MXPA00004761A - Single-chain bifunctional glycoprotein hormones - Google Patents

Abstract

Single-chain agonists and/or antagonists of the glycoprotein hormones are disclosed. These proteins are of the formulae:(1) b1-(linker1)m-a-(linker2)n-b2;or (2)b1-(linker1)m-b2-(linker2)n-a;or (3) a-(linker1)m-b1-(linker2)n-b2, wherein each of b1 and b2 has the amino acid sequence of the b subunit of a vertebrate glycoprotein hormone or a variant thereof;"a"designates the a subunit of a vertebrate glycoprotein hormone or a variant thereof;"linker"refers to a covalently linked moiety that the spaces b1 and b2 subunits at distances from the a subunit and from each other effective to retain said activity, and each of m and n is independently 0 or 1.

Description

SINGLE CHAIN BIFUNCTIONAL GLICOPROTEIN HORMONES Technical Field The invention relates to the field of protein design, specifically to the modified forms of certain glycoprotein hormones which are normally present as heterodimers. The invention relates to the modified single chain forms of chorionic gonadotropin (CG), thyroid stimulating hormone (TSH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) which it can provide two effects or functions, or they can generally behave as agonists and / or antagonists of the natural hormones.

Background of the Technique In humans, four heterodimers of glycoprotein hormones (LH, FSH, TSH and CG) have identical subunits and different β subunits. Three of these hormones are present in virtually the Ref .120095 totality of other vertebrate species as well; GC has already 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, discloses a number of modified forms of these hormones. One important modification is the C-terminal extension of the β subunit by the carboxy terminal peptide (CTP) of human chorionic gonadotropin or a variant thereof. Other muteins of these hormones are also described. TCC is the amino acid sequence extending from any of positions 112-118 to position 145 of the subunit of human chorionic gonadotropin. The PCT application describes the variants of the extension of CTP obtained by the conservative amino acid substitutions in such a way that the capacity of the CTP to alter the characteristics of the spacing is not destroyed. In addition, PCT application 094/24148 published October 27, 1994, incorporated herein by reference, describes the modification of these hormones by the extension or insertion of CTP at locations other than the C-terminus and the shorter CTP fragments that the sequence that extends from positions 112-118 to 145.

The extended β subunit of CTP is also described in two articles by the applicants here: LaPolt, P.S. et al .; Endocrinology (1992) 131: 2514-2520 and Fares, F.A. et al .; Proc. Nati Acad. Sci. USA (1992) ^ 9: 4304-4308. Both of these articles are incorporated here for reference. The crystal structure of the heterodimeric form of human chorionic gonadotropin has now been published in more or less contemporary 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. Na ture (1994) 369: 438-439. PCT application W091 / 16922 published November 14, 1991 describes a multiplicity of chimeric forms and forms modified in other ways of the heterodimeric glycoprotein hormones. In general, the description is focused on the chimeras of subunits a or subunits β that involve portions of several chains α or β respectively. A construction simply listed in this application, and not otherwise described, substantially fuses the entire ß chain of human chorionic gonadotropin to the preprotein of subunit a, ie, including the sequence of the secretory signal for this subunit. Two additional published PCT applications describe single chain forms of these hormones wherein units a and β are covalently linked to lead to a fusion peptide of the general formula: β (linker) na or (linker) n β where n is O olyay ß represent the respective subunits of these hormones: Moyle, WR, PCT application WO95 / 22340 published on August 24, 1995 and the inventor's application here, WO96 / 05224 published on February 22, 1996. The description of These documents are also incorporated here for reference. The forms of the single chain glycoprotein hormones described above in which the number of cysteine bridges has been depleted, are disclosed in US Application Serial No. 08 / 933,693 filed September 19, 1997, and incorporated herein by reference. It has now been found possible to construct single chain forms of the glycoprotein hormones which have improved agonist and / or antagonist activity and / or which are bifunctional, including two β subunits in a single chain so that they share a subunit a common. These forms may contain several extensions of CTP and insertions as well as variants of the natural forms of the α and β subunits and of the CTP as described in the documents described above.

