MXPA01003947A - Multiple domain glycoprotein hormones and methods of using - Google Patents

Abstract

Forms of differentially acting glycoprotein hormones are disclosed. These compositions are of the formula:(1):&bgr;1-(linker1)m-&agr;-(linker2)n-&bgr;2;(2):&bgr;1-(linker1)m-&bgr;2-(linker2)n-&agr;;(3):&agr;-(linker1)m-&bgr;1-(linker2)n-&bgr;2;(4):&bgr;2≈&agr;-(linker)m-&bgr;1;or (5):&bgr;1-(linker)m-&agr;≈&bgr;2 wherein each of&bgr;1 and&bgr;2 has the amino acid sequence of the&bgr;subunit of a vertebrate glycoprotein hormone or a variant of said amino acid sequence, as variants are defined herein."&agr;"designates the&agr;subunit of a vertebrate glycoprotein hormone or a variant thereof;"linker"refers to a covalently linked moiety that spaces the&bgr;1 and&bgr;2 subunits at appropriate distances from the&agr;subunit and from each other."≈"is a noncovalent link. Each of m and n is independently 0 or 1.

Description

GLUCOPROTE1CAS HORMONES WITH MULTIPLE DOMAINS AND METHODS OF USE RECOGNITION OF GOVERNMENT SUPPORT This invention was made in part with government support under contract number NIH NOI-HD-9-2922, awarded by the National Institutes of Health. The American government has certain rights in this invention.

FIELD OF THE INVENTION The invention relates to the field of protein engineering, specifically to the modified forms of certain glycoprotein hormones having native forms that normally occur as hedimers. The invention relates to multiple complex domains of chorionic gonadotropin (CG), thyroid stimulating hormone (TSH), luteinizing hormone (LH), and follicle stimulating hormone (FSH), wherein a subunit covalently linked to a β subunit can associate with an additional β subunit or can be covalently linked to two β subunits. These hormones with multiple glycoprotein domains can provide two or more effects or functions, or they can behave as agonists and / or antagonists of the native hormones.

RELATED BACKGROUND TECHNIQUE In humans, four important glycoprotein hormones (LH, FSH, TSH, and CG) are hedimers that have identical subunits and different β subunits. Three of these hormones are also present in virtually all vertebrate species; CG has so far been found only in primates and in the placenta and urine of pregnant mares. PCT application WO90 / 09800, published September 7, 1990, and incorporated herein by reference, describes a number of modified forms of these hormones. A major modification is the C-inal extension of the β-subunit by the carboxy-inal peptide (CTP) of human chorionic gonadotropin or a variant thereof. Other muteins of these hormones are also described. CTP is the amino acid sequence that extends from one of positions 112-118 to position 145 of the β subunit of human chorionic gonadotropin. The PCT application describes variants of the CTP extension obtained by conservative amino acid substitutions such as the ability of CTP to althe characstic elimination tests of the hormone that is not destroyed. In addition, PCT application W094 / 24148 published October 27, 1999, incorporated herein by reference, describes modifications to these hormones by extension or insertion of CTP at sites other than C-inal and shorCTP fragments than the sequence extending from positions 112-118 to 145. The extended CTP from the β subunit of FSH is also described in two articles by the applicants here: LaPolt, PS et al .; Endocrinology (1992) 131: 2514-2520 and Fares, F.A. et al .; Proc. Nati Acad Sci E.U.A. (1992) 89: 4304-4308. Both articles are incorporated here as references. The crystal structure of human chorionic gonadotropin has been published in more or less contemporary articles; one by Lapthorn, A.J. ef al. Nature (1994) 369: 455 ^ 61 and the other by Wu, H. et al. Structure (1994) 2: 545-558. The results of these articles are summarized by Patel, DJ. Nature (1994) 369: 438-439. PCT application W091 / 16922 published November 14, 1991 describes a multiplicity of chimeric and other modified forms of glycoprotein hormones. In general, the description focuses on the chimeras of subunits a or subunits β that involve the portions of several a or β chains respectively. A construct simply listed in this application, and not otherwise described, substantially fuses all β-chains of the human chorionic gonadotropin of the preprotein of subunit a, ie, including the secretory signal sequence for this subunit. Two additionally published PCT applications describe the single chain forms of these glycoprotein hormones wherein the a and β subunits are covalently linked to result in a compound of the general formula: β (linker) na; or a (linker) nß Where n is 0 or 1 and a and ß represent the respective subunits of these hormones: Moyle, W.R., PCT application WO95 / 22340 published on August 24, 1995 and the inventor's application herein, WO96 / 05224 published February 22, 1996. The description of these documents is also incorporated herein by reference. The pattern of single chain forms described above for these hormones in which the number of cystine bonds have been exhausted is described in E.U.A. serial number 08 / 933,693 filed September 19, 1997, and incorporated herein by reference. The a subunit of a single chain forms a glycoprotein hormone, CGß-a, which is found to be covalently linked to an FSHß subunit as described by the applicants in Society for the Study of Reproduction, Abstract 193, 1996. Recently, it was found that the a subunit of the single chain glycoprotein hormone, FSHß-a, forms a non-covalent bond with the GCß subunit as described by the applicants in Endocrine Society, Abstract OR28-3, 1998. It has now been possible to use these glycoprotein hormones which have enhanced agonist and / or antagonist activity and / or which are multifunctional either by the covalent attachment of an additional β subunit to a subunit to a single chain hormone that mimics a natural hormonal profile and / or controls the hormonal relationships These differentially active glycoprotein hormones and their therapeutic uses to treat disorders such as polycystic ovary disease, infertility, and ovarian hyperstimulation are described below.

DETAILED DESCRIPTION OF THE INVENTION The invention provides differentially acting glycoprotein hormones containing a subunit covalently linked to a β subunit to form a single chain hormone and an additional β subunit either covalently bound to the single chain hormone or non-covalently bound to the subunit a coupled to the single chain hormone. The compositions of the invention may be either glycosylated, partially glycosylated, or non-glycosylated and the α- and β-fused chains that occur in the native glycoprotein hormones or variants thereof may optionally be linked through a linker portion. Particularly preferred linker moieties include the carboxyterminal peptide unit (CTP) either as a whole unit or a variant including variants that represent only a portion thereof. If the β subunits are the same, the compositions containing a non-covalently linked subunit may act as agonists or antagonists, but the degree of activity may vary with time. This variation in activity is due to differences between the circulating half-lives of the ß covalently bound and non-covalently bound subunits. The circulating half-life of the non-covalently linked subunit will inherently be shorter than that of the β-subunit covalently bound to the subunit a. This is due to the dissociation of the complex over time in the physiological environment; however, the portion covalently bound to the molecule remains pharmaceutically effective. For example, a composition having a FSHß subunit covalently bound to a subunit that is not covalently linked to other FSHß subunit may have higher activity during the circulating half-life of the complex. However, the activity may decrease after the short half-life of the unattached FSHß subunits ends. A composition having an FSHß subunit covalently linked to a subunit that is not covalently linked to a CGβ subunit could exhibit a higher circulating half life for FSH activity and a lower circulating half life for CG activity. For the duration of the lower circulating half-life, both the FSHß and CGß subunits could act on their respective receptors. During the longer half-life, only the FSHß subunit covalently bound to the a subunit could be active.

