycoprotein hormone, FSH&bgr;-&agr;, was found to form a noncovalent link with a GC&bgr; subunit as disclosed by the applicants in Endocrine Society, Abstract OR28-3, 1998.
It has now been found possible to use these glycoprotein hormones which have enhanced agonist and/or antagonist activity and/or which are multi-functional by either covalently linking an additional &bgr; subunit to a single-chain hormone or noncovalently linking an additional &bgr; subunit to the tethered a subunit of a single-chain hormone to mimic a natural hormone profile and/or control hormone ratios. These differentially acting glycoprotein hormones and their therapeutic uses for treating disorders such as polycystic ovarian disease, infertility, and ovarian hyperstimulation are disclosed hereinbelow.
DISCLOSURE OF THE INVENTION
The invention provides differentially acting glycoprotein hormones containing an &agr; subunit covalently linked to a &bgr; subunit to form a single-chain hormone and an additional &bgr; subunit either covalently linked to the single-chain hormone or noncovalently linked to the tethered a subunit of the single-chain hormone. The compositions of the invention may either be glycosylated, partially glycosylated, or nonglycosylated and the fused &agr; and &bgr; chains that occur in the native glycoprotein hormones or variants of them may optionally be linked through a linker moiety. Particularly preferred linker moieties include the carboxy terminal peptide (CTP) unit either as a complete unit or a variant including variants which represent only a portion thereof.
If the &bgr; subunits are the same, the compositions containing a noncovalently linked &bgr; subunit can act as agonists or antagonists, but the degree of activity may vary with time. This variation in the activity is due to the difference between the circulating half-lives of the covalently linked and the noncovalently linked &bgr; subunits. The circulating half-life of the noncovalently linked &bgr; subunit will inherently be shorter than that of the &bgr; subunit covalently linked to the &agr; subunit. This is due to dissociation of the complex over time in the physiological environment; however, the covalently linked portion of the molecule remains an effective pharmaceutical.
For example, a composition having a FSH&bgr; subunit covalently linked to an &agr; subunit which is noncovalently linked to another FSH&bgr; subunit would have a greater activity during the circulating half-life of the complex. However, the activity would decrease after the shorter half-life of the non-tethered FSH&bgr; subunit ends.
A composition having a FSH&bgr; subunit covalently linked to an &agr; subunit which is noncovalently linked to a CG&bgr; subunit would exhibit a longer circulating half-life for FSH activity and a shorter circulating half-life for CG activity. For the duration of the shorter circulating half-life, both the FSH&bgr; and CG&bgr; subunits would act upon their respective receptors. During the longer half-life, only the FSH&bgr; subunit covalently. linked to the &agr; subunit would be active.
In all cases, if the &bgr; subunits are different, the compositions are bifunctional as agonists and/or antagonists. It will be noted that differential ratios of activity can be obtained by increasing or decreasing the agonist activity of one component relative to the other. For example, one could enhance the FSH/LH ratio by utilizing an FSH subunit with enhanced agonist activity and/or an LH subunit with decreased agonist activity.
In one aspect, the invention is directed to a method to provide different glycoprotein hormone activities to a subject in need of hormone regulation.
By a glycoprotein hormone “activity” is meant the ability to behave as an agonist or antagonist of a corresponding native hormone with the same or differe
Ben-Menahem David
Boime Irving
Spector Lorraine
Washington University
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