Description of the invention The invention provides single chain forms of glycoprotein hormones that contain two β subunits that may be the same or different. The single chain forms of the invention may be either glycosylated, partially glycosylated, or non-glycosylated and the α and β chains which may be present in the natural glycoprotein hormones or variants thereof may optionally be linked through a portion linker Particularly preferred linker portions include the carboxy terminal peptide unit (CTP) either as a complete unit or a variant that includes variants that represent only a portion thereof. The resulting single-chain hormones either retain or enhance the activity of the non-modified heterodimeric forms or are antagonists of this activity. If the two β subunits are different, they are bifunctional as agonists and / or antagonists. Accordingly, in one aspect, the invention is directed to a glycosylated or non-glycosylated protein of the formula β1- (linker1) m-a- (linker2) n-β2 (1); or ß1- (linker'- -ß2- (linker2) na (2); or a- (linker ^ m-ß1- (linker2) n-ß2 (3) where each of ß1 and ß2 has the amino acid sequence of the β-subunit of a vertebrate glycoprotein hormone or a variant of the amino acid sequence, wherein said variants are as defined herein, "a" designates the a subunit of a vertebrate glycoprotein hormone or a variant thereof "linker" refers to a covalently linked portion that spaced the β1 and β2 subunits at the appropriate distances from the subunit au to each other, each of m and n independently 0 or 1. In all of the foregoing cases, the form of a single chain preserves the conformation so that the inclusion of the complete subunits in the form of a single chain is unnecessary. Accordingly, the invention includes compounds of formulas (1), (2) and (3) comprising the fragments of subunits a and / or ß where you are forms retain the biological activity exhibited by the corresponding forms which contain the complete subunits. In other aspects, the invention is directed to recombinant materials and methods for producing the proteins of the invention, to the pharmaceutical compositions containing them; to the specific antibodies for them; and the methods for its use.

Brief Description of the Drawings Figure 1 shows the binding of the compound CGß-a-CTP-FSHß to the LH receptor in competition with hCG. Figure 2 shows the binding of the compound shown in Figure 1 to the FSH receptor in competition with FSH.

Ways to Carry Out the Invention Four "glycoprotein" hormones in humans provide a family which includes human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), luteinizing hormone (LH), and thyroid stimulating hormone (TSH) . When used here, the "glycoprotein hormones" refer to all of the members of this family. All of these hormones are heterodimers comprised of the subunits to which, for a given species, they are identical in the amino acid sequence between the group, and the β subunits which differ according to the family member. Accordingly, normally these glycoprotein hormones are present as heterodimers composed of the a and β subunits that are associated but not covalently linked. Most vertebrates produce FHS, THS and LH; Chorionic gonadotropin has been found only in primates, including humans, and in pregnant mares. In animals, the a and ß subunits of each hormone are encoded in different genes and are synthesized separately and then assembled in the non-covalent heterodimeric complex. In the compounds of the invention the β subunits are directly linked to a subunit a in a single chain molecule which is essentially linear in its primary structure. The three-dimensional structure conferred by secondary and tertiary structural considerations and conformation is apparently sufficiently similar to the heterodimeric form to allow the functionality of the heterodimer represented by the β subunits to be displayed. Nevertheless, by suitable variation of the structures of the subunits, the compounds of the invention may have an agonistic or antagonistic activity; for example, if the β subunits are different, the compounds may exhibit an antagonist activity with respect to one receptor for one of the glycoprotein hormones but an agonist activity for the other receptor, or 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 α and β subunits as well as the nature of the linker portions and the orientation of the a and β subunits. In the most preferred embodiment of the invention, the compounds of formula (1), (2) or (3) are fusion proteins wherein subunits a and β are linked head to tail either directly or through the linkers of the peptides. Where only the amino acids encoded by the genes comprise the sequence, the compound can be recombinantly synthesized. However, it is unnecessary to restrict the compounds of the invention in this way; the a and ß subunits as well as the linkers can include amino acids that are not encoded by the genes. 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, the subunits can be linked either directly or through a linker in a head-to-head or tail-to-tail configuration as well as a head-to-tail configuration as might be required in a fusion protein. 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 reasons the most preferred form is a head-to-tail configuration wherein the standard peptide bonds are sufficient and the compound can be prepared as a fusion protein recombinantly or using synthetic peptide techniques either in a single sequence of reactions or, preferably, by ligating or joining the individual portions of the complete sequence. Whatever the modality, the subunits a and ß are joined to the rest of the molecule in the positions near their ends or terminations N and C. It is preferred that these subunits are linked directly in their terminations, however this link can be simply " next". In general, "near" indicates a position which is within 10 amino acids, preferably within five amino acids, more preferably within two amino acids of the terminus or extremity, and even more preferably at the extremity or termination per se.

The Components of the Subunit When used herein, the common subunit, and the β subunits of FSH, LH, TSH, and CG as well as the heterodimeric forms have their conventional definitions and refer to proteins having the amino acid sequences known in the art per se. , or the allelic variants thereof, regardless of the glycosylation configuration exhibited or other derivation of the amino acid side chains. The "natural" forms of these peptides are those which have the amino acid sequences that have been isolated from the relevant vertebrate tissue, and have these sequences known per se, or those of their allelic variants.