In all cases, if the β subunits are different, the compositions are bifunctional as agonists and / or antagonists. It will be evident that differential activity relationships can be obtained by increasing or decreasing the agonist activity of one component relative to the other. For example, one can improve the FSH / LH ratio by using an FSH subunit that improves agonist activity and / or an LH subunit that decreases agonist activity. In one aspect, the invention is directed to a method for providing different activities of glycoprotein hormones to a subject in need of hormonal regulation. By an "activity" of glycoprotein hormone refers to the ability to behave as an agonist or antagonist of a corresponding native hormone with identical or different biological half-life. Thus, "two different activities of glycoprotein hormones" means that the activities conferred on the compositions for each subunit β differ in one or more ways. One can be an agonist, the other an antagonist; one can be modified in a way that provides improved activity; one can be modified in a way that provides diminished activity; one may correspond to the activity of LH and the other to that of FSH, or one may have a long circulating half-life and the other a short circulating half-life. Thus, by providing the different native β subunits in the composition of the formulas (1) - (5) or by providing variants of these β subunits, a wide variety of different hormone glycoprotein activities can be obtained. In another aspect, the invention is directed to a glycosylated or non-glycosylated protein of the formula: β1 - (linker1) m-a- (linker2) n-β2 (1); ß1 - (linker1) m-ß2- (linker) n-a (2); a- (linker1) m-ß1 - (linker2) n-ß2 (3); ß2 * a- (linker) m-ß1 (4); or ß '- (linker1) ma «ß2 (5) wherein each ß1 and ß2 has the amino acid sequence of the β subunit of a vertebrate glycoprotein hormone or of a variant of said amino acid sequence, wherein said variants are defined here, "a" designates the a subunit for a vertebrate glycoprotein hormone or a variant thereof; "linker" refers to a covalently linked portion that divides the β and β2 subunits at appropriate distances from subunit a and from each. "" "Is a non-covalent link. Each of m and n is independently 0 or 1. In all the above cases, the compositions of the invention preserve the conformation so that the inclusion of the complete subunits in the composition is not necessary. Thus, the invention includes compositions of formulas (1) - (5) comprising fragments of the α and / or β subunits wherein these forms retain the biological activity exhibited by the corresponding forms containing the complete subunits. It will be evident that the compounds of formulas (1) - (5) can further be modified to contain additional β-subunits covalently linked. Thus, the compounds of formulas (2) or (3) may not be covalently associated with an additional β subunit; the compositions of formulas (4) or (5) may contain additional β subunits in the covalent chain. In addition, other non-covalent associations may be employed, such as the ß-β dimers with a or an a-a dimer with β. In other aspects, the invention is directed to methods for producing the compositions of the invention, to pharmaceutical formulations containing the compositions of formulas (1) - (5), and to methods for their uses. Antibodies specific for these compositions are also included in the invention.

Modes for carrying out the invention Four "glycoprotein" hormones in humans provide a family that includes human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), luteinizing hormone (LH), and thyroid stimulating hormone (TSH) . As used herein, "glycoprotein hormones" refers to all members of this family as they occur in humans and in other vertebrates. All of these native hormones are heterodimers comprised of ct subunits which, for given species, are identical in amino acid sequence between groups, and β subunits that differ according to the members of the family. Thus, normally these native glycoprotein hormones occur as heterodimer compounds of subunits a and β that are associated but not covalently linked. Most vertebrates produce FSH, TSH and LH; Chorionic gonadotropin has been found only in primates, including humans, and pregnant mares. In animals, the a and ß subunits of each hormone are encoded in different genes and synthesized separately and then assembled into the non-covalent heterodimeric complex. In the compounds of the invention, at least one β subunit is directly linked to subunit a in a single chain primary structure. The three-dimensional conformation conferred by the secondary and tertiary structural considerations is sufficiently similar to the native heterodimeric form to allow the functionality of the glycoprotein hormone represented by the β subunit to be exhibited. An additional β subunit is linked to this particular chain either covalently (formulas (1) - (3)) or by a non-covalent bond of the subunit a coupled to an additional β subunit. By suitable variation of the structures of the β subunits, the compositions of the invention may have agonist and / or antagonist activity "corresponding" to that of the native hormone; for example, the compounds may exhibit antagonist activity with respect to one receptor for one of the glycoprotein hormones, but agonist activity for the receptor for another, or they may have agonist or antagonist activity for both. The spectrum of activities exhibited by the compounds of the invention will depend on the selection of the individual subunits a and β and the variants employed as well as on the nature of the linker portions and the orientation of the a and β subunits. In the compounds of formulas (1), (2) or (3), all three subunits are covalently linked; the compositions of formulas (4) and (5) contain a single chain dimer β-a or a-β covalently linked. The covalent binding in each case is proximal to the N- or C-terminal of each subunit and can, in the case of any pair of subunits, be head-to-head (ie, proximal to the N-terminus of both components), cola-acola (ie, proximal to the C-terminus of both components), or, more preferably, head-to-tail, wherein the N-terminus of one subunit is covalently linked to the C-terminus of the other. The fusion proteins comprising the head-to-tail junctions can be easily prepared using standard recombination techniques by providing all the amino acids of the subunits and any linkers encoded by the gene. Alternatively, the compounds of the invention can be prepared synthetically in which case, in addition to the head-to-tail configuration, linkers can be used to attach the proximal subunits to their respective terminals. Bifunctional linkers include both heterobifunctional and homobifunctional types, and are available from Pierce Chemical Company, Rockford, Illinois. Linkers that provide the ability to link two amino groups, or two carboxyl groups, or a carboxyl group and an amino group are available. If the binding is not precise at the N-terminus, an amino acid that provides a functional group containing side chain will be required in the proximal position of the terminal to be bound. A) Yes, in preferred embodiments of the invention, the compounds of formulas (1), (2) or (3) are fusion proteins wherein subunits a and ß are linked head-to-tail either directly or through peptide linkers, in where only the amino acids encoded by the gene comprise the sequence. These can be synthesized recombinantly. In another preferred embodiment of the invention, the compositions of formulas (4) and (5) comprise a single-chain form where subunits a and β are joined head-to-tail either directly or through peptide linkers and a β subunit. additional non-covalently bound to the coupled subunit, wherein only the amino acids encoded by the gene comprise the multiple domain complex. This complex, too, can be recombinantly synthesized. However, it is not necessary to restrict the compositions of the invention in this way; the a and ß subunits as well as the linkers may include amino acids that are not encoded by the gene. In addition, the linkers may be different from peptides such as dicarboxylic acids or anhydrides, diamines, or bifunctional linkers such as those sold by Pierce Chemical Co., Rockford, IL and the like. In addition, units in the form of a single chain can be linked either directly or through a linker in a head-to-head or tail-to-tail configuration as well as head-to-tail configuration as may be required in the protein of fusion. Under these circumstances, for a head-to-head configuration, two amino groups can be linked through an anhydride or through any dicarboxylic acid derivative; two carboxyl groups can be linked through diamines or diols using standard activation techniques. However, for convenience the most preferred form is a head-to-tail configuration wherein the standard linker peptide is sufficient and the single chain form can be prepared as a fusion protein recombinantly or using synthetic peptide techniques either in a sequence single reactions or, preferably, ligand individual portions of the entire sequence. Whatever the mode, the subunits a and ß are joined to the remainder of the molecule at the positions proximal to the N and C terminals. It is preferred that these subunits are attached directly at their terminals, however this junction can simply be "proximal" . In general, "proximal" indicates a position that is within the 10 amino acids, preferably within the 5 amino acids, more preferably within the two terminal amino acids, and more preferably in the terminal per se. As evidenced above, where the junction is different from that of the N- or C-terminal, a side-chain functional group can be provided in the proximal position of the appropriate terminal.

