MXPA00002716A - Mutants of thyroid stimulating hormone and methods based thereon - Google Patents

Abstract

The present invention is based upon the discovery that mutant&agr;subunits and mutant&bgr;subunits each comprising amino acid substitutions relative to the wild type can be produced and assembled to form a mutant TSH heterodimer or TSH analog that possesses higher bioactivity in vitro and longer half life in vivo. Accordingly, the present invention provides methods for using mutant TSH heterodimers, TSH analogs, fragments, and derivatives thereof for treating or preventing diseases of the thyroid, in particular thyroid cancer. The invention also relates to methods of diagnosis, prognosis and monitoring for thyroid-related functions. Pharmaceutical and diagnostic compositions, methods of using mutant TSH heterodimers and TSH analogs with utility for treatment and prevention of metabolic and reproductive diseases are also provided.

Description

MUTANTS OF THYROID STIMULATING HORMONE AND METHODS BASED ON IT 1. FIELD OF THE INVENTION The present invention relates to mutants of the thyroid stimulating hormone, and derivatives and analogs thereof. Methods for producing mutants of the thyroid stimulating hormone, derivatives and the like are also provided. The invention also relates to pharmaceutical compositions and methods for diagnosis and treatment. 2. BACKGROUND OF THE INVENTION [0002] Glycoprotein hormones are a group of evolutionarily conserved hormones that are involved in the regulation of reproduction and metabolism (Pierce and Parsons, 1981, Endocr. Rev. 11: 354-385). This family of hormones includes follicle stimulating hormone (FSHL), luteinizing hormone (LH), thyroid stimulating hormone (TSH), and chorionic gonadotropin (C6). TSH is a heterodynamic glycoprotein with 28-30 kDa produced in thyrotopes of the anterior pituitary gland. The differences in the molecular weight of TSH are mainly due to the heterogeneity of the carbohydrate chains. The thyrotropin-releasing hormone stimulates its synthesis and secretion, and inhibits thyroid hormone in a classic endocrine feedback loop. TSH controls the function of the thyroid by interacting with the TSH receptor coupled to the G protein (TSHRU) (Vassant and Dumont, 1992, Endocr, Rev. 13, 596611). TSH that binds to its receptor in thyroid cells leads to the stimulation of second messenger pathways that predominantly involve adenosine, 3 '5' -cyclic monophosphate (cAMP), inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) and, ultimately, produces the modulation of thyroid gene expression. The physiological roles of TSH include the assimilation of related thyroid functions, such as iodine uptake and uptake, the release of thyroid hormone from the gland, and the promotion of thyroid growth (Wondisford et al, 1996, Thyrotropin. et al, (en) Werner and Ingbar The Thyroid, Lippencott-Raven, Philadelphia, pp. 190-207). In the structural aspect, the glycoprotein hormones are related heterodimers comprising a subunit a and a specific β subunit of the hormone. The common human subunit contains an apoprotein core of 92 amino acids that includes 10 middle cystine residues, all of which are found in the bisulfide bond. It is encoded by a single gene located on chromosome 6 in humans, and therefore, identical to the amino acid sequence within a given species (Fiddes and Goodman, 1981, J. Mol. Appl. Gen. 1: 3- 18). The hormone-specific β-subunit genes differ in length, structural organization and chromosomal location (Shupnik et al, 1989, Endocr, Rev. 10: 459475). The gene of the β subunit of human TSH predicts a mature protein of 118 amino acid residues and is located on chromosome 1 (Wondisford et al, supra). The various β subunits can be aligned according to 12 invariant residues of half cystine forming 6 disulfide bonds. Despite an amino acid sequence identity of 30 to 80%, the β subunit is sufficiently different to direct the differential receptor link with high specificity (Pierce and Parsons, supra). An important structural element of these hormones is their half of carbohydrate which makes up of 5-35% by weight. The common subunit has two oligosaccharides linked with asparagine (N), and the subunit β one (in the TSH the LH) or two (in the CG and the FSH). In addition, the β-subunit CG has a unique 32-residue carboxyl terminal extension peptide (CTEP) with four glycosylation sites, of linked O-serine. (Baenziger, 1994, Glycosylation and glycoprotein hormone function, in Lustbander et al (eds.) Glycoprotein Hormones: Structure, Function and Clinical Implications, Springer-Verlag, New York, pages 167-174). Traditionally, the structure-function relationships of the human glycoprotein hormones have been carried out fundamentally with gonadotropins, particularly hCG. Recently, the crystal structure of partially deglycosylated hCG has been resolved and this has revealed two relevant structural features that may also be relevant for other glycoprotein hormones (Lapthorn et al., 1994, Nature 369: 455461; Wu et al. 1994, Structure 2: 548-558). Both subunit a and subunit β have a similar topology of hCG (each subunit has two ß-fork structures (Ll and L3) on one side of a central knot of cystine (formed by three bisulfide bonds) and a long circuit (L2) on the other Molecular biology studies on human TSH have facilitated the cloning of cDNA and the TSH subunit gene (Joshi et al., 1995, Endocrinol 136: 3839-3848) , cloning of the TSH receptor cDNA (Parmentier et al., 1989, Science 246: 1620-1622, Nagayama et al., 1990. Biochem Biophys. Res. Commun. 166: 394403), and the recombinant expression of TSH (Cole et al., 1993, Bio / Technol 11: 1014-1024, Grossiriann et al., 1995, Mol Endocrinol 9: 948-958, Szkudlinski et al, 1996 supra.) Previous studies on structure-function of the TSH were mainly concentrated in highly conserved regions and in the creation of chimeric subunits, however, these approaches did not produce Ones with greater biological activity in vitro (Grossmann et al, 1997, Endocr. Rev. 18: 476-501).