The "variant" forms of these proteins and of the CTP units (see below) are those which have deliberate alterations, including truncated portions, in the amino acid sequences of the natural protein produced, for example, by site-specific mutagenesis. site or by other recombinant manipulations, or which are prepared synthetically. These alterations consist 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 natural hormone - that is, either they must retain the biological activity of the natural hormone to behave as agonists, or they must behave as antagonists, generally in virtue of being able to bind to receptors for natural hormones but lack the ability to effect signal transduction. "Conservative analogue" means, in the conventional sense, an analogue in which the substituted residue is of the same general amino acid category as that for which the substitution is made. Amino acids have been classified into such groups, as understood in the art, for example, by Dayhoff, M. et al., Atlas of Protein Sequences and Structure (1972) 5: 89-99. In general, acidic amino acids fall into a group; the basic amino acids in another; neutral hydrophilic amino acids in another; and etc. More specific classifications are described in WO 96/05224 incorporated for reference above. A set of preferred variants is one in which the glycosylation sites of either the α or β subunits or both have been altered. Some useful variants of the hormone quartet described herein are described in U.S. Pat. No. 5,177,193 issued January 5, 1993 and incorporated herein for reference. As shown therein, the glycosylation configurations can be altered by destroying the relevant sites or, in the alternative, by selection of the host cells in which the protein is produced. Alterations in the amino acid sequence also include both insertions and deletions. Accordingly, truncated forms of the hormones are included among the variants, for example, subunit mutants to which they are lacking some or all of the amino acids at positions 85-92 at the C-terminus. , subunits a with 1-10 amino acids deleted from the N-terminus are included. Variants also include those with non-critical regions altered or removed. Such deletions and alterations can comprise complete loops, so that sequences of considerably more than 10 amino acids can be deleted or changed. The resulting variants, however, must retain at least the binding domains of the receptor and / or the regions involved in signal transduction. There is considerable literature on the variants of the glycoprotein hormones and it is clear that a large number of possible variants which result in both agonist and antagonist activity can be prepared. 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. Glycoprotein Hormones, Lusbader, J. W. et al. EDS, Springer 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 Bielinska, M. et al. J Cell Biol (1990) 111: 330a (Abstract 1844).

Other variants include those in which one or more cystine junctions are deleted or deleted, typically replacing a neutral amino acid with one or both cysteines which participate in the binding. Particular preferred cystine bonds which 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 hormone quartet can be constructed in chimeric forms to provide the functions biological factors of both components of the chimera, or, in general, the hormones of the altered biological function. Accordingly, chimeric molecules which exhibit activities of both FSH and LH / CG 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, substituent amino acids 101-109 of FSH-β for the corresponding residues in the subunit of CG-β produce an analog with activity of both hCG and FSH. When used here, "peptide" and "protein" are used interchangeably, since the length distinction between them is arbitrary. As stated above, the "variants" employed as the α and β subunits in the formation of the compound of the invention with or without the linking portions may represent the complete amino acid sequences of the subunits or only portions thereof. The "variants" also include the α and / or β chains which contain a CTP (or a variant of CTP) inserted in a non-critical region. The "non-critical" regions of subunits a and ß are those regions of molecules not required for biological activity (including agonist and antagonist activity). In general, these regions are removed from the binding sites, the precursor cleavage sites, and the catalytic regions. Critical regions for inducing proper folding or bending, binding to receptors, catalytic activity and the like should be evaluated. It should be noted that some of the regions which are critical in the case of the dimer become non-critical in single-chain forms since the conformational restriction imposed by the molecule can avoid the need for these regions. The inquiry into the non-critical regions is easily carried out by deleting or modifying the candidate regions and carrying out an appropriate assay for the desired activity. The regions where the modifications lead to the loss of activity are critical; the regions where the alteration leads to the same activity or to a similar activity (including the antagonist activity) are considered non-critical. Again, it should be emphasized, that "Biological activity" means the activity which is either agonist or antagonist with respect to those of the natural hormones. Accordingly, 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 subunit a, positions 33-59 are thought to be necessary for signal transduction and stretch of 20 amino acids at the carboxy terminus or limit is necessary for receptor binding / transduction of the signal. 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 "close" to the N or C termination, the insertion is at any location within the 10 amino acids of the terminator or boundary, preferably within the 5 amino acids, and even more preferably at the termination or limit per se. When used herein, the "CTP unit" refers to an amino acid sequence found at the carboxy terminus or boundary of human chorionic gonadotropin ß which extends from amino acids 112-118 to residue 145 at the termination or limit of C or a portion thereof. Therefore, each "complete" CTP unit contains 28-34 amino acids, depending on the NTP end or terminus. By a "partial" CTP unit is meant an amino acid sequence which occurs between positions 112-118 to 145 inclusive, but which has at least one amino acid deleted from the shortest "complete" CTP unit possible (ie say 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. Some non-glycosylated forms of the hormones are antagonists and are useful as such. 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 the CTP useful in the agonists will contain one or more of these arranged or distributed sites in the order in which they appear in the natural CTP sequence, although the intermediate sites can be omitted. In some cases, CTP units can be inserted or used as serial linkers. By inserts or extensions "in series" it is understood that the insert or extension contains at least two "units of CTP". Each unit of CTP can be complete or a fragment, and natural or a variant. The totality of the CTP units in the extension or series insert may be identical, or they may be different from each other. The "linker portion" is a portion that binds the sequences to and ß without interfering with the activity that could otherwise be exhibited by the same chains a and ß as members of a heterodimer, or which alter this activity to convert it from an agonist activity to an antagonist. The level of activity can change within a reasonable range, but the presence of the linker can not be such that it deprives the single-chain form of a substantial agonist as well as a substantial antagonist activity. The single chain form does not represent a propeptide but the mature protein does and must exhibit a relevant activity with respect to the hormonal activity of the heterodimer, the elements of which they form their components.