The components of the subunit As used herein, the common subunit, and the subunits FSH, LH, TSH and CG β as well as the compositions of the invention have their conventional definitions and refer to proteins having amino acid sequences known in the art. technique per se, or allelic variants thereof, without considering the glycosylation pattern exhibited or other derivations of the amino acid side chains. The "native" forms of these peptides are those that have the amino acid sequences as isolated from the relevant vertebrate tissue, and have these sequences known per se, or others of allelic variants. "* The" variant "forms of these proteins and the CTP subunits are those that correspond to the native subunit but have deliberate alterations, including truncations, in amino acid sequences of the native protein, produced by, for example, site-specific mutagenesis or by other recombinant manipulations, or which is prepared synthetically The resulting "variants" can behave as agonists or antagonists.Agonists can have improved activity compared to the native form or decreased activity.As the activity level is adjusted in the two ß subunits included in the compositions of the invention, variations in effective hormone ratios can be achieved.For example, by supplementing an LH activity with decreased activity but a FSHß subunit with native or enhanced activity, the ratio of FSH / LH activity can be improved. alterations that result in "variants" consisting of 1-10, preferably 1-8 and more preferably 1-5 amino acid changes, including deletions, insertions, and substitutions, more preferably conservative amino acid substitutions. The resulting variants must retain an activity that affects the corresponding activity of the native hormone, that is, they must retain either the biological activity of the native hormone to which they correspond in order to behave as agonists, or they must behave as antagonists, generally in virtue of its ability to bind receptors to native hormones but lacking the ability to effect signal transduction. "Conservative substitution" means, in the conventional sense, a substitution in which the substituted residue is of the same general category of amino acids from which the substitution was made. Amino acids have been classified in such groups, as understood in the art, by, for example, Dayhoff, M, et al., Atlas of Protein Sequences and Structure (1972) 5: 89-99. In general, acidic amino acids fall within a group; the basic amino acids in another; the neutral hydrophilic amino acids within another; and so on. The more specific classifications are set forth in WO96 / 05224 unofficially referred to above.

A set of preferred variants is one in which the glycosylation sites of either the a or β subunits or both have been altered. Some useful variants of the hormonal quartet described herein are set forth in the U.S. patent. No. 5,177,193 issued January 5, 1993, and incorporated herein by reference. As shown here, glycosylation patterns can be altered by destroying the relevant sites or, alternatively, by choosing a host cell in which the protein is produced. Alterations of the amino acid sequence also include both insertions and deletions. Thus, truncated forms of the hormones are included among the variants, for example, mutants of the subunit a that lack some or all of the amino acids at positions 88-92 at the C-terminus. In addition, subunits a with 1-10 amino acids deleted from the N-terminus are included. The variants also include those with non-critical regions altered or removed. Said deletions and alterations may include whole loops, so that sequences of considerably more than 10 amino acids can be deleted or changed. The resulting variants must, however, retain at least the receptor binding domains with or without the regions involved in signal transduction. There is considerable literature on glycoprotein hormone variants and it is clear that a large number of possible variants can be prepared which result in both agonist and antagonist activity. Such variants are described, for example, in Chen, F. et al. Molec Endocrinol (1992) 6: 914-919; Yoo, J. et al. J. Biol Chem (1993) 268: 13034-13042; Yoo, J. et al. J Biol Chem (1991) 266: 17741-17743; Puett, D. et al Glvcoprotein Hormones, Lusbader, J. W. et al. EDS, Sprinqer Verlag New York (1994) 122-134; Kuetmann, H.T. et al. (ibid.) pages 103-117; Erickson, L.D. et al. Endocrinology (1990) 126: 2555-2560. and Bieliska, M. et al J Cell Biol (1990) mi 330a (Summary 1844). Other variants include those in which one or more cystine bonds are deleted, typically by substitution of a neutral amino acid by one or both of the cysteines involved in the binding. Particularly preferred cystine bonds that can be deleted are those between positions 26 and 110 and between positions 23 and 72. Furthermore, it has been shown that the β subunits of the hormonal quartet can be constructed in chimeric forms so as to provide biological functions of both components of the chimera, or, in general, hormones of altered biological function. Thus, chimeric molecules that exhibit both FSH and LH / CG activities can be constructed as described by Moyle, Proc Nati Acad Sci (1991) 88: 760-764; Moyle, Nature (1994) 368: 251-255. As described in these articles, replacing amino acids 101-109 of FSH-β with the corresponding residues of the CG-β subunit produces an analog with both hCG and FSH activities.As used herein, "peptides" and "proteins" are used interchangeably, since the length distinction between them is arbitrary. As stated above, the "variants" employed as subunits a and β in the formation of the compound of the invention with or without binding portions may represent the complete amino acid sequences of the subunits or only portions thereof. The "variants" also include α- and / or β-chains that contain a CTP (or a variant of CTP) inserted within a non-critical region. The "variants" can be agonists or antagonists of the hormone containing the corresponding native β subunit ie a "variant" of the LHß subunit that will confer LH agonist or antagonist activity. The agonist activity may be the same as that of the native β subunit or may be improved or decreased. The "non-critical" regions of the a and ß subunits are those regions of the molecules that are not required for biological activity (including agonist and antagonist activity). In general, these regions are removed from the binding sites, precursor breaking sites, and catalytic regions. Critical regions should be evaluated to induce proper folding, binding to receptors, catalytic activity and the like. It should be evident that some of the regions that are critical e? the case of the interaction a and ß in the dimer become non-critical in units of a single chain since the conformational restriction imposed by the molecule can obviate the need for these regions. The determination of non-critical regions is easily achieved by deleting or modifying candidate regions and conducting an appropriate assay for the desired activity. The regions where the modifications result in loss of activity are critical; the regions where the alterations result in the same or similar activity (including antagonistic activity) are considered non-critical. It should be emphasized again that by "activity" reference is made to the activity either agonist or antagonist of the corresponding native hormones. Thus, certain regions are critical for the behavior of a variant as an antagonist, even when the antagonist is unable to directly provide the physiological effect of the hormone. For example, for the subunit a, it is thought that the positions 33-59 are necessary for the transduction of the signal and the row of 20 amino acids at the carboxyl terminus is needed for the signal / receptor transduction binding. The critical residues for the assembly with the β subunit include at least residues 33-58, particularly 37-40. Where the non-critical region is "proximal" to N or C terminal, the insertion is anywhere within the terminal 10 amino acids, preferably within 5 amino acids, and more preferably in the terminal per se. As used herein, the "CTP unit" refers to an amino acid sequence that is located on the carboxyl terminal of the β-subunit of human chorionic gonadotropin extending from amino acids 112-118 to residue 145 at the C-terminal or a portion thereof. Thus, each "complete" CTP unit contains 28-34 amino acids, depending on the N-terminus of the CTP. By a "partial" CTP unit it refers to a sequence of amino acids occurring between positions 112-118 to 145 inclusive, but which has at least one amino acid deleted from the shortest "complete" CTP unit possible (i.e. from positions 118-145). These "partial" sequences are included in the definition of "variants". The "partial" CTP units preferably contain at least one O-glycosylation site. The CTP unit contains four glycosylation sites in the serine residues at positions 121 (site 1); 127 (site 2); 132 (site 3); and 138 (site 4). The partial forms of useful CTP in the agonists will contain one more of these arranged sites in the order in which the native CTP sequence appears, although the intervention sites can be omitted. Some non-glycosylated forms of the hormones are antagonists and are useful as such. In some cases, CTP units can be inserted or used as tandem linkers. By "tandem" inserts or extensions it is understood that the insert or extension contains at least two "CTP units". Each CTP unit may be complete or a fragment, and may be native or a variant. All CTP units in the tandem or insert extension may be identical, or may be different from each other. The "linker" is a portion that binds the a and ß sequences without interfering with activity that could otherwise be exhibited by the same α and β chains as members of a hormone, or which alters that activity to convert it from agonist to antagonist activity. The level of activity can change within a reasonable range, but the presence of the linker can not be such that it deprives the hormone of a single chain of substantial agonist or substantial antagonist activity. The single-chain forms must exhibit activity relevant to the hormonal activity of the native hormones, the elements that make up their components. As used herein, "*" or "non-covalent bond" means a non-covalent bond that exists between the subunit covalently linked to the β1 subunit and an additional β2 subunit.