Strategies have been devised to prolong the hormonal half-life of glycoprotein hormones in the circulation. In gene fusion experiments, the carboxyl terminal extension peptide of the β hCG subunit, which contains several O-linked carbohydrates, was added to the β subunit of human TSH (Joshi et al, 1995, Endocrinol., 136: 3839- 3848; Grossmann et al., 1997, J. Biol. Chem. 272: 21312-21316). Although the in vitro activity of these chimeras was not altered, their circulatory half-lives were prolonged, thus producing greater biological activity in vivo. In addition, by expressing the ß and Oi subunits genetically fused as a single strand stability was improved and the plasma half-life was prolonged, compared to the wild type of the glycoprotein hormone (Sugahara et al, 1995, Proc. Nati. Acad. Sci. USA 92: 2041-2045; Grossmann et al., 1997, J. Biol. Chem. 272: 21312-21316). 2.1 USE OF THE TSH IN THE DIAGNOSIS AND SURVEILLANCE OF THE THYROID CARCINOMA TSH has been tested for the stimulation of 131 I absorption and Tg secretion in the diagnosis and follow-up of up to 19 patients with differentiated thyroid carcinoma, thus avoiding the side effects of thyroid hormone withdrawal (Meier et al. et al., J. Clin Endocrinol, Metab. 78: 188-196). The preliminary studies of this first trial are very encouraging. The incidence of thyroid carcinoma in the United States is approximately 14,000 cases per year. Most of these are differentiated, and the most common subtypes are papillary or follicular cancer. Given that the 10 and 20 year survival rate of differentiated thyroid carcinomas is 90% and 60%, respectively, long-term surveillance for local recurrence and distant metastases becomes essential in the management of such patients. , especially since the tumor can recur even decades after the first therapy. The main methods of follow-up are radioactive iodine exploration and thyroglobulin (Tg) measurements of the whole body. For the optimal sensitivity of these diagnostic procedures, residual thyroid tissue by TSH is used to increase the absorption of 131 Iodo or Tg secretion, respectively. However, patients with thyroid cancer are treated after thyroid hormone thyroidectomy to suppress endogenous TSH to avoid the possible stimulatory effects of TSH on residual thyroid tissue, as well as to maintain euthyroidism. Generally, consequently, levo-T4 or, less commonly used T3 is removed 4-6 and 2 weeks before the radioactive iodine scan and determination of Tg to stimulate endogenous TSH secretion. This simultaneous but transient hypothyroidism leads to a considerable deterioration in the quality of life of these patients, and may interfere with their ability to work. In addition, since TSH can act as a growth factor in malignant thyroid tissue, prolonged periods of increased endogenous secretion of TSH may pose a possible risk for these patients. In the 1960s, bovine TSH (bTSH) was used to stimulate residual thyroid tissue to overcome the need to elevate endogenous TSH (Blahd et al., 1960, Cancer 13: 745-756). However, several disadvantages soon became apparent, which led to the discontinuation of its application in clinical practice. Compared to the withdrawal of hormones, bTSH proved to be less effective in detecting malignant and residual thyroid tissue and its metastasis. In addition, allergic reactions were frequently recognized as well as the appearance of neutralizing antibodies that may further limit the effect of the subsequent administration of bTSH as well as interference with endogenous TSH determinations (Braverman et al., 1992, J. Clin. Endocrinol, Metab 74.1135-1139). From the diagnostic or therapeutic perspective, there is, therefore, considerable interest in the creation of hTSH analogues with desirable properties. In general, the activity of the hormones can be increased by prolonging the hormonal half-life (analogues of lasting duration) or by increasing their intrinsic activity (superactive analogues). The present inventors have created mutant TSH and analogs, and have shown that these novel molecules have biological activity in vitro and in vivo that exceeds that of wild-type TSH. His discovery is described below. 3. COMPENDIUM OF THE INVENTION The present invention relates to mutants of the thyroid stimulating hormone (TSH), including mutants of the subunit to common glycoprotein hormones, mutants of the β-subunit of TSH, analogs, derivatives and fragments of TSH, preferably TSH heterodimers having mutant subunits with one or more substitutions of amino acid residues or modified TSH heterodimers (as described below) to increase the half-life in vivo. Mutant subunits, mutant TSH heterodimers, analogs, derivatives and TSH fragments are more active than wild-type TSH in the TSH receptor (TSHR) that binds and stimulates TSHR signal transduction, and have prolonged hormonal lifetimes in the circulation. In a preferred embodiment, the present invention provides mutant TSH or TSH analogue with mutations at amino acid positions located near or within the ß Ll hairpin structure in the subunit a and at amino acid positions located near or within the structure in ß L3 hairpin of the β subunit, and more preferably, the mutant TSH is modified to increase the half-life in vivo, for example, but not limited to, fusing the β subunit of TSH to the carboxyl terminal extension peptide (CTEP) FROM the human chorionic gonadotropin β-subunit (hCG), or to fuse the β-subunits already to produce a single-chain TSH analog. Methods for performing chemical synthesis or recombinant DNA technology are within the scope of the invention. In another preferred embodiment the present invention provides a mutant subunit of the TSH heterodimer with a mutant OI subunit with an amino acid substitution at position 22 of subunit a as shown in Figure 1 (SEQUENCE DEFAULT NUMBER: 1) . Nucleic acid sequences encoding mutants of subunit a and of the β subunit, and fusion analogues of TSH and its subunits, biological assays, and host-vector host systems for the production of hormones are also provided. The present invention further provides diagnostic and therapeutic application methods of mutant TSH and TSH analogs, TSH derivatives and fragments thereof, in hypothyroidism and cancer, especially thyroid carcinoma.

In a preferred embodiment, the mutant TSH is used to stimulate the absorption of radioactive iodine and thyroglobulin secretion in the diagnosis and follow-up surveillance of patients with thyroid cancer. In another preferred embodiment, the heterodimers of the mutant TSH of the invention can be used in the inhibition assays of the TSH receptor binding to detect the presence of antibodies against the TSH receptor, for example, autoantibody antibodies. TSHR characteristic of diseases and conditions, for example, but not limited to, Graves' disease. Also provided are pharmaceutical and diagnostic compositions comprising mutant TSH or TSH analogs. 3.1 DEFINITIONS As used herein, the following terms shall have the indicated meanings: TSH = human thyroid stimulating hormone, unless another species is designated TSHR = human thyroid stimulating hormone receptor hCG = chorionic gonadotropin human CTEP = carboxyl terminal extension peptide of the β subunit of the hCG subunit CÍ = subunit a of the common human glycoprotein hormone, unless otherwise denoted β subunit = subunit β of human TSH, unless point out something else. Conventional one-letter codes are used to denote amino acid residues. As used herein, mutations within the subunits of TSH are indicated by the TSH subunit, followed by the wild-type amino acid residue, the position of the amino acid and the mutant amino acid residue. For example ßl58R will mean a mutation of isoleucine to arginine at position 58 in the β subunit of TSH. 4. DESCRIPTION OF THE FIGURES Figure 1. Amino acid sequence (SEQUENCE IDENTIFICATION NUMBER: 1) OF THE HUMAN COMMON glycoprotein hormone subunit OI. The amino acid residues (positions 8-30) located on or near the aLl circuit are indicated by a line on top of the sequence. The numbers above the sequence indicate the positions of the amino acids where the mutation is preferred. Figure "X. Amino acid sequence (NUMBER OF SEQUENCE IDENTIFICATION: 2) of the β subunit of human TSH. The amino acid residues (positions 52-87) located on or near the ßL3 circuit are indicated by a line on top of the sequence. The numbers on the sequence indicate the amino acid positions where the mutation is preferred. Figure 4. Stimulation of cAMP production in CHO-JP26 cells by a heterodimer of mutant TSH (continuous-line triangles) comprising a mutant subunit with the Ag22r and A wild-type β subunit; and a TSH of wild type (circles in continuous line). Figure 5. Stimulation of cAMP production in JP09 cells by the mutant TSH heterodimer comprising an a4K subunit with the mutations O1ql3 + ael4k + pl6k + aq20k and a protein fusion of the β subunit and the CTEP (a4K + ßCTEP; strikethrough boxes); the heterodimer of the mutant TSH comprising the aql3 + ael4k + apl6k + aq20k mutations and a wild-type β TSH subunit (o4K, open frames); and wild-type TSH (continuous-line circles.) Figure 6. Mutations in the ßLl cycle of the specific TSH subunit ß The line graph illustrates that ßl3E and ßR14E had no biological activities significantly different from that of wild-type TSH , but that the heterodimers that incorporated any of the mutant subunits BF1R, BE6N or BA17R showed significantly higher biological activities in the normal in vitro assay for the production of cAMP Figure 7. The combinations of the mutations in the specific TSH subunit ß showed activities hormones considerably greater than wild-type TSH in the normal in vitro assay for cAMP production, heterodimers included any of the combinations of BF1R BE6N mutations or BF1R mutations, BE6N 0 BA17R. Figure 8a-8B. The line graph shows the in vivo activity of the hTSH analogues. Figure 8a shows the levels of T4 in the blood of mice with T3 previously suppressed 6 hours' after intraperitoneal injection of either wild-type hTSH (hTSH-weight) or TSH analogs. The values are the mean normal error ± of the mean of five mice per data point. Figure 8B is a bar graph showing the total ratios of T4 with respect to TSH for the individual elements. The units obtained by dividing the total mean of T4 in the serum (μg / dl) by the mean hTSH in the serum (ng / ml) both determined 6 hours after the intraperitoneal injection. The recovery of 200 or 20 ng of material injected 6 hours after the intraperitoneal injection was similar (2%, 1% and% for the wild type, a4K and 4K / ß3R, respectively). 5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to mutant TSH proteins, nucleic acid molecules encoding mutant TSH proteins, and methods for manufacturing and diagnosing therapeutic methods thereof. The present inventors have designed and created thyroid stimulating hormones (TSH), TSH derivatives, TSH analogs and fragments thereof, which have mutations (preferably amino acid substitutions) in subunits a and β which increase the biological activity of the heterodimer of TSH composed of these subunits in relation to the biological activity of wild-type TSH and that are modified to increase the hormonal half-life in the circulation. The present inventors have discovered that these mutations to increase the biological activity and the strategies to raise the hormonal half-life are synergized in such a way that the heterodimers of the TSH that have both the superactive mutations and the long-acting modifications have a much biological activity. greater than would be expected from the additional activity fostered by the superactive mutations and the lasting action of the modifications individually. The present inventors have also discovered that an amino acid substitution at amino acid 22 of subunit a (as presented in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1), preferably a substitution of a basic amino acid, such as lysine or arginine, more preferably arginine, increases the biological activity of TSH in relation to wild-type TSH.