Preferred Modes of Bifunctional Hormones The bifunctional hormones of the invention are more efficiently and economically produced using recombinant techniques. Therefore, the fusion proteins comprising those forms of the α and β chains, the CTP units and other linker portions which include only the amino acids encoded by the gene, are preferred. However, it is possible, as described above, to construct at least portions of the single chain hormones using synthetic peptide techniques or other organic synthesis techniques and therefore the variants which contain the amino acids not encoded by the .gen and Linkers based on substances other than peptides are also within the scope of the invention. In the most preferred embodiment, the termination or boundary C of the β1 subunit is bound covalently, optionally through a linker, to the N terminus or boundary of the mature subunit which in turn is optionally covalently linked to through a linker to the ß2 subunit. The link can be a direct peptide linkage where the C-terminal amino acid of a subunit is directly linked through the binding of the peptide to the N terminus or boundary of the other; however, in many cases it is preferable to include a linker portion between the terminations or boundaries. In many cases, the linking portion will provide at least one turn or turn β between the two chains. The presence of the proline residues in the linker can thus be advantageous. (It should be understood that in the description of the links between the terminations or limits of the units comprising the single-chain forms, one or more terminations or limits may be altered by substitution and / or deletion as described above). In a particularly preferred set of embodiments, the bond is from head to tail and the linker portion will include one or more units and / or variants of CTP or truncated forms thereof. The preferred forms of the CTP units used in such linker portions are described hereinafter. In addition, the linker portion may include a drug or drug covalently linked, but preferably releasably, to the linker portion. The means for coupling or binding the drug to the linker portion and for providing its release are conventional. In addition to its presentation in the linker portion, CTP and its variants can also be included in any non-critical region of the subunits that make up the single chain hormone as described above. Although the CTP units are preferred inclusions in the linker portion, it is understood that the linker can be any suitable covalently bonded material which provides the appropriate spatial relationship between the subunits a and β. Accordingly, for head-to-tail configurations the linker can generally be a bivalent moiety such as a peptide comprising an arbitrary number, but typically less than 100, more preferably less than 50 amino acids which have the appropriate hydrophilicity / hydrophobicity ratio to provide the proper spacing and conformation in the solution or a different linker of the peptides which confers these characteristics. In general, the linker must be in hydrophilic balance to reside in the surrounding solution and out of the way of the interaction between subunits a and β of the two β subunits. It is preferable that the linker includes ß turns or turns provided by the proline residues in the peptide linker, or comprising residues of serine and / or glycine. Any suitable polymer, including peptide linkers, with the correct characteristics described above, can be used. Particular preferred embodiments of the bifunctional hormones of the invention include the head to tail configuration: ßFSH-a-ßFSH; a-ßFSH-ßLH; ßFSH-Ot-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 (5115-123) -a-ßFSH; ßLH (5115-123) -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 (5115-123) -CTP-a; and similar. Particularly preferred are the human forms of the subunits. In the above constructions, "CTP" refers to the CTP or its variants including the truncated portions as described in the document No. 96/05224. Although for human use, the human forms of subunits a and ß are desirable, it should be noted that the corresponding forms in other vertebrates are useful in veterinary contexts. Therefore, the subunits of FSH, TSH, and LH characteristic of cattle, sheep, horses, pigs, felines, canines, and other species are appropriate for indications affecting these species per se. Suitable drugs that may be included in the linker portion include peptides or proteins such as insulin-like growth factors; the epidermal growth factors; the growth factors of acidic and basic fibroblasts; growth factors derived from platelets; the various stimulating factors of the colonies, such as the granulocyte CFS, the CSF of macrophages, and the like; as well as several cytokines such as IL-2, IL-3 and the large amount of additional interleukin proteins; the various interferons; the tumor necrosis factor; and similar. Suitable cleavage sites for the release of these drugs may be included, such as target or target sequences for proteases whose target or target sites are not present in the a and beta subunits. Peptide or protein based drugs have the advantage that the complete construct can be easily produced by the recombinant expression of a single gene. Also, small molecule drugs such as antibiotics, anti-inflammatories, toxins, and the like can 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. The. Proper provision for drug release from inclusion within the linker will be provided, for example, by also including sites for enzyme-catalyzed lysis as further described under the section entitled "Methods of Preparation" hereinafter.