Preferred Modalities of the Glycoprotein Hormones Which Act Differently The compounds of the invention are produced more efficiently and economically using recombinant techniques. Therefore, single chain proteins comprising those forms of the α and β chains, CTP units and other linker portions that include only the amino acids encoded by the gene are preferred. It is therefore possible, as stated above, to construct at least portions of the single chain hormones using synthetic peptide techniques or other organic synthesis techniques and therefore variants within the scope of the invention are also within the scope of the invention. amino acids not encoded by the gene and linkers not based on peptides. In a preferred embodiment, the C-terminus of the β1 subunit is covalently linked, optionally through a linker, to the N-terminus of the mature subunit which in turn is optionally covalently linked through an aa linker the ß2 subunit. The linkage can be a direct peptide bond where the C-terminal amino acid of a subunit is directly linked through a peptide bond to the N-terminus of the other; however, in many examples it is preferred that a linker portion be included between the two terminals. In many cases, the linking portion will provide at least one ß-turn between the two chains. Therefore the presence of a proline residue in the linker can be advantageous. (It should be understood that when discussing the links between the terminals of the subunits comprising the form of a single chain, one or more terminals may be altered by substitution and / or deletion as described above). In a particularly preferred set of embodiments, the joint is head-to-tail and the linker portion will include one or more CTP units and / or variants or truncated forms thereof. The preferred forms of the CTP units used in said linker portions are described hereinafter. In addition to its occurrence in the linker portion, CTP and its variants can also be included in a noncritical region of the subunits making the single chain hormone as described above. While the CTP units are preferred inclusions in the linking portion, it is understood that the linker can be any covalently bound material that provides the appropriate spatial relationship between the a and ß subunits. Thus, for the head-to-tail configurations the linker can generally be a bivalent portion such as a peptide comprising an arbitrary number, but typically less than 100, more preferably less than 50 amino acids which has the appropriate proportion of hydrophilicity / hydrophobicity to provide the appropriate space and conformation in solution or a non-peptidic linker which confers these characteristics. In general, the linker must be in hydrophilic equilibrium to reside in the solution that surrounds it and outside the path of interaction between subunits a and β or the two subunits β. It is preferable that the linker includes β-turns typically provided by the proline residues in the peptide linkers, or comprising serine and / or glycine residues. Any suitable polymer, including peptide linkers, can be used with the correct characteristics described above. Particularly preferred embodiments of the single chain forms of the invention include a head-to-tail configuration: ßFSH-a-ßFSH; a-ßFSH-ßLH; ßFSH-a-ßLH; ßLH-a-ßLH; a-ßLH-ßFSH; ßLH-a-ßFSH; ßTSH-a-ßTSH; ßTSH-ßFSH-a; ßTSH-a-ßFSH; ßCG a-ßCG; a-ßCG-ßFSH; a-ßCG-ßTSH; ßCG-ßFSH-a; ßCG-a-ßTSH; ßFSH-CTP-a ßFSH; a-ßFSH-CTP-ßLH; ßFSH-CTP-a-ßLH; ßLH-CTP-a ßLH; a-ßLH-CTP-ßFSH; ßLH-a-CTP-ßFSH; ßLH (d115-123) -a- ßFSH; ßLH (d115-123) -CTP-a-ßFSH; ßCG-CTP-a CTP-ßFSH-CTP-CTP; ßTSH-CTP-CTP-a ßFSH-CTP-CTP; ßFSH-CTP-CTP-a-ßLH; ßLH-CTP-CTP-ßLH-a; ßCG-CTP-CTP-a-ßTSH; ßCG-CTP-CTP-ßLH-a; ßFSH-CTP-ßLH (d115-123) -CTP-a; and similar. Also particularly preferred are the human forms of the subunits. In the above constructions, "CTP" refers to CTP or its variants including truncations as described in PCT application WO96 / 05224. In one embodiment, the C-terminus of the β1 subunit is covalently linked, optionally through a linker, to the N-terminus of the mature subunit which in turn is not covalently linked to an additional β2 subunit. The α and β subunits in the single chain form of the present invention allow non-covalent attachment of an additional β subunit to the coupled subunit. Particularly preferred embodiments of the compositions of formulas (4) and (5) for use in the method of the invention include tail-to-head configurations (for the single chain component): ßFSH-a * ßFSH; ßFSH-a »ßCG; ßFSH-a «ßLH; ßFSH-a * ßTSH ßCG-a * ßCG; ßCG-a * ßFSH; ßCG-a * ßLH; ßCG-a * ßTSH ßLH-a * ßLH; ßLH-a * ßFSH; ßLH- < x «ßCG; ßLH-a * ßTSH ßTSH-a * ßTSH; ßTSH-a * ßCG; ßTSH-a «ßLH; ßTSH-a * ßFSH; ßFSH «a-ßFSH; ßCG * a-ßCG; ßLH * a-ßFSH; ßTSH «a-ßFSH; ßCG * a-ßCG; ßFSH * a-ßCG; ßLH «a-ßCG; ßTSH * a-ßCG; ßLH * a-ßLH; ßFSH * a-ßLH; ßCG «a-ßLH; ßTSH * a-BLH; ßTSH «a-ßTSH; ßCG * a-ßTSH; ßLH »a-ßTSH; ßFSH * a-ßTSH and the like. Therefore, in one embodiment of the invention, the coupled β subunit and the additional β subunit will differ from one another. For example, if the coupled β subunit is a β subunit of FSH or a variant and the non-covalently linked β subunit is the β subunit of CG or a variant, the resulting compound will have the ability to act on either the FSH and the CG receptors simultaneously. The non-covalently linked subunit can have agonist or antagonist activity, independently of the activity of the coupled β subunit. Additionally, the additional β subunit may have a different circulating half life than that of the coupled β subunit. This difference in the circulating half-lives of the β subunits allows variations in the degree of activity with respect to time. Another preferred embodiment of the invention is when the additional β subunit and the coupled β subunit are the same or variants of each. For example, a covalently linked FSHß subunit and an additional non-covalently bonded βFSH subunit. A modality of this type could have the effect of increasing the agonist or antagonist activity. The activity could increase during the duration of the short circulating half-life. Due to the longer circulating half-life of the coupled β subunit (when the β subunits are otherwise identical), when the non-covalently bound β subunit is no longer effective, the single-chain form will still be active but to a lesser extent . Another embodiment of the invention is when a β subunit is mutated to reduce or increase the activity of the other β subunit. For example, a ß-coupled subunit having an LH antagonist activity in combination with the β-subunit having an FSH agonist activity could have the effect of increasing the FSH / LH ratio suitable for follicle development and fertility. If a shorter circulating half-life of LH activity is desired, then the ß-coupling subunit may have FSH activity and the other β-subunit may have LH activity.