The present inventors have designed mutant subunits by combining individual mutations within a single subunit and modifying subunits and heterodimers to increase the half-life of the heterodimer in vivo (as described below). In particular, the inventors have designed (SEQUENCE IDENTIFICATION NUMBER:) mutant CÍ TSH and mutant β that have mutations, particularly mutations in specific domains. These domains include the ß Ll fork structure of the common OI subunit (as presented in Figure 1), and the ß L3 hairpin structure of the β subunit of the wild-type subunit (as presented in Figure 2). In a preferred embodiment, the present invention provides subunits to mutants, mutant β subunits, and TSH heterodimers that comprise either a mutant subunit or a mutant β subunit, wherein the subunit a comprises single or multiple amino acid substitutions. , preferably located within or near the hairpin structure ß Ll of subunit a, and wherein the β subunit comprises individual or multiple amino acid substitutions, preferably located at or near the ß L3 hairpin structure of the β subunit (preferably these mutations increase the biological activity of the TSH heterodimer that comprises the mutant subunit and the TSH heterodimer that has the mutant subunit has also been modified to increase the serum half life relative to the TSH heterodimer. Wild type amino acid sequence with the invention, a subunit β comprising subst Individual or multiple locations, preferably located on or near the ß L3 hairpin structure of the β subunit, can be fused with its carboxyl terminal to the CTEP. Said β subunit, CTEP subunit may be expressed together or assembled with either a wild-type or mutant Oi subunit to form a functional TSH heterodimer having a biological activity and a serum half-life greater than that of wild-type TSH. In another preferred embodiment, a mutant β subunit comprising individual or multiple amino acid substitutions, preferably located in or near the β L3 hairpin structure of the β subunit, and a mutant subunit comprising individual or multiple amino acid substitutions, preferably located at or near the hairpin structure ß Ll of the OI subunit, they fuse to form a single chain TSH analog. Said fusion of subunit ß-mutant subunit OI has a biological activity and a serum half-life greater than that of wild-type TSH.

In yet another preferred embodiment, the mutant β subunit comprising individual or multiple amino acid substitutions, and further comprising CTEP at the carboxyl terminus, and a mutant OI subunit comprising individual or multiple amino acid substitutions, preferably located at or near the hairpin structure ß Ll of the subunit, they fuse to form an analog of the single chain TSH. Also provided are fusion proteins, analogs and nucleic acid molecules encoding said proteins and analogs, and the production of the above proteins and analogues, for example, by recombinant DNA methods. In particular aspects, the invention provides amino acid sequences of subunits a and β mutants, and fragments and derivatives thereof that are otherwise active in their function. The subunits a and ß of the mutant TSH "active in function", as used herein, refer to the material that shows one or more known functional activities related to the wild-type subunit, for example, link to TSH, trigger the translation of the TSH signal, antigenicity (binding with an anti-TSH antibody), immunogenicity, etc. In particular embodiments, the invention provides fragments of subunits a and β mutants of TSH that consist of at least 6 amino acids, 10 amino acids, 50 amino acids or at least 75 amino acids. In several preferred embodiments subunits to mutants comprise or consist essentially of a hairpin domain aLl; the mutant β subunits comprise or consist essentially of a mutated βL3 hairpin domain. The present invention further provides nucleic acid sequences encoding subunits o1 and β mutants and subunits a and β mutants modified (e.g. subunit fusions β-CTEP or subunit fusions β-mutant-subunit mutant), and methods for applying the sequences of nucleic acid. Mutations in subunits a and in the β subunits are described in more detail in Sections 5.1 and 5.2 below, respectively. The present invention also relates to therapeutic and diagnostic methods and to compositions based on mutant subunits, mutant β subunits, mutant TSH heterodimers, and TSH analogs, derivatives and fragments thereof. The invention provides for the application of the mutant TSH and analogs of the invention for the diagnosis and treatment of thyroid cancer by the administration of mutant TSH and analogs that are more active and have a longer biological activity in the circulation than wild-type TSH . The invention further provides methods for diagnosing diseases and conditions characterized by the presence of autoantibodies against the TSH receptor by applying the TSH heterodimers and analogs of the invention in inhibition assays of the TSH receptor binding. The invention also provides diagnostic kits. The invention also provides methods of treatment for conditions of the thyroid gland such as thyroid cancer. For clarity in the presentation, and without this being by way of limitation, the detailed description of the invention is divided into the following subsections. 5.1 MUTANTS FROM SUBUNITY TO COMMON The common human OI subunit of glycoprotein hormones contains 92 amino acids as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1), including 10 semi-cysteine residues, all of which are in the bisulfide bonds. The invention relates to mutants of the OI subunit of human glycoprotein hormones wherein the subunit comprises single or multiple amino acid substitutions, preferably located at or near the hairpin structure ß1L of subunit a. The amino acid residues located in or near the structure Ll, from position 8-30 as shown in Figure 1 are important for effecting receptor binding and signal transduction. Amino acid residues located in the aL1 structure, such as those in position 11-22, form a set of basic residues in all vertebrates except in hominoids, and have the ability to promote receptor binding and transduction. the signal. In particular, the amino acid residue of position 22 is one of the residues that include in the power of TSH. In accordance with the invention, subunits to mutants have substitutions, deletions or insertions of one, two, three, four or more amino acids in the wild-type protein. In a preferred embodiment, the mutant subunits have one or more substitutions of amino acid residues relative to the wild-type a subunit, preferably, one or more amino acid substitutions at the amino acid residues selected from the residues at position 8-30, 11-22, 8-22, 11-30 , 11-16, 14-22, 13-14 or 16-17. In another preferred embodiment of the invention, the mutant subunit has a single amino acid substitution at positions 11, 13, 14, 16, 20 and 22 of the sequence of subunit a. In several preferred embodiments, the substitution of the amino acid is done with a positively charged residue or a basic residue of a group consisting of lysine, arginine and, less preferably, histidine. In a preferred embodiment, subunit a of the invention has a single amino acid substitution at position 22, where the glycine residue is replaced with an arginine, that is, aG22R. A mutant subunit having an aG22R mutation can have at least one or more additional amino acid substitutions, for example, but not limited to, OTITIK, aQ13K, aE14K, aP16K, aF17R and aQ20K. In other embodiments, the a subunit has one, two, three, four or more amino acid substitutions selected from a group consisting of aTllK, cQQK, aEl4K, aP16K, aFl7R and aQ20K and aG22R. For example, a preferred mutant subunit (which will be used in conjunction with a modification to increase the biological activity of the heterodimer serum of the wild type having the mutant subunit), also referred to herein as a4K, provides for the following mutations: aTHK + aQ13K + aE14K + cíP16K + aQ20K. The mutant OI subunits of the invention are active in function, that is, capable of displaying one or more functional activities related to the wild-type OI subunit. Preferably, the mutant subunit is capable of non-covalently associating with a wild type subunit or mutant β subunit to form a heterodimer of wild-type a subunit that binds to the wild-type subunit. Preferably, such a heterodimer of wild-type TSH also triggers signal transduction. More preferably, the TSH heterodimer comprising an a subunit has an in vitro biological activity or in vivo biological activity greater than the wild-type TSH. In the present invention it is contemplated that more than one mutation may be combined within a subunit to mutant to produce a subunit to superactive, which together with a wild-type or mutant β subunit forms a heterodimer of TSH, which has a substantially biological activity greater than wild-type TSH. It is also contemplated that mutations of the α-subunit will be combined with strategies to increase the serum half-life of the TSH heterodimer having subunit α (ie, a heterodimer of TSH with a β-CTEP subunit fusion or a fusion of subunit ß-subunit a). Mutations within a subunit and long-acting modifications act synergistically to produce an unexpected increase in biological activity. As another example, subunits to mutants having the desired immunogenicity or antigenicity can be used, for example, in immunological assays, for immunization, to inhibit transduction of the TSH signal, etc. The mutant subunit can be tested for the desired activity by methods known in the art, including, but not limited to, the assays described in Section 5.8. 