Other Modifications The single chain proteins of the invention can also be conjugated or derived in the generally understood ways to derive the amino acid sequences, such as phosphorylation, glycosylation, deglycosylation of the normally glycosylated forms, acylation, modification of the side chains of amino acids (for example, the conversion of proline to idroxyproline) 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 the non-glycosylated form either by producing them in prokaryotic hosts or by mutation of the glycosylation sites normally present in the subunits and / or any CTP units that may be present. Both the non-glycosylated versions and the partially glycosylated versions of the hormones can be prepared by manipulating the glycosylation sites. Normally, glycosylated versions, of course, are also included within the scope of the invention. As is generally known in the art, single chain proteins of the invention can also be coupled to labels, carriers, solid supports, and the like, depending on the desired application. Labeled forms can be used to track your metabolic fate; labels suitable for this purpose include, especially labels of radioisotopes such as iodine 131, technetium 99, indium 111, and the like. Labels 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 enzyme labels, fluorescent labels, chromogenic labels, and the like. The use of such tags allows the location of the relevant receptors since they can be used as target location agents for such receptors. The proteins of the invention can also be coupled or linked to the carriers to improve their immunogenicity in the preparation of antibodies specifically immunoreactive with these new modified forms. Suitable carriers for this purpose include keyhole-shaped limpet hemocyanin (KLH), bovine serum albumin (BSA) and diphtheria toxoid, and the like. Standard coupling or binding techniques for linking the modified peptides of the invention to carriers, which include the use of bifunctional linkers, may be employed. Similar binding techniques, in the company of others, can be employed to couple or bind the proteins of the invention to the solid supports. When they are coupled or joined, these proteins can then be used as affinity reagents for the separation of the desired components with which the specific reaction is displayed. Accordingly, they are useful in the purification and isolation of the receptors with which the appropriate β subunit interacts.

Preparation Methods Methods for building the proteins of the invention are well known in the art, if only the amino acids encoded by the gene are included, and the single chain is in a head-to-tail configuration, the most practical approach in the present is to synthesize these recombinantly by the expression of the DNA encoding the desired protein. The DNA containing the nucleotide sequence encoding the single chain forms, including the variants, can be prepared from natural sequences, or synthesized de novo or using combinations of these techniques. The techniques for site-directed mutagenesis, binding or ligation of additional sequences, PCR amplification, and construction of suitable expression systems are all, for now, well known in the art. Portions or all of the DNA encoding the desired protein can be synthetically constructed using standard solid phase techniques, preferably to include restriction sites for ease of binding or ligation. Suitable control elements for transcription and translation of the included coding sequence can be provided to the DNA coding sequences. As is well known, expression systems are now available in a manner 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 Neurosporus, plant cells, insect cells, mammalian cells such as CHO cells, bird cells, and the like. The choice of host is particularly pertinent to posttranslational events, more particularly including glycosylation. The localization of glycosylation is controlled mostly for the nature of the glycosylation sites within the molecule; however, the nature of the sugars that occupy this site is largely controlled by the nature of the host. Accordingly, a fine tuning of the properties of the hormones of the invention can be achieved by the proper choice of the host. A particularly preferred form for the subunit a portion, whether the subunit is modified or not, is the "minigen" construct. When used herein, the a subunit of the "minigene" refers to the construction of the gene described by Matzuk, MM, et al., Mol Endocrinol (1988) 2: 95-100, in the description of the construction of pM2 / CG ao pM2 / I. For recombinant production, the modified host cells using the expression systems are used and cultured to produce the desired protein. These terms are used herein as follows: ', A "modified" recombinant host cell, ie, a cell "modified to contain" the recombinant expression systems of the invention, refers to a host cell which has been altered to that contains this expression system by any convenient way of introducing it, including transfection, viral infection, and so on. "Modified cells" refer to cells that contain this expression system whether 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 temporally. In summary, recombinant host cells "modified" with the expression system of the invention refer to cells which include this expression system as a result of their manipulation to include it, when they do so naturally, regardless of the manner to effect this incorporation. "Expression system" refers to a DNA molecule which includes a coding nucleotide sequence to be expressed and those that accompany the control sequences 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 mechanism to regulate the expression. The control sequences are those which are designed to be functional in a particular target or target recombinant host cell, and therefore the host cell must be chosen to be compatible with the control sequences in the expression system built.