While for human use, the human forms of the a and ß subunits are desired, it should be demonstrated that the forms that correspond to other vertebrates are useful in veterinary contexts. Thus, the FSH, TSH and LH subunits characteristic of bovine, ovine, equine, porcine, feline, canine, and other species are appropriate for indications that affect these species per se. In some embodiments, an additional drug may be included in the linker portion. Such drugs can be peptides or products such as insulin-like growth factors; epidermal growth factors; acidic and basic fibroblastic growth factors; growth factors derived from platelets; the various stimulating factors of the colony, such as CSF granulocytes, CSF macrophages, and the like; as well as the various cytokines such as IL-2, IL-3 and the plethora of additional interleukin proteins; the various interferons; the tumor necrosis factor; and similar. Suitable break sites for the release of these drugs can be included, such as target sequences for proteases whose target sequences are not present in the a and beta subunits. Peptide or protein-based drugs have the advantage that the total construct can be easily produced by recombinant expression of a single gene. Small molecule drugs such as antibiotics, anti-inflammatories, toxins and the like can also be used.

In general, the drugs included within the linker portion will be those desired to act in the vicinity of the receptors to which the hormones ordinarily bind. Proper provision for the release of the drug from inclusion with the linker will be provided, for example, by also including sites for enzyme-catalyzed lysis as will be described in more detail under the section headed "Preparation Methods" below. In addition, if desired, the amount of time the drug is active and in circulation can be limited to the shortest circulating half-life of the non-covalently bound β-subunit. This can be achieved by including the drug within the non-covalently bound β subunit instead of the single chain form.

Other modifications The compounds of the invention can further be conjugated or derivatized in a generally understood form for derivatized amino acid sequences, such as phosphorylation, glycosylation, deglucosylation of ordinarily glycosylated forms, acylation, modification of amino acid side chains (eg, proline conversion) to hydroxyproline) and similar modifications analogous to those post-translational events which have been found to occur generally. The glycosylation status of the hormones of the invention is particularly important. Hormones can be prepared in non-glycosylated form either by producing them in prokaryotic hosts or by mutating the glycosylation sites normally present in the subunits and / or any CTP unit that may be present. Both non-glycosylated and partially glycosylated versions of the hormones can be prepared by manipulating the glycosylation sites. Normally, glycosylated versions are, of course, also included within the scope of the invention. As is generally known in the art, the compounds of the invention can also be coupled to labels, carriers, solid supports, and the like, depending on the desired application. The marked forms can be used to trace their metabolic fate; markers suitable for this purpose include, in particular, radioisotope labels such as iodide 131, technetium 99, indium 111, and the like. Markers can also be used to mediate the detection of single chain proteins in assay systems; in this case, radioisotopes can also be used as well as enzymatic markers, fluorescent labels, chromogenic labels, and the like. The use of said markers allows the location of the relevant receptors since they can be used as target agents for said receptors. The compounds of the invention can also be coupled to vehicles to improve their immunogenicity in the preparation of antibodies specifically immunoreactive with these new modified forms. Suitable vehicles for this purpose include keyhole lock key (KLH) hemocyanin, bovine serum albumin (BSA) and diphtheria toxoid, and the like. Standard coupling techniques for binding the modified peptides of the invention to vehicles include the use of bifunctional linkers, which may be employed. Similar adaptation techniques may be employed, along with others, to couple the proteins of the invention to said supports. When coupled, these proteins can be used as affinity reagents for the separation of desired components with which specific reaction is exhibited. Thus, they are useful in the purification and isolation of the receptors with which the β subunit interacts.

Methods of Preparation Methods for building the compounds of the invention are well known in the art. As stated above, only the amino acids encoded by gene are included, and the single-chain forms are in a head-to-tail configuration, the most practical method so far is to synthesize these materials recombinantly by the expression of DNA encoding the protein or proteins desired. The DNA containing the nucleotide sequence codes for the single chain forms included in the compositions of the invention, including variants, which can be prepared from native sequences, or synthesized de novo or using combinations of these techniques. Techniques for site-directed mutagenesis, ligation of additional sequences, amplification such as by PCR, and construction of suitable expression systems are all, up to now, well known in the art. Portions or all of the DNA encoding the desired protein can be constructed synthetically using standard solid phase techniques, preferably including restriction sites to facilitate ligation. The control elements suitable for the transcription and translation of the coding sequences that are included can be provided by the coding sequences of the DNA. As is well known, expression systems are currently available and are compatible with a wide variety of hosts, including prokaryotic hosts such as E. coli or B. subtilis and eukaryotic hosts such as yeast, other fungi such as Aspergillus and Neurospora, cells plants, insect cells, mammalian cells such as CHO cells, bird cells, and the like. The choice of the host is particularly relevant to post-translational events, more particularly including glycosylation. The localization of glycosylation is controlled mainly by the nature of the glycosylation sites within the molecule; however, the nature of the sugars that occupy this site is controlled mainly by the nature of the host. Accordingly, a fine adjustment of the properties of the hormones of the invention can be achieved by the proper choice of the host. A particularly preferred form of the gene of the subunit a portion, wherein the a subunit is modified or unmodified, is the "minigene" construct. As used herein, the a subunit of the "mipigen" refers to the gene construct described in Matzuk, MM, et al., Mol Endocrinol (1988) 2: 95-100, in the description of the construction of pM2 / CGa or pM2 / a. For recombinant production, modified host cells using the expression system to produce the desired protein are used and cultured. These terms are used herein as follows: A "modified" recombinant host cell, i.e., a "modified" cell containing the recombinant expression system of the invention, refers to a host cell that has been altered to contain this system of expression by any convenient way to introduce it, including transfection, viral infection, and others. "Modified cells" refer to cells that contain this expression system where the system is integrated into the chromosome or is extrachromosomal. The "modified cells" can be either stable with respect to the inclusion of the expression system or the coding sequence can be expressed transiently. Briefly, recombinant host cells "modified" with the expression system of the invention refers to cells that include this expression system as a result of their manipulation to include it, where they do not contain it in a native manner, regardless of the way to effect this incoforation. The "expression systems" refer to a DNA molecule that includes a coding nucleotide sequence that is expressed and those control sequences that accompany it necessary to effect the expression of the coding sequence. Typically, these controls include a promoter, regulatory sequences of the termination, and, in some cases, an operator or other mechanisms to regulate expression. The control sequences are those that are designated as functional in a recombinant host cell that is a particular target and therefore the host cell must be chosen to be compatible with the control sequences in the constructed expression system. Generally, the secretion of the produced protein is desired. Thus, nucleotide sequences encoding a signal peptide are also included so as to produce the signal peptide operably linked to the desired single-chain hormone to produce the preprotein, which prior to secretion, breaks down to release the hormone from a single mature chain or the desired β subunit. Glycoprotein hormones are normally secreted proteins and the signal sequence included may be that associated with hormones per se or may be heterologous to it. Although not preferred, the intracellular production of hormones can be effected by proper manipulation of the coding genes. As used herein "cells", "cell cultures" and "cell lines" are used interchangeably without particular attention to nuances of meaning. Where the distinction between these is important, it will be clear from the context. Where anyone can apply, it is intended that all of them be included.

