Single chain forms of the glycoprotein hormone quartet

Details Single chain forms of the glycoprotein hormone quartet Single chain forms of the glycoprotein hormone quartet

1997-08-25

2001-05-29

Spector, Lorraine (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S325000, C435S360000, C435S252300, C435S320100, C536S023400, C530S398000

Reexamination Certificate

active

06238890

ABSTRACT:

TECHNICAL FIELD
The invention relates to the field of protein engineering and the glycoprotein hormones which occur normally as heterodimers. More specifically, the invention concerns single-chain forms of chorionic gonadotropin (CG), thyroid stimulating hormone (TSH), luteinizing hormone (LH), and follicle stimulating hormone (FSH).
BACKGROUND ART
In humans, four important glycoprotein hormone heterodimers (LH, FSH, TSH AND CG) have identical &agr; subunits and differing &bgr; subunits. Three of these hormones
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 of human chorionic gonadotropin or a variant thereof. Other muteins of these hormones are also described. The relevant positions for the CTP are 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 is not destroyed. In addition, U.S. Serial No. 08/049,869 filed Apr. 20, 1993, 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 the heterodimeric form of human chorionic gonadotropin has now 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.
At least one instance of preparing a successful single-chain form of a heterodimer is now known. The naturally occurring sweetener protein, monellin, is isolated from serendipity berries in a heterodimeric form. Studies on the crystal structure of the heterodimer were consistent with the proposition that the C-terminus of the B chain could be linked to the N-terminus of the A chain through a linker which preserved the spatial characteristics of the heterodimeric form. Such a linkage is advantageous because, for use as a sweetener protein, it would be advantageous to provide this molecule in a form stable at high temperatures. This was successfully achieved by preparing the single-chain form, thus impeding heat denaturation, as described in U.S. Pat. No. 5,264,558.
PCT application WO91/16922 published Nov. 14, 1991 describes a multiplicity of chimeric and otherwise modified forms of the heterodimeric 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. This construct falls outside the scope of the present invention since the presence of the signal sequence intervening between the &bgr; and &agr; chains fails to serve as a linker moiety as defined and described herein.
It has now been found that the normally heterodimeric glycoprotein hormones retain their properties when in single-chain form, including single-chain forms that contain the various CTP extensions and insertions described above.
DISCLOSURE OF THE INVENTION
The invention provides single-chain forms of the glycoprotein hormones, at least some of which hormones are found in most vertebrate species. The single-chain forms of the invention may either be glycosylated, partially glycosylated, or nonglycosylated and the &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 only as a portion thereof, as well as shorter linkers of 1-16 amino acids. The resulting single-chain hormones either retain the activity of the unmodified heterodimeric form or are antagonists of this activity.
Thus, in one aspect, the invention is directed to a glycosylated or nonglycosylated protein which comprises the amino acid sequence of the &agr; subunit common to the glycoprotein hormones linked covalently, optionally through a linker moiety, to the amino acid sequence of the &bgr; subunit of one of said hormones, or variants of said amino acid sequences wherein said variants are defined herein.
The availability of single-chain forms preserves conformation so that the entire portions of the subunits that make up the single-chain forms are unnecessary. Thus, the invention includes single-chain forms of fragments of the subunits wherein the single-chain forms retain the biological activity exhibited by the single-chain forms of the complete subunits.
In other aspects, the invention is directed to recombinant materials and methods to produce the single-chain proteins of the invention, to pharmaceutical compositions containing them; to antibodies specific for them; and to methods for their use.


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Biosynthesis of a biologically active single peptide chain containing the human common alpha and chorionic gonadotropin beta subunits in tandem, Sugahara T. et al,Proc. of the Natl Acad of Sci of the U.S.., (Mar. 14, 1995) 92 (6) 2041-5.
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