If the secretion of the produced protein is desired, additional nucleotide sequences encoding a signal peptide are also included to produce the signal peptide operably linked to the desired single-chain hormone to produce the preprotein. During secretion, the signal peptide is cleaved to release the mature single-chain hormone. When used here "cells", "cell cultures", and "cell lines" are used interchangeably without particular attention to the nuances of meaning. Where the distinction between them is important, it will be clear from the context. Where any of them can be understood, all of them are proposed to be included. The produced protein "can be recovered from the lysate of the cells if it is produced intracellularly, or from the medium if it is secreted." The techniques for recovering the recombinant proteins from the cell cultures are well understood in the art, and these proteins can be purified using the known techniques such as chromatography, electrophoresis of the gene, selective precipitation, and the like All or a portion of the hormones of the invention can be synthesized directly using the techniques of synthesis of the peptides known in the art. can be linked or linked, and the release sites for any drug contained in the linker portion introduced by the standard chemical means, for those modalities that contain amino acids which are not encoded by the genes and those modalities where the configuration is used. head with head or tail with tail, p Of course, the synthesis must be at least partially at the level of the proteins. Head-to-head joints at natural N termini or boundaries or positions close to N terminations or boundaries may be effected by linkers which contain functional groups with amino groups, such as dicarboxylic acid derivatives . The tail-to-tail configurations at the terminations or boundaries of C or the positions near the terminations or boundaries of C may be effected through linkers which are diamines, diols, or combinations thereof.

Antibodies The proteins of the invention can be used to generate antibodies specifically immunoreactive with these new compounds. These antibodies 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 concentrated as polyclonal antisera to ensure adequate immunization. Polyclonal antisera can then be collected such as 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 selected for the production of monoclonal antibodies immunospecific with the proteins of the invention. '"Antibodies" includes any fragment which retains the required immunospecificity, such as Fat > r ab '/ F (ab') 2r Fv and etcetera. Accordingly, antibodies can also be prepared using recombinant techniques, typically by isolation of the nucleotide sequences encoding at least the variable regions of the monoclonal antibodies with appropriate specificity and the construction of appropriate expression systems. This approach allows any desired modification such as the production of Fv forms, chimeric forms, "humanized" forms, and the like. By "immunospecific for the proteins of the invention" are meant antibodies which specifically bind to the reference compound of the invention, but not heterodimers or any of the subunits included per se or any single chain forms which include only a single β subunit within the general parameters considered to determine affinity or non affinity. It is understood that specificity is a relative term, and an arbitrary limit could be chosen, such as a difference in specific binding of 100 times or larger. Accordingly, an immunospecific antibody included within the invention is at least 100 times more reactive with the single chain protein than with the corresponding heterodimers, the single chain forms of the prior art or the separated subunits. Such antibodies can be obtained, for example, by selecting those which bind the compounds of the invention and discarding those which also bind the heterodimers, subunits or single chain forms of the prior art described in WO95 / 22340 and WO96 / 05224.