The protein produced can be recovered from lysate of the cells if it is produced intracellularly, or from the medium if it is secreted. Techniques for the recovery of recombinant proteins from cell cultures are well understood in the art, and these proteins can be purified using known techniques such as chromatography, gel electrophoresis, selective precipitation, and the like. With respect to the recombinant production in the compounds of formulas (1) - (3), a unique expression system comprising the nucleotide sequence encoding the compounds of these formulas will be employed. For the compositions of formulas (4) and (5), in general, two expression systems, both contained within the recombinant host, are preferably used. Thus, an expression system for the a- (linker) m-ß1 or ß1- (linker) ma portion of the compound will be constructed containing the nucleotide sequence coding for this single-chain peptide and a system will also be included in the cell. of additional expression encoding ß2. These two expression systems can be contained in a single vector, within the chromosome of the host cell, in separate vectors, or in one expression system that can reside on the chromosome and the other in an extrachromosomal replication vector. Alternatively, a dicistronic expression system may be employed which contains both the required coding nucleotide sequences, either in an extrachromosomal replication vector or contained within the chromosome of the host cell. In yet another method, the two components of the non-covalent bond can be prepared separately and associated under suitable conditions in vitro. The conditions that favor the assembly of the compositions of formulas (4) or (5) may be familiar to those skilled in the art and could mimic intracellular conditions. In addition, all or a portion of the compounds of the invention can be synthesized directly using peptide synthesis techniques known in the art. The synthesized portions can be ligated, and release sites for any drug contained in the linker portion can be introduced by standard chemical means. For those modalities that contain amino acids that are not encoded by the gene and those modalities where the head-to-head or tail-to-tail configurations are used, of course, the synthesis must be at least partially at the protein level. Head-to-head and N-terminal natural or proximal N-terminal positions can be made through linkers containing functional groups reactive with amino acids, such as dicarboxylic acid derivatives. The head-to-head configurations in the C-terminal position or in positions proximal to the C-terminal can be effected through linkers that are diamines, diols, or combinations thereof.

Antibodies The proteins of the invention can be used to generate antibodies specifically immunoreactive with the multiple domain glycoprotein hormones described herein. These anticuefos are useful in a variety of diagnostic and therapeutic applications. The antibodies are generally prepared using standard immunization protocols in mammals such as rabbits, mice, sheep, or rats, and the antibodies are titrated as a polyclonal antibody to ensure adequate immunization. The polyclonal antiserum can be harvested as such for use in, for example, immunoassays. Cells that secrete antibodies from the host, such as spleen cells, or peripheral blood leukocytes, can be immortalized using known techniques and screened for the production of monoclonal antibodies immunospecific with the proteins of the invention. The "antibodies", which can be of any animal species, including humans, include any fragment that retains the required immunospecificity, such as Fab, Fab-, or F (ab-) 2 Fv and others. Thus, the antibodies can also be prepared using recombinant techniques, typically by isolating nucleotide sequences encoding at least the variable regions of the monoclonal antibodies with appropriate specificity and appropriate constructs of expression systems. This method allows any desired modification such as the production of Fv forms, chimeric forms, "humanized" forms, and the like. By "immunospecificity" of the proteins of the invention "refers to antibodies that specifically bind to the subject compounds of the invention, but not to the native glycoprotein hormones or to any of the subunits included per se or any of the units of a single chain which includes only a subunit ß within the general parameters considered to determine affinity or non affinity.Specificity is understood to be a relative term, and an arbitrary limit may be chosen, such as a difference in specific binding of 100- Thus, an immunospecific antibody included within the invention is at least 100-fold more reactive with the multiple domain complex than with the corresponding native hormone, single chain form in the prior art or separate subunits. they can be obtained, for example, by selecting for those that bind the compounds of the invention and discarding those that also bind to the native hormones, subunits or single-chain forms of the prior art set forth in WO95 / 22340 and WO96 / 05224.

Formulation and methods of use The proteins of the invention are formulated and administered! methods comparable to those known for the heterodimers that generally correspond. Thus, the methods of formulation and administration vary according to the particular hormone or combination of hormones used. However, the dose level and frequency of administration can be altered in comparison with the native heterodimers, especially if the CTP units are present in view of the extended biological half-life due to their presence.

Formulations for the proteins of the invention are those typical for protein or peptide drugs such as those found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Company, Easton, PA. Generally, proteins are administered by injection, typically, intramuscularly, subcutaneously, or intraperitoneally, or using formulations for transmucosal or transdermal administration. Other modes for administration such as suppositories can also be employed. These formulations also include a detergent or penetrant such as bile salts, fusidic acid, and the like. These formulations can be administered as aerosols or suppositories in the case of transdermal administration, in the form of skin patches. Oral administration is also possible by providing the formulation with peptide protectors of the invention from the degradation in the digestive system. The optimization of the dosage regimen and formulation is conducted as a routine matter and has been generally carried out in the art. These formulations can also be modified to include those common veterinary uses. The compositions of the invention can be used in many forms, most obviously as substitutes for the native forms of the hormones. Thus, the compositions of the invention can be used in the treatment of infertility, as an aid to in vitro fertilization techniques and other therapeutic methods associated with the native hormones or their subunits. These techniques are applicable for humans as well as for other animals. The choice of composition in terms of its derivation of species will depend, of course, on the subject to whom the method will be applied. One will realize that the ability to act differentially that is conferred to the compositions of the invention confers opportunities for therapies that were previously unavailable. The compositions of the invention are also useful as reagents in a manner similar to that employed with respect to native heterodimers. In addition, the compounds of the invention can be used as diagnostic tools to detect the presence or absence of antibodies that bind to native proteins to the extent that antibodies bind to the relevant portions of these multiple domain compounds in biological samples. . They are also useful as control reagents in test equipment to assess the level of these hormones in various samples. The protocols for evaluating the levels of the hormones themselves or of the antibodies raised against them are standard immunoassay protocols commonly known in the art. Various competitive methods and direct assay method can be used that involve a variety of labeling techniques including radioisotope labeling, fluorescent labeling, enzyme labeling and the like. The compounds of the invention are also useful for detecting and purifying receptors to which the native hormones bind. Thus, the compounds of the invention can be coupled to solid supports and used in affinity chromatographic preparations for antihormonal receptors or anticuefos. The resulting receptors themselves are useful for evaluating hormone activity for candidate drugs to select therapeutic trials and candidate reagents. Of course, the dual specificity of the β subunits in any of these compounds where the β subunits are different should be taken into account. Nevertheless, where the two β subunits are identical, they offer a powerful affinity purification tool for the relevant receiver. Finally, antibodies only reactive with the compounds of the invention can be used as purification tools to isolate these materials in their subsequent preparations. These can also be used to monitor the levels of these compounds administered as drugs. The following examples are intended to illustrate but not limit the invention.