5.2 SUBSTANCE MUTANTS ß OF TSH The human common ß subunit of glycoprotein hormones contains 118 amino acids as shown in Figure 2 (SEQUENCE IDENTIFICATION NUMBER: 2. The invention relates to mutants of the ß subunit of human glycoprotein hormones wherein the subunit comprises single or multiple amino acid substitutions, preferably located at or near the ß L3 hairpin structure of the β subunit where the mutant β subunits are fused to the β-subunit of the β-subunit of the hCG or are part of a heterodimer of TSH that has a subunit to mutant with an amino acid substitution at position 22 (as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1)), or is a fusion of β-subunit subunit The amino acid residues located in or near the βL3 structure positions 52-87 of the β subunits of human TSH are inserted in the amino acid residues in hCG that are located peripherally and appear to be exposed to the surface of the crystal structure. Of particular interest is the set of basic residues of hCG that is not present in TSH (from position 58-69). Substitutions of basic or positively charged residues in this TSH domain lead to an additive and considerable increase in binding affinity of TSH as well as intrinsic activity. The heterodimers of the mutant TSH have substitutions, deletions or insertions of one, two, three, four or more amino acids in the wild-type protein. In a preferred embodiment, the mutant β subunits have one or more substitutions of amino acid residues in relation to the β-subunit of wild type, preferably, one or more amino acid substitutions at the amino acid residues selected among the residues at position 52-87, 52-69 or 58-87 of the β-subunit as shown in Figure 2 (NUMBER IDENTIFICATION OF SEQUENCE: 2). In another embodiment of the invention, the mutant β subunit has a single amino acid substitution at positions 58, 63 or 69 of the β subunit sequence as shown in Figure 2 (SEQUENCE IDENTIFICATION NUMBER: 2) . In yet another embodiment of the invention, the mutant β subunit has multiple amino acid substitutions at the amino acid residues selected from the residues at positions 58, 63 or 69 as shown in Figure 2. (SEQUENCE IDENTIFICATION NUMBER) : 2) . In various embodiments, the substitution of the amino acid is done with a positively charged residue or a basic residue of a group consisting of lysine, arginine and, less preferably, histidine. In a preferred embodiment, the mutant β subunit has two, three or more amino acid substitutions selected from a group consisting of ßl58R, BE63R and ßL69R. For example, a preferred β subunit, also referred to herein as β3R, comprises three mutations βl58R + BE63R + ßL69R. The mutant TSH, the analogs, derivatives and fragments of the TSH of the invention having β subunits also have a subunit a with an amino acid substitution at position 22 (as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1)) or a serum half-life that is greater than a of the wild-type TSH. In one embodiment, a mutant β subunit comprising one or more amino acid residue substitutions in the β TSH subunits is covalently linked to the carboxyl terminal extension peptide (CTEP) of the hCG. CTEP, which comprises the amino acids of the carboxyl terminus 32 of the β subunit of hCG (as shown in Figure 3), is covalently linked to the β-glycoprotein subunit, preferably the carboxyl terminus of the β-glycoprotein subunit is linked covalently with the amino terminus of CTEP. The β subunit of CTEP can be covalently linked to CTEP by any method known in the art, for example, a peptide bond or by a heterobifunctional reagent capable of forming a covalent bond between the amino terminus and the carboxyl terminus of a protein, for example, but not limited to, a peptide linker. In a preferred embodiment, the mutant β subunit and the CTEP are linked by means of a peptide bond. In several preferred embodiments, the mutant β-CTEP subunit fusions may comprise one, two, three or more amino acid substitutions selected from the group consisting of βl58R, BE63R and βL69R. In another embodiment, a β subunit is fused, for example, by covalent bond, with a subunit a, preferably a glycoprotein subunit (eg, as described in Section 5.2 above). The mutant β subunits of the invention are active in their function, that is, capable of displaying one or more functional activities related to the wild-type β subunit. Preferably, the mutant β subunit is capable of non-covalently associating with a β-subunit of wild type or mutant β subunit to form a heterodimer of wild-type TSH that binds to TSHR. Preferably, such a heterodimer of the wild type also triggers signal transduction. More preferably, the TSH heterodimer comprising a subunit ß mutant has an in vitro biological activity or in vivo biological activity greater than wild-type TSH. In the present invention it is contemplated that more than one mutation may be combined within a mutant β subunit to produce a heterodimer of the mutant TSH having a biological activity considerably greater than the wild-type TSH. The inventors discovered that multiple mutations within a subunit and modifications to increase the half-life of the TSH heterodimer (this is, β-CTEP subunit fusion or β subunit fusion-subunit fusion) can act synergistically to achieve biological activity that is greater than the sum of the increase in mutations and lasting action modifications. (It is also contemplated that mutations of the OI subunit are combined with strategies to increase the serum half-life of the TSH heterodimer having subunit a (ie, a heterodimer of TSH with a β-CTEP subunit fusion or a fusion of subunit β-subunit a) Mutations within a subunit and the long-acting modifications act synergistically to produce an unexpected increase in biological activity The mutant C i subunit can be tested for the desired activity by means of procedures known in the art, including, but not limited to, the assays described in Section 5.8 5.3 HERMEROMERS OF MUTATING AND ANALOG TSH TSH The present invention provides heterodimers of human TSH and human TSH analogs comprising a subunit or mutant and a mutant β subunit, wherein the mutant subunit comprises individual or multiple amino acid substitutions, preferably located in or near the fork structure ß Ll of the subunit a (as described in Section 5.1), and where the mutant β subunit comprises individual or multiple amino acid substitutions, preferably located on or near the structure in ß L3 hairpin of the ß subunit (as described in Section 5.2), whose heterodimer or analogue is also modified to increase the serum half-life (for example by ß-CTEP subunit fusion or ß-subunit subunit fusion) . Individual or multiple amino acid substitutions in the β subunit of the mutant TSH can be made in amino acid residues selected from positions 52-87, and preferably positions 58-69, of the amino acid sequence of the β subunit of human TSH . A non-limiting example of said mutant TSH comprises a heterodimer of the mutant subunit, OI4K, and the mutant β subunit, β3R, as described above. In one embodiment, the invention provides TSH heterodimer comprising a subunit a, preferably a subunit a mutant and a subunit β, preferably a subunit β mutant, wherein the subunit a mutant or subunit β mutant is fused with the CTEP of the β subunit of hCG (as described in Section 55.2) The term "fusion protein" refers herein to a protein that is the product of the covalent linkage of two peptides. Covalent linkages include any method known in the art for lig two