Formulation and Methods of Use The proteins of the invention are formulated and administered using methods comparable with those known for the corresponding heterodimers. Accordingly, the formulation and methods of administration will vary according to the particular hormone or combination of hormones used. However, the dosage level and frequency of administration can be altered when compared to the heterodimer, especially if the CTP units are present in view of the prolonged biological half-life due to their presence. Formulations for the proteins of the invention are those typical of protein or peptide drugs such as those found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Company, Easton, PA. In general, the proteins are administered by injection, typically intravenous, intramuscular, subcutaneous, or intraperitoneal injection, or using the formulations for transmucosal or transdermal delivery. These formulations generally include a detergent or penetrant such as bile salts, fusidic acids, and the like. These formulations can be administered as aerosols or suppositories or, in the case of transdermal administration, in the form of skin patches. Oral administration is also possible as long as the formulation protects the peptides of the invention from degradation in the digestive system. The optimization of the dosing and formulation regimen is carried out as a matter of routine and as was generally done in the art. These formulations can also be modified to include those suitable for veterinary use. The compounds of the invention can be used in many ways, most obviously as substitutes for the heterodimeric forms of the hormones. Accordingly, similarly to heterodimers, the agonist forms of the single chain hormones of the invention can be used in the treatment of infertility, as adjuncts in the techniques of in vitro fertilization, and other associated therapeutic methods. with the natural hormones. These techniques are applicable to humans as well as to other animals. The choice of the single chain protein in terms of its derivation from the species will depend, of course, on the subject to whom the method is applied. It will be noted that the double functionality which is conferred on these compounds containing two different β subunits confers opportunities for therapies that previously have not been available. The compounds of the invention are also useful as reagents in a manner similar to that employed with respect to the 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 natural proteins to the extent that such antibodies bind to the relevant portions of these single-chain compounds in the samples biological They are also useful as control reagents in sets or assay sets to evaluate the levels of these hormones in various samples. The protocols for evaluating the levels of the hormones themselves or of antibodies raised against them, are the standard immunoassay protocols commonly known in the art. Various competitive and direct assay methods can be used that involve a variety of labeling techniques including radioisotope labeling, fluorescence labeling, enzyme labeling and the like. The compounds of the invention are also useful in detecting and purifying receptors to which natural hormones bind. Accordingly, the compounds of the invention can be coupled to the solid supports and used in the affinity chromatographic preparation of the hormone receptors or antibodies. The resulting receptors are themselves useful in the evaluation of hormonal activity for candidate drugs in screening tests for therapeutic and reagent quantities. Of course, the double specificity of the β subunits in any of these compounds in which the β subunits are different should be taken into account. However, where the two β subunits are identical, they offer a powerful affinity purification tool for the relevant receptor. Finally, the antibodies reactive only with the compounds of the invention can be used as purification tools for the isolation of these materials in their subsequent preparations. They can also be used to verify the levels of these compounds administered as drugs. The following examples are proposed to illustrate but not to limit the invention.

Example 1 Preparation of CGß-a-CTP-FSHß A nucleotide sequence encoding the title compound was prepared using the nucleotide sequences available for the relevant portions of the subunits. The CGß region encodes the 145 amino acids of human CGß; the nucleotide sequence encoding the a subunit encodes the 92 amino acids of the human variant as the minigene; the coding sequence of CTP encodes the 28 amino acids representing positions 118-145 of human chorionic gonadotropin; and the coding region of FSHß encodes the 111 amino acids of the human FSHß subunit. An amplified fragment containing exon 3 of CGβ, minigene a, CTP and βFSH is inserted into the Sali site of pM2HA-CGßexonl, 2 an expression vector which is derived from pM2 and contains exons 1 and 2 of CGß in the manner described by Sacháis, ß Biol Chem (1993) 268: 2319. The pM2 that contains exons 1 and 2 of CGß is described in Matzuk, M.M. et al. Proc Nati Acad USA (1987) 8_4: 6354-6358 and Matzuk, M.M. et al. J Cell Biol (1988) 106: 1049-1059. First, a fragment containing the minigene downstream of CG3 exon 3 is inserted into this vector to obtain pM2-HACGβ. PM2-HACGßa was then cleaved with Scal and ligated with pBIIKS (+) to-CTP-FSH restricted in Scal. The resulting pM2-HACGβ-a-CTP-FSH expression vector produces the title compound when inserted into a suitable host.