EXAMPLE 1 Preparation of CGB-a-CTP-FSHB A nucleotide sequence encoding the titled compound was prepared using the nucleotide sequence available from the relevant portions of the subunits. The CGß region that encodes 145 amino acids of human CGβ; the subunit a that encodes the nucleotide sequence of 92 amino acids of a as a minigene; the sequence encoding CTP encoding 28 amino acids representing positions 118-145 of human chorionic gonadotropin; and the region encoding FSHß that encodes the 11 amino acids of the human FSHß subunit. An amplification fragment containing exon 3 CGβ, minigene, CTP and ßFSH was inserted into the Salí site of pM2HA-CGßexon1, 2 an expression vector derived from pM2 and containing exons 1 CGß and 2 from the manner described by Sacháis, ß Biol Chem (1993) 268: 2319. pM2 containing the CGß exons 1 and 2 is described in Matzuk, MM ef al. Proc Nati Acad USA (1987) 84: 6354-6358 and Matzuk, M.M. et al. J. Cell Biol (1988) 106: 1049-1059. First, a fragment containing the minigene to the 3 'end of CG 3 exon 3 was inserted into this vector to obtain pM2-HACGβ. pM2-HACGßa was then disrupted with Seal and ligated with pBIIKS (+) to-CTP-FSH restricted with Seal. The resulting expression vectors pM2-HACGß-a-CTP-FSH produce the titled compound when inserted into a suitable host.

EXAMPLE 2 Production and activity of CGß-a-CP-FSHß The expression vector constructed in Example 1 was transferred to Chinese hamster ovary (CHO) cells and the production of the protein was evaluated by immunoprecipitation of the radiolabeled protein on SDS gels. The culture medium was collected, concentrated and tested for human LH receptor binding (which was expected to bind to the ßCG-a moiety). For this assay, the LH receptor was prepared by inserting the cDNA encoding the entire human LH receptor into the pCMX expression vector (Oika a, J. XC ef al Mol Endocrinol (1991) 5: 759-768. 293 exponentially growing were transfected with this vector using the method of Chem, C. et al., Mol Cell Biol (1987) 7: 2745-2752, resulting in the expression of the LH receptor on the surface.In this assay, the cells expressing to the human LH receptor (2 x 105 / tube) were incubated with 1 ng of hCG in competition with incremental concentrations of unlabeled hCG or incremental amounts of the sample to be tested at 22 ° C for 18 hours. The presence of the samples measured the binding capacity in the sample In this assay, with respect to the human LH receptor in 293 cells, the heterodimeric hCG had a typical wild-type activity as previously determined and CGB-a-CTP-FSHβ -contained in the middle too showed activity These results are shown in Figure 1. As shown, both heterodimeric hCG (solid squares) and the single-chain bifunctional protein of the invention (solid circles) successfully competed with hCG for LH receptors. The bifunctional compound is less potent due to modification of the α-subunit at the carboxyl terminus. Also shown in Figure 1 are the test results where various amounts of a culture supernatant derived from cells modified to contain the two expression systems were evaluated. An expression system produced a single chain FSHß-a; the other produced the β subunit of hCG. The resulting non-covalently associated single-chain FSHa-β / CGß (solid triangles) complex also competed successfully for binding. Similarly, the supernatant of the culture medium containing CGß-α-CTP-FSHβ was evaluated for binding to the FSH receptor, expressed in 293 cells. The assay was conducted in the manner described above, except that the cells that Expressed the human FSH receptor were replaced by those expressing the human LH receptor and the labeled FSH was used as the competitor. The results of this test are shown in Figure 2. As shown, the single-chain compound (solid circle) successfully competed with FSH (solid square) for binding. In a non-related experiment, also shown in Figure 2, the mixture of a different type of complex ie, FSHβ-a from a single chain non-covalently associated with CGβ - which is mixed with an excess of FSHβ-a from a single chain that is not in complex (solid triangle) was an excellent competitor.

EXAMPLE 3 Construction of additional expression vectors In a manner similar to that established in example 1, the expression vectors for the production of FSHβ-CTB-α-CGβ; a- FSHß-CTP-CG ß, CG ß- ßFSH-CTP-a, and dysfunctional single-strand ßLH-CTP- ßFSH-CTP-α were prepared and transfected into CHO cells. Culture supernatants were cultured and evaluated as described above with respect to both LH and FSH receptors. These compounds also show ability to bind to both receptors.

EXAMPLE 4 Preparation of FSHß-a «CGB To create the compound, FSHß-a «CGß, expression vectors were prepared for the production of a single-chain FSHβ-a, and a CGβ subunit and were co-transfected into Chinese hamster ovary (CHO) cells in a manner similar to the methods described in the PCT application of the invention herein, WO96 / 05224 published February 22, 1996. The CGβ subunit combined with FSHβ-a to form the non-covalent complex FSHβ-a "CG. The production and activity of the FSHß-a complex "non-covalent CG was evaluated by immunoprecipitation of the radiolabelled protein on SDS gels. The culture medium was collected, concentrated and tested for the binding of the human LH receptor (which was expected to bind to the ßCG portion) and the human FSH receptor (which was expected to bind FSHß-a). The results indicate that the FSHß-a complex "non-covalent CG exhibits a binding for the specific receptor to CG and FSH. These data indicate that the a subunit of the complex, although covalently bound to the FSHß domain, can functionally interact with a different β subunit and the presence of this configuration does not prevent bioactivity. Another multiple domain complex such as ßFSH-a «ßLH, ßCG-a« ßLH, ßLH-a «ßTSH and ßTSH x« ßFSH can also be generated in a similar manner.

EXAMPLE 5 Use of compounds that act differently to regulate hormonal relationships Compounds that act differently are formulated and administered using methods comparable to those known for the native hormones corresponding thereto.