peptides covalently in their amino and carboxyl terms, respectively, said methods include, but are not limited to, binding through a peptide bond or through a reagent heterobifunctional, for example, but not by way of limitation, a peptide linker. In a preferred embodiment, the heterodimer of the mutant TSH may comprise a subunit to human mutant and a β subunit of the human mutant TSH, wherein the β subunit of the human mutant TSH is covalently linked at its carboxyl terminus with the amino terminus of CTEP. the present invention also relates to single chain human TSH analogues comprising a human mutant subunit covalently linked (as described above for the fusion of the β-CTEP subunit) to a β subunit of the human mutant TSH , wherein the mutant human subunit OI - and subunit β of the human mutant TSH each comprise at least one amino acid substitution in the amino acid sequence of the respective subunit. In a preferred embodiment, the mutant β subunit is bound via a peptide linker to a mutant subunit. In a more preferred embodiment, the CTEP of hCG which has a high content of serine / proline and lacks considerable secondary structure, is the peptide linker. Preferably, the mutant OI subunit comprising individual or multiple amino acid substitutions, preferably located at or near the ßL1 hairpin structure of the a subunit (as described in Section 5.1, supra) is covalently linked to a β subunit. mutant comprising individual or multiple amino acid substitutions, preferably located at or near the ß L3 hairpin structure of the β subunit (as described in Section 5.2, supra). In one embodiment, the β subunit of the human mutant TSH comprising at least one amino acid substitution at the amino acid residues selected from positions 52-87, preferably positions 58-69, of the amino acid sequence of the ß Subunit of human TSH is covalently linked at its carboxyl terminus to the amino terminus of a subunit a of wild-type human TSH or an a subunit of mutant TSH comprising at least one amino acid substitution, wherein one or more substitutions are given in the amino acid residues selected between positions 8-30 and preferably 11-22 of the amino acid sequence of the human subunit. The mutant subunit or β subunit of the human mutant TSH may lack its signal sequence. The present invention also provides a human TSH analog that comprises a β subunit of the human mutant TSH covalently linked to the CTEP which is, in turn, covalently linked to a subunit to a mutant, where the subunit to mutant and the β subunit of the human mutant TSH each comprise at least one amino acid substitution in the amino acid sequence of each of the subunits. In a particular embodiment, a mutant β-subunit fusion-CTEP is covalently linked with a subunit to a mutant, such that the carboxyl terminus of the mutant β subunit is linked to the amino terminus of the mutant OI subunit through the CTEP or the hCG. Preferably, the carboxyl terminus of the mutant β subunit is covalently linked to the amino terminus of the CTEP, and the carboxyl terminus of the CTEP is covalently linked to the amino terminus of a mutant subunit without the signal peptide. Accordingly, in a particular embodiment, the human TSH analog comprises a β subunit of the human mutant TSH comprising at least one amino acid substitution at amino acid residues selected from positions 58-69 of the sequence of amino acids of the β subunit of human TSH covalently linked at the carboxyl terminus of the β subunit of the human mutant TSH with the amino terminus of the CTEP which is covalently bound at the carboxyl terminus of the carboxyl terminal extension peptide mentioned with the amino terminus of the mutant subunit comprising at least one amino acid substitution, wherein one or more of the substitutions are at amino acid residues selected from positions 11-22 of the amino acid sequence of the human subunit. In another preferred embodiment, the heterodimer of the mutant TSH comprises a subunit a having an amino acid substitution at position 22 of the sequence of the human subunit (as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1)), preferably a substitution with a basic amino acid (eg, arginine, lysine and, less preferably, histidine), more preferably with arginine. In particular embodiments, the TSH mutant heterodimer comprising at least one mutant subunit or the single chain TSH analog described above is active in function, that is, capable of showing one or more functional activities related to wild-type TSH, such as TSHR binding, TSHR signaling and extracellular secretion. Preferably, the heterodimer of the mutant TSH or the single chain TSH analog is capable of binding to the TSHR, preferably with higher affinity than that of the wild-type TSH. In addition, it is also preferable that the heterodimer of the mutant TSH, or the single chain TSH analog, triggers the signal transduction. More preferably, the heterodimer of the mutant TSH comprising at least one mutant subunit or the single chain TSH analog of the present invention has a biological activity in vitro or in vivo greater than the wild-type TSH and has a life serum media longer than wild-type TSH. The heterodimers of the mutant TSH and the single chain TSH analogs of the invention can be tested for the desired activity by methods known in the art, including, but not limited to, the assays described in Section 5.8.

In Section 6, working examples of heterodimers of the mutant TSH are described. 5.4 POLYUCLEOTIDES OF MUTATING TSH AND MUTANT ANALOGUES The present invention also relates to nucleic acid molecules comprising sequences encoding mutant subunits of human TSH and TSH analogs of the invention, wherein the sequences contain at least one insertion , deletion or basic substitution, or combinations of these that cause additions, deletions and individual or multiple substitutions of amino acids, in relation to wild-type TSH. The basic mutation that does not alter the reading frame of the coding region is preferred. As applied herein, when it is claimed that two coding regions are fused the 3 'end of the nucleic acid molecule is ligated to the 5' end (or by means of a nucleic acid encoding a peptide linker) of the other nucleic acid molecule, such that the translation proceeds from the coding region of the nucleic acid molecule within the other without a frame change. Due to the degeneracy of the nucleotide coding sequences, any other DNA sequence encoding the same amino acid sequence for a mutant a or β subunit can be used in the practice of the present invention. These include, but are not limited to, nucleotide sequences comprising all or parts of the coding region of the a or β subunit that are altered by the substitution of different codons that encode the same amino acid residue within the sequence, producing so a silent change. In one embodiment, the present invention provides nucleic acids comprising sequences encoding subunits to mutants, wherein subunits to mutants comprise individual or multiple amino acid substitutions, preferably located within or near the hairpin structure. the subunit a (as described in Section 5.1). In a particular embodiment, the invention provides nucleic acids encoding subunits to mutants having an amino acid substitution at position 22 of the amino acid sequence of subunit a as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER) : 1); preferably a substitution with a basic amino acid, more preferably a substitution with arginine. The present invention further provides nucleic acid molecules comprising sequences that encode mutant β subunits that comprise single or multiple amino acid substitutions, preferably located within or near the ß L3 hairpin structure of the β subunit. Nucleic acid molecules encoding a single chain TSH analog are also provided, wherein the carboxyl terminus of the mutant β subunit is linked to the amino terminus of the mutant subunit via the CTEP of the β subunit of the hCG . In a preferred embodiment, the nucleic acid molecule encodes a single chain TSH analog, wherein the carboxyl terminus of a mutant β subunit is covalently linked to the amino terminus of CTEP, and the carboxyl terminus of CTEP it is covalently linked to the amino terminus of a subunit to a mutant without the signal peptide. The single chain analogs of the invention can be made by ligating the nucleic acid sequences encoding the α and β subunits with each other through methods known in the art, within the appropriate coding framework, and expressing the fusion protein. through methods commonly known in the art. Alternatively, the fusion protein can be made with protein synthesis techniques, for example, with the use of a peptide synthesizer. 