Example 2 Production and Activity of CGß-a-CTP-FSHß The expression vector constructed in Example 1 was transfected into the cells of the Chinese hamster ovary (CHO) and the production of the protein was evaluated by immunoprecipitation of the radiolabeled protein on the SDS gels. The culture medium is collected, concentrated and tested for binding to the human LH receptor (which is expected to bind to the ßCG-a moiety). For this assay, the LH receptor was prepared by inserting the cDNA encoding the complete human LH receptor into the pCMX expression vector (Oikawa, J. X-C et al., Mol Endocrinol (1991) 5: 759-768). 293 cells that grow exponentially were transfected with this vector using the method of Chen, C. et al. Mol Cell Biol (1987) 1_: 21 5-2152, leading to expression of the LH receptor on the surface. In the assay, cells expressing the human LH receptor (2 x 10 5 / tube) were incubated with 1 ng of hCG labeled in competition with increasing concentrations of unlabeled hCG or increasing amounts of the sample to be tested at 22 ° C for 18 hours. The reduction in the label in the presence of the sample measures 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 the medium containing the CGß-a-CTP-FSHβ also showed activity. These results are shown in Figure 1. As shown, both the heterodimeric hCG (filled squares) and the single chain bifunctional protein of the invention (full circles) successfully competed with the hCG labeled for the LH receptor. The bifunctional compound is less potent due to the modification of the carboxy terminus or limit of the a subunit. Also shown in Figure 1 are test results where the variable amounts of a culture supernatant derived from cells modified to contain two expression systems were tested. 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 complex (solid triangles) also competed successfully for binding. In a similar manner, the supernatant of the culture medium containing the CGß-a-CTP-FSHβ was tested for binding to the receptor for FSH, expressed in 293 cells. The assay was carried out in the manner described above, except that the cells expressing 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 titer compound (full circles) successfully competed with FSH (full squares) for binding. In an unrelated experiment, also shown in Figure 2, the mixture of a different type of complex - that is, FSHß-a from a single chain associated non-covalently with CGß-el, which is mixed with FSHß-a from a single chain, in excess, that has not formed a complex (full triangles), was an excellent competitor.

Example 3 Construction of Additional Expression Vectors In a manner similar to that described in Example 1, the expression vectors for the production of the single-stranded bifunctional FSHß-CTP-a-CGß; α-FSHβ-CTP-CG ß, CG ß- ßFSH-CTP-a, and ßLH-CTP-ßFSH-CTP-a are prepared and transfected into CHO cells. Culture supernatants are cultured and tested as described above with respect to both LH and FSH receptors. These compounds also show ability to bind to both receptors.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (16)

1. A glycosylated or non-glycosylated protein having agonist and / or antagonist activity, of the formula: ß1- (linker1) m-a- (linker2) n-ß2 (1); or ß1- (linker1) m-ß2- (linker2) n-a (2); or a- (linker1) m-ß1- (linker2) n-ß2 (3) characterized in that each of ß1 and ß2 has the amino acid sequences of the β subunit of a vertebrate glycoprotein hormone or a variant thereof; "a" designates the a subunit of a vertebrate glycoprotein hormone or a variant thereof; "linker" refers to a covalently linked portion that spans the β1 and β2 subunits to the effective distances from the a subunit a and to each other, to retain the activity, and each of m and n is independently 0 or 1.

2. The protein according to claim 1, characterized in that m and n are 1.

3. The protein according to claim 1, characterized in that at least one of the linker portions includes a drug that is to be targeted for the glycoprotein hormone receptor, or because at least one linker is CTP or a variant thereof .

4. The protein according to claim 1, characterized in that β1 is the β subunit of FSH, LH or TSH extended to a position close to its termination or limit C by a partial or complete CTP unit or a variant thereof.

5. The protein according to claim 1, characterized in that the subunit to or one or more subunits β or both are modified by the insertion of a CTP unit or a variant thereof in a non-critical region thereof and / or because the linker portion includes a CTP unit or a variant thereof.

6. The protein according to claim 1, characterized in that the variants contain 1-5 conservative amino acid substitutions when referring to the natural forms or are truncated forms of said sequences or both.

7. A pharmaceutical composition, characterized in that it comprises the protein according to claim 1 mixed with a suitable pharmaceutical excipient.

8. The protein according to claim 1, characterized in that it is attached or coupled to a solid support.

9. Antibodies, characterized in that they are immunospecific for the protein according to claim 1.

10. A DNA or RNA molecule, characterized in that it comprises a nucleotide sequence that encodes the protein according to claim 1.

11. An expression system for the production of an agonist and / or an antagonist of a glycoprotein hormone, the expression system is characterized in that it comprises a first nucleotide sequence encoding the protein of claim 1 operatively linked to control the sequences capable of effecting the expression of the first nucleotide sequence.

12. The expression system according to claim 11, characterized in that it also contains a second nucleotide sequence encoding a peptide of the signal operably linked to the protein encoded by the first nucleotide sequence.

13. A host cell, characterized in that it is modified to contain the expression system according to claim 11.

14. A host cell, characterized in that it is modified to contain the expression system according to claim 12.

15. A method for producing a single chain protein which is an agonist and / or antagonist of a glycoprotein hormone, the method is characterized in that it comprises culturing the cells of . / according to claim 14 under conditions wherein the protein is produced; and recover the culture protein.

16. A method for producing a single chain protein which is an agonist and / or antagonist of a glycoprotein hormone, the method is characterized in that it comprises culturing the cells of claim 14 under the conditions wherein the protein is produced; and recover the culture protein.