A. Increased fertility by increasing the FSH / LH ratio and / or increasing CG. It was possible to increase fertility by increasing the relationship FSH / LH with a compound of the formula (1) - (5) wherein one β subunit had a weaker LH agonist activity or an LH antagonist activity and the other β subunit had (optionally enhanced) FSH agonist activity. The LHß subunit can be modified to have less agonist or antagonist activity by eliminating glycosylation of the LHß chain or by point mutations, where the FSHß subunit can be used in its native or modified form to have a higher agonist activity. The resulting compound will have the ability to increase the hormone levels of FSH while simultaneously decreasing the hormone levels of LH when a therapeutic dose of this compound is administered to a mammal during the follicular phase of the menstrual cycle, which results in increased fertility. Additionally, it may be advantageous to make the circulating half-life of the FSH subunit longer than the circulating half-life of LHß. This is helped by using a compound FSHß-a «LHß. Increased fertility can also be achieved by increasing CG hormone levels and decreasing LH hormone levels by administering a compound of formula (1) - (5) wherein a β subunit has LH antagonist activity or LH antagonist activity and the other β subunit has CG agonist activity. Additionally, the CGβ subunit can be modified to have a binding affinity greater than LHβ for the CG / LH receptor. When administered at appropriate times and doses, the FSH / LH ratio will increase the favorable ratio for fertility and CG activity will also increase favorable pregnancy levels. Similarly, fertility can be increased by a compound of formula (1) - (5) wherein one β subunit has FSH agonist activity and the other β subunit has CG agonist activity. The administration of these complexes will increase the FSH / LH ratio favorable for fertility and will also increase the CG activity for favorable levels for pregnancy. It may also be advantageous to make the circulating half-life of a β subunit longer than the other, which is helped by using a composition of formula (4) or (5). B. Induction of infertility by decreasing the FSH / LH ratio and / or decreasing CG. Infertility can be induced by decreasing the FSH / LH ratio with the administration of a compound of the formula (1) - (5) wherein one β subunit has LH agonist activity and the other β subunit has FSH antagonist activity or reduced agonist activity. For this application, the LHß subunit can be modified to have improved agonist activity by altering the glycosylation of the LHß chain or by point mutations, where the FSHß subunit can be modified to have a lower agonist activity. The resulting compound will have the ability to increase LH activity while simultaneously decreasing FSH activity, thereby decreasing the FSH / LH ratio to a level not favorable for fertility. Infertility can also be induced by decreasing the FSH / LH ratio with the administration of a compound of the formula (1) - (5) wherein one β subunit has LH agonist activity and the other β subunit has CG agonist activity. The administration of this compound can result in a low FSH / LH ratio and a high hormonal CG level, both not favorable for activity and pregnancy. As mentioned above, it may be advantageous to make the circulating half-life of a β-subunit longer than the other, by using a composition of the formula (4) or (5). It may also be advantageous to vary the binding affinities of the β subunits. C. Treatment of polycystic ovary syndrome by decreasing the LH / FSH ratio. Polycystic ovarian syndrome is characterized by incomplete follicular development and abnormal ovulation. Women who suffer from this disease have elevated androgens and a high proportion of LH / FSH in relation to normal fertile women. Thus, at the time of the menstrual cycle when it is assumed that follicular development has started normally, the administration of a compound of the formula (1) - (5) wherein one β subunit has less LH agonist activity or antagonist activity and the other subunit β has FSH agonist activity will promote follicular development and will induce ovulation. Since the administration of an FSH agonist has the risk of causing hyperstimulation, it is preferable that the FSHß subunit is not covalently linked to the β subunit that has a shorter circulating half-life and / or the FSHβ subunit is manipulated to have an affinity of decreased union.

Claims (20)

1. NOVELTY OF THE INVENTION
2. CLAIMS ß2 «a- (linker) m-ß1 (4); or ß1- (linker) m-a «ß2 (5); wherein each of ß1 and ß2 have the amino acid sequence of the β subunit of a vertebrate glycoprotein hormone, or a variant thereof; "a" has the amino acid sequence of the a subunit of a vertebrate glycoprotein hormone or a variant thereof; "linker" is a linker portion; and "» "is a non-covalent union between a and ß2; each of m and n is independently 0 or 1; for the manufacture of a pharmaceutical composition for providing a subject with glycoprotein hormones of different activities, wherein each of β1 and β2 confers a different activity to said composition. 2. The use as claimed in claim 1, wherein ß1 and ß2 correspond to different native ß subunits.
3. 3. The use as claimed in claim 1, wherein ß1 and ß2 exhibit different biological half-lives.
4. 4. The use as claimed in claim 1, wherein one of ß1 and ß2 confers agonist activity and the other confers antagonistic activities.
5. 5. - The use as claimed in claim 1, wherein said subject needs improved fertility.
6. 6. The use as claimed in claim 5, wherein both ß1 and ß2 confer agonist activity to FSH in said composition: or wherein both ß1 and ß2 confer CG agonist activity; or wherein both β1 and β2 confer antagonistic activity to LH; or wherein one of ß1 and ß2 confers FSH agonist activity and the other confers LH antagonist activity or lower LH antagonist activity; or wherein one of ß1 and ß2 confers FSH agonist activity and the other confers CG agonist activity; or wherein one of ß1 and ß2 confers LH-antagonistic activity or less LH agonist activity and the other confers CG agonist activity.
7. 7. The use as claimed in claim 1, wherein said subject needs to become or remain infertile.
8. 8. The use as claimed in claim 7, wherein both β1 and β2 confer FSH antagonist activity in said composition; or wherein both ß1 and ß2 confer CG antagonist activity or where both ß1 and ß2 confer LH agonist activity; or wherein one of ß1 and ß2 confers FSH antagonist activity or lower FSH agonist activity and the other confers LH agonist activity; or wherein one of ß1 and ß2 confers FSH antagonist activity or reduced FSH agonist activity and the other confers CG antagonist activity or reduced CG agonist activity; or where one of ß1 and ß2 confers LH agonist activity and the other confers CG antagonist activity or less CG agonist activity.
9. 9. The use as claimed in claim 1, wherein the subject needs treatment for polycystic ovarian disease.
10. 10. The use as claimed in claim 9, wherein one of ß1 and ß2 confers FSH agonist activity and the other confers LH antagonist activity or LH agonist activity in said composition; or wherein both β1 and β2 confer FSH agonist activity; or where both ß1 and ß2 confer LH antagonist activity.
11. 11. A glycosylated or non-glycosylated composition of the formula ß2"a- (linker) m-ß1 (4); or ß - (linker) m-a «ß2 (5) wherein each of ß1 and ß2 has the amino acid sequence of the β subunit of a vertebrate glycoprotein hormone, or a variant thereof; "a" has the same amino acid sequence of the a subunit of a vertebrate glycoprotein hormone or a variant thereof; "linker" is a linker portion; and "» "is a non-covalent union between a and ß2; m is 0 or 1; wherein each of β1 and β2 confers a different activity in said composition; and with the proviso that if ß1 is CG then ß2 is not FSH.
12. 12. A pharmaceutical composition that regulates the concentrations of glycoprotein hormone in a mammal comprising an effective amount of the composition of the formula ß2"a- (linker) m-ß1 (4); or ß1- (linker) m-a «ß2 (5) wherein each of ß1 and ß2 has the amino acid sequence of the β subunit of a vertebrate glycoprotein hormone, or a variant thereof; V has the same amino acid sequence of the a subunit of a vertebrate glycoprotein hormone or a variant thereof; "linker" is a linker portion; and "» "is a non-covalent union between a and ß2; each of m or n is 0 or 1 independently; wherein each of β1 and β2 confers an activity different from said composition; and with the proviso that if ß1 is CG then ß2 is not FSH.
13. 13. A modified recombinant host cell containing a nucleic acid comprising an initial expression system comprising a nucleotide sequence encoding a- (linker) m-β1 or β1- (linker) ma operatively linked to a control sequence for the expression thereof and a nucleic acid comprising a second expression system comprising a nucleic acid sequence encoding β2 operably linked to a control sequence for the expression thereof; wherein a, β1, β2, linker and m are as defined in claim 11.
14. 14. Cells according to claim 13, further characterized in that the first expression system and the second expression system share the same control sequence .
15. 15. - The cells according to claim 13, further characterized in that the first expression system and the second expression system reside in separate extrachromosomal replication vectors.
16. 16. The cells according to claim 13, further characterized in that the first expression system and the second expression system reside on a chromosome of the host cell.
17. 17. The cells according to claim 13, further characterized in that one of said first and second expression systems resides in the chromosome of said cells and the other is in an extrachromosomal replication vector.
18. 18. The cells according to claim 13, further characterized in that the first and second expression systems reside in the same extrachromosomal replication vector.
19. 19. A method for producing a composition of formula (4) or (5) whose method comprises culturing the cells according to claim 13 under conditions wherein said composition is produced; and recovering said culture composition.
20. 20. The antibodies specifically immunoreactive with the composition according to claim 11.