5.5 PREPARATION OF SUBUNITIES AND ANALOGUES OF MUTATING TSH The production and utilization of subunits to mutants, mutant ß subunits, mutant TSH heterodimers, TSH analogs, single chain analogs, derivatives and fragments thereof of the invention are found within of the field of application of the present invention. In this document, methods for manufacturing the above are described. . 5.1 CLONING OF THE GENE OF TSH The nucleotide sequences of the cDNA and the gene coding of the common human subunit (Fiddes and Goodman, 1981, J. Mol. Appl. Gen. 1: 3-18) and of the ß subunit of human TSH (Hayashizaki et al., 1985, FEBS Lett, 88: 394-400, Wondisford et al., 1988, J. Bio, Chem. 263-12538-12542, Wondisford et al., 1988, Mol. Endocrinol 2: 32-39). The coding regions for the subunits can be obtained by means of standard procedures known in the art from cloned DNA (eg, a "library" of DNA), by chemical synthesis, by cloning cDNA, or by cloning Genomic DNA, or fragments thereof, purified and taken from the desired cell (see, for example, Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, Glover, DM (ed.), 1985, • DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, United Kingdom, Vol. I, II). The polymerase chain reaction (PCR) can be used to amplify the sequences encoding the common o o or β subunits of TSH in a genomic or cDNA library. Synthetic oligonucleotides can be used as primers to amplify the PCR sequences from a source (RNA or DNA), preferably a cDNA library. The DNA that is amplified can include cDNA or genomic DNA of any human being. After successful isolation or amplification of a segment of a subunit, that segment can be cloned and sequenced in molecular terms, and used as a probe to isolate a complete cDNA or genomic clone. This, in turn, will allow the characterization of the nucleotide sequence of the gene, and the production of its protein product for functional analysis or therapeutic or diagnostic use, as described infra. They stand out among the alternatives for isolating the coding regions, but they are not limited to synthesizing the sequence of the gene itself from the published sequence by chemical means. Other methods are possible and are within the scope of the invention. It is not intended that the above methods limit the following general description of the methods by which the mutants of the subunits of the hormones can be obtained. The identified and isolated gene can be inserted into an appropriate cloning vector for the amplification of the gene sequence. A large number of vector-host systems known in the art can be used. Possible vectors include, but are not limited to, modified plasmids or viruses, bacteriophages such as lambda derivatives, or plasmids such as the plasmid derivatives pBR322 or pUC or the BLUESCRIPT vector (Stratagene). The insertion into a cloning vector can, for example, be achieved by ligating the DNA fragment in a cloning vector having complementary cohesive terms. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may have a different enzymatic composition. Alternatively, any desired site can be produced by ligating nucleotide (linker) sequences with the terms of the DNA; these ligated linkers may comprise oligonucleotides in particular synthesized by chemical means encoding restriction endonuclease recognition sequences. In an alternative method, the dissociated vector and the gene of the mutant subunit can be modified by homopolymer tail. Recombinant molecules can be introduced into the host cells through transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated. In an alternative method, the desired gene can be identified and isolated after insertion into an appropriate cloning vector following a "gun shot" approach. The enrichment of the desired gene, for example, by fractionation, can be done before insertion into the cloning vector. In specific embodiments, the transformation of the host cells with recombinant DNA molecules comprising the mutant subunit gene, cDNA, or the synthesized DNA sequence allows the generation of multiple copies of the gene. Thus, the gene can be obtained in large quantities by means of growth transformers, isolating the recombinant DNA molecules from the transformers and, when necessary, recovering the inserted gene from the isolated recombinant DNA. Gene copies are used in mutagenesis experiments to study the structure and function of the mutant, heterodimer subunits of TSH and TSH analogs. 5.5.2 MUTAGENESIS Mutations present in mutant subunits a and β, heterodimers of mutant TSH, TSH analogs, single chain analogues of the TSH of the invention can be produced by various methods known in the art. Manipulations that occur in their production can occur in genes or proteins. For example, the cloned coding region of the subunits can be modified by any of the numerous strategies known in the art (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring , Harbor, New York).

The sequence can be dissociated at appropriate sites with restriction endonuclease (s), followed by additional enzymatic modification, if desired, isolated and ligated in vitro. In the production of a mutant subunit, care must be taken to ensure that the modified gene remains within the same translation reading frame, uninterrupted by translation stop signals, in the region of the gene where the subunit is encoded. In addition, the nucleic acid sequence encoding the subunits can be mutated in vivo or in vivo, to create variations in the coding regions for example, amino acid substitutions), or to create or destroy the translation, initiation or termination sequences, or form new restriction endonuclease sites or destroy those that existed before, to further facilitate in vitro modification. Any method for mutagenesis known in the art can be applied, including, but not limited to, chemical mutagenesis, site-directed in vitro mutagenesis (Hutchinson, C, et al., 1978, J. Biol. Chem 253: 6551), extension of overlap based on PCR (Ho et al., 1989, Gene 77:51:59), mutagenesis of mega-initiator based on PCR (Arkar et al., 1990, Biotechniques, 8: 404-407), etc. Mutations can be confirmed by sequencing dideoxy DNA with double helix. One or more residues within a subunit can be replaced by another amino acid, preferably with one having different properties, to generate a range of functional differentials. Substitutes of an amino acid within the sequence can be selected from members of a different class to which the amino acid belongs. Non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The amino acids with positive charge (basic) include arginine, lysine and histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid. Manipulations of the mutant subunit sequence can also be done on the proteins. Included within the scope of the invention are the mutant subunits, the heterodimer of the mutant TSH, the TSH analogs, single chain analogues which are differentially modified during or after translation, for example, by glycosylation, acetylation, phosphorylation, amidation, derivation by means of known protection / blocking groups, proteolytic cleavage, binding to an antibody molecule or other cellular ligand, etc. Various chemical modifications can be made by known techniques, including but not limited to, chemical cleavage in particular by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBh; acetylation, formylation, oxidation, reduction; Metabolic synthesis in the presence of tunicamycin, etc. In addition, mutant subunits and single chain analogues of TSH can be synthesized by chemical means. For example, a peptide corresponding to a portion of a mutant subunit comprising the desired mutated domain can be synthesized by the application of a peptic synthesizer. Furthermore, if desired, non-classical amino acids, amino acid chemical analogs can be introduced as substitution or addition within the sequence of the mutant subunit. Non-classical amino acids include, but are not limited to, common amino acid D-isomers, isobutyric acid or -isobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, and-Abu, e-Ahx, 6- amino hexanoic, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citruiline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ß-alanine, fluoro acids -amino, designer amino acids such as β-methyl amino acid, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. In addition, the amino acid may be D (dextrorotatory) or L (levorotatory).

In particular embodiments, the mutant or analogue subunit of TSH is a fusion protein comprising, for example, but not limited to, mutant CTEP subunit and the CTEP of the β subunit of hCG or a β subunit mutant and a mutant subunit. In one embodiment, said fusion protein is produced by the recombinant expression of a nucleic acid encoding a mutant or wild-type subunit bound in frame to the coding sequence of another protein, for example, but not limited to , toxins, for example ricin or diphtheria toxin. Said fusion protein can be made by ligating the appropriate nucleic acid sequences encoding the amino acid sequences to each other, through methods known in the art, in the appropriate coding framework, and expressing the fusion protein by means of commonly known methods in the technique. Alternatively, said fusion protein can be made with synthetic protein techniques, for example, by the use of a peptide synthesizer. Chimeric genes comprising portions of the α or β subunit fused to any heterologous protein coding sequence can be created. One particular embodiment refers to a single chain analog comprising subunit or mutant fused to a mutant β subunit preferably with a peptide linker between the mutant OI subunit and the mutant β subunit. 5.6 EXPRESSION THE GENES OF THE MUTATING SUBUNITY The nucleotide sequence coding of a mutant subunit of TSH, or an analog or fragment or other derivative thereof, active in function (see Section 5.4), can be inserted into a appropriate character vector, that is, a vector containing the necessary elements for the transcription and translation of the protein coding sequence. The cDNA or the common gene of the a subunit, or the cDNA or gene of the β subunit of the human TSH can also supply the necessary transcription and translation signals or the genomic sequences that flank these two genes. A variety of host-vector systems can be used to express the protein coding sequence. They stand out among these, but are not limited to, mammalian cell systems infected with viruses (eg, vaccinia virus, adenovirus, etc.); insect cell systems infected with viruses (for example, baculovirus); microorganisms for example yeast containing yeast vectors. The expression of vector elements varies in their strength and their specifics. Depending on the host-vector system used, various elements of transcription and translation may be used. In particular embodiments, a subunit is expressed to a human mutant that encodes a region or a β subunit of the human mutant TSH that encodes a region, or a sequence that encodes a mutated and active portion in terms of function. Any of the methods described above for inserting DNA fragments into a vector can be applied to construct expression vectors containing a chimeric gene consisting of the transcription / translation control signals and the appropriate protein coding sequences. These methods include recombinant DNA in vitro and synthetic and recombinant techniques in vivo (genetic recombination). The expression of the nucleotide sequence encoding a mutant OI subunit or β subunit of the mutant TSH, or peptide fragments thereof can be regulated by a second nucleotide sequence, such that the mutant subunit (s) or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, the expression of a subunit to mutant or β-subunit of the TSH or peptide fragments thereof can be controlled with any promoter / scavenger element known in the art. The promoters that can be used, but are not limited to, are the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter contained in the terminal repeat of the 3 'long Rous sarcoma virus (Yamamoto, et al. al., 1980, Cell 22: 787-797), the herpes thymidine quinidase promoter (Wagner et al., 1981, Proc. Nati, Acad. Sci. USA 78: 1441-1445), the regulatory sequences of the metallothionein gene (Brlnster et al., 1982, Nature 296: 39-42). In a particular embodiment, a vector comprising one or more promoters functionally linked to the coding region of a mutant subunit or a β subunit of the mutant TSH or both, one or more origins of replication is used. and, optionally, one or more selectable markers (eg, a gene with resistance to antibiotics). The expression of two subunits within the same eukaryotic host cell is preferred since said co-expression favors the proper assembly and glycosylation of a functional heterodimer of TSH. Thus, in a preferred embodiment, said vectors are used to express both the mutant subunit and the β subunit in a host cell. The coding region of each of the mutant subunits can be cloned into different vectors; the vectors are introduced into a host cell in sequence or simultaneously. Alternatively, the coding regions of both subunits can be inserted into a vector to which the appropriate promoters are functionally linked. A host cell strain can be chosen that modulates the expression of the inserted sequences, or that modifies and processes the genetic product in the desired manner. The expression of certain promoters can be raised in the presence of certain inducers; thus, the expression of mutant subunits created by genetic engineering can be controlled. Moreover, the different host cells have characteristic and specific mechanisms for processing and translation modification and subsequent to it (eg, glycosylation, protein phosphorylation). Appropriate cell lines or host system can be chosen to ensure the desired modification and processing of the expressed foreign protein. Expression in mammalian cells can be used to ensure the "native" glycosylation of a heterologous protein. Moreover, the different vector / host expression may affect the processing reactions in different measures. When a recombinant host cell expressing the genetic sequence of the a or β subunit of the mutant TSH is identified, the genetic product can be analyzed. This is achieved through tests based on the physical or functional properties of the product, including radioactive labeling of the product followed by analysis by gel electrophoresis, immunological assay, etc. 5.7 GENERATION OF ANTIBODIES TO MUTATING AND ANALOGUE SUBUNITIES OF IT In accordance with the invention, the subunits a and ß mutants, the heterodimers. of the mutant TSH, the TSH analogs, the single chain TSH analogues, their fragments or other derivatives thereof, can be used as an immunogen to generate antibodies that immunologically bind specifically with said immunogen. They stand out among the antibodies, but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an expression library. In a particular embodiment, antibodies of a mutant TSH are produced. In another embodiment, antibodies of a domain of a subunit a or β mutants are produced. A number of methods are known in the art for producing polyclonal antibodies to a or β subunits, mutant TSH heterodimers, TSH analogs, single chain TSH analogs, fragments thereof or other derivatives thereof. For the production of the antibody, various host animals can be immunized by injection of the subunits, the heterodimer, the single chain analog and derivatives thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants can be used to increase the immune response, depending on the species of the host, and even but not limited to, Freund's (complete and incomplete), mineral gels, for example, aluminum hydroxide, surface-active substances, for example lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, hemocyanins, keyhole limpet hemocyanins, dinitrofen, and possibly useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and corynebacterium parvum. For the preparation of monoclonal antibodies directed towards subunits and mutant ß, heterodimers of mutant TSH, TSH analogs, single chain TSH analogs, fragments thereof or other derivatives thereof, any technique that establishes the production of antibody molecules by continuous cell lines in cultures. For example, the hybridoma technique originally devised by Kohier and Milstein (1975, Nature 256: 495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV hybridoma technique for producing monoclonal antibodies (Colé et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In a further embodiment of the present invention monoclonal antibodies can be produced in germ-free animals using recent technology (PCT / US90 / 02545). According to the invention, human antibodies can be used using human hybridomas (Cote et al., 1983, Proc. Nati, Acad. Sci. USA 80: 2026-2030) or by transforming human B cells with the EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, according to the invention, the techniques invented for the production of "chimeric antibodies" can be applied (Morrison et al., 1984, Proc. Nati Acad.Sci.U.S.A. 81: 6851-6855; Neubergeret al., 1984, Nature 312: 604-608; Takeda et al., 1985, Nature 314: 452-454) by splicing genes from a mouse antibody molecule specific for the epitope together with the genes of a human antibody molecule with the appropriate biological activity; such antibodies are within the scope of the present invention. In accordance with the invention, the techniques described for the production of single chain antibodies (US Pat. No. 4,946,778) can be adapted to produce specific single chain antibodies against the antibodies. subunits, heterodimers, TSH analogs their fragments or other derivatives thereof. A further embodiment of the invention applies the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246: 1275-1281) to allow rapid and easy identification of the monoclonal Fab fragments with specificity desired.

Antibody fragments that contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include, but are not limited to, the F (ab ') 2 fragment that can be produced by the digestion of the pepsin of another antibody molecule; the Fab 'fragments that can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments. In the production of antibodies, the selection of the desired antibody can be achieved by methods known in the art, for example ELISA (immunological absorption assay linked with enzymes, for its acronym in English). For example, to select antibodies that recognize a specific domain of a mutant subunit, hybridomas generated for a product that binds to a fragment of a mutant subunit containing said domain can be tested. For the selection of an antibody that specifically binds to a mutant TSH subunit, the mutant TSH or a single chain analog but which does not bind specifically to wild-type TSH, can be selected based on the positive link to the mutant and the lack of binding to the wild-type protein. Antibodies specific to a domain of a mutant TSH or a single chain analog are also provided. The above antibodies can be used in methods known in the art related to the location and activity of the subunits of the mutant TSH, the mutant TSH or a single chain analog of the invention, for example, to copy the image of these proteins, measure their levels in the appropriate physiological samples, in diagnostic methods, etc. 5.8 ANALYSIS OF MUTATING TSH SUBUNITIES This document describes methods for determining the structure of mutant TSH subunits, mutant heterodimers and TSH analogues, and for analyzing in vitro activities and biological functions in vivo. of the above. When a subunit or β of the TSH is identified, it can be isolated and purified by normal methods, including chromatography (eg ion exchange chromatography, affinity chromatography and sizing column), centrifugation, differential solubility or by of any other normal technique for protein purification. The functional properties can be evaluated by applying any relevant assay (including the immunological assays described below). Alternatively, when an α or β subunit of TSH is produced by a recombinant host cell, the amino acid sequence of the subunit (s) can be determined by standard techniques for protein sequencing, for example, with an automatic amino acid sequencer The sequence of the mutant subunit can be characterized by a hydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Nati. A'cad. Sci. USA 78: 3824) . A hydrophilic profile can be used to identify the hydrophobic and hydrophilic regions of the subunit and the corresponding regions of the genetic sequence encoding said regions. A secondary structural analysis can also be made (Chou, P. and Fasman, G., 1974, Biochemistry 13: 222) to identify the regions of the subunit that assume specific secondary structures. Other methods of structural analysis can also be used. Among these, X-ray crystallography (Engstom, A., 1974, Biochem. Exp .. Biol. 11: 7-13) and computer modeling (Fletterick, R. and Zoller, M. (eds.), 1986, Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). In addition, prediction of structures, analysis of crystallographic data, sequence alignment, as well as the creation of homology models, using computer software programs available in the art can be achieved., for example, BLAST, CHARMm version 21.2 for Convex, and QUANTA v.3.3, (Molecular Simulations, Inc., York, United Kingdom). The activity of the mutant subunits, the mutant β subunits, the mutant TSH heterodimers, the TSH analogs, the single chain analogs, the derivatives and fragments thereof can be assayed by various methods known in the art. For example, if a test of the ability of a mutant subunit or mutant TSH is made to bind or compete with the wild-type TSH or its subunits to bind to an antibody, several immunological assays known in the art may be applied, but not limited to competitive and non-competitive testing systems applying techniques such as radioactive immunological assays, ELISA (enzyme-linked immunological absorption assay), sandwich immunological assays, radiometric immunological assays, precipitin reactions gel diffusion, immunological diffusion assays, in situ immunological assays (using colloidal gold labels, enzymes or radioactive isotopes, for example), western blot, precipitation reactions, agglutination assays (eg, gel agglutination assays, blood clumping tests), complementary fixation tests, immunological tests Fluorescence assays, protein A assays, and immunoprecipitation assays, etc. The antibody binding can be detected by detecting the primary antibody label. Alternatively, the primary antibody is detected by detecting the binding of a second antibody or reagent to the primary antibody, particularly in the case that the secondary antibody is labeled. Many means are known in the art to detect binding in an immunological assay and are within the scope of the present invention. The binding of subunits to mutants, mutant β subunits, mutant TSH heterodimers, TSH analogues, single chain analogues, derivatives and fragments thereof, to the thyroid stimulating hormone receptor ( TSHR) can be determined by methods well known in the art such as, but not limited to, bovine TSH, as described by Szkudlinski et al. (1993, Endocrinol, 133: 1490-1503). The biological activity of the mutant TSH heterodimers, TSH analogs, single chain analogs, derivatives and fragments thereof can also be measured, for example, by assays based on cyclic AAMP stimulation in cells expressing the TSHR, for example, but not limited to, the assays described in Section 6.2.3 mfra and by Grossmann et al. (1995, Mol.Endocrinol., 9: 948-958); and the stimulation of thymidine uptake in thyroid cells, for example, but not limited to, as described by Szkudlmski et al. (1993, Endocrinol, 133: 1490-1503). Biological activity in vivo can be determined by means of binding ratios of binding TSHR in animal models, such as measurement of secretion of mice in the post-injection heterodimer of the mutant TSH, the analog of TSH, or the single chain analog, for example as described in East-Palmer et al. (1995, Thyroíd 5: 55-59) and in Section 6.2, supra. For example, wild-type TSH and mutant TSH are intrapeptoneally injected into male albino Swiss Crl: CF-1 mice with endogenous TSH previously suppressed by administering 3 μg / ml T3 in drinking water for 6 days. Blood samples are taken 6 hours after the orbital sinus and serum T4 and TSH levels are measured by respective chemical luminescence tests (Nichols Institute). The half-life of a protein is a measure of the stability of the protein and indicates the time needed for a reduction in the concentration of the protein by half. The half-life of a mutant TSH can be determined by any method for measuring TSH levels in samples taken from a subject over time, for example, but not limited to, immunological assays with the use of antibodies. anti TSH to measure the levels of the mutant TSH present in the samples taken during a period of time after the administration of the mutant TSH or the detection of a mutant TSH with radioactive labeling in samples taken from a subject after the administration of the Mutant TSH with radioactive labeling. The person skilled in the art will know other methods that are within the scope of the invention. 5.9 APLICATIONS FOR DIAGNOSIS AND THERAPEUTICS The invention provides treatment or prevention of various diseases and conditions by administration of the therapeutic compound (hereinafter referred to as the "Therapeutic") of the invention. Therapeutic heterodimers which have a subunit a with at least one amino acid substitution at position 22 of the subunit stand out among said Therapeutics as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1) and either a subunit ß mutant or wild type, preferably with one or more amino acid substitutions at or near the Ll structure (amino acids 8-30 as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1)) and a β subunit, preferably with one or more amino acid substitutions at or near the L3 structure (amino acids 52-87 as shown in Figure 2 (SEQUENCE IDENTIFICATION NUMBER: 2)) and covalently linked to the CTEP of the β subunit of the hCG; heterodimers of the TSH with a subunit a, preferably with one or more amino acid substitutions at or near preferably with one or more amino acid substitutions at or near the Ll structure and a β subunit, preferably with one or more amino acid substitutions at or near the L3 structure, where the mutant subunit and the mutant β subunit,. are covalently linked to form a single chain analog, including a TSH heterodimer where the subunit a and the β subunit and the CTEP of the a subunit of hCG are covalently linked to a single chain analog, other derivatives, analogs and fragments thereof (eg, as described above) and the nucleic acids encoding the TSH heterodimers of the invention and derivatives, analogs and fragments thereof. The subject to whom the Therapeutic is administered is preferably an animal, but is not limited thereto, for example, cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal. In a preferred embodiment, the subject is a human being. In general, the administration of products that originates in a species that is the same as the subject is preferred. Thus, in a preferred embodiment, a mutant or modified human heterodimer of the TSH, derivative or analog, or a nucleic acid, is administered to a human patient, therapeutically or to make the diagnosis. In a preferred aspect, the Therapeutics of the invention is substantially purified. Many conditions manifested as hypothyroidism can be treated with the methods of the invention. Conditions in which TSH is absent or less in relation to normal or desired levels are treated or prevented by administration of the heterodimer or TSH analog of the invention. Conditions in which the TSH receptor is absent or less or non-sensitive or less sensitive than TSHR to wild-type TSH can also be treated by administration of a heterodimer of the mutant TSH or the analog of TSH. An active TSHR by constitution can cause hyperthyroidism and it is contemplated that the heterodimers of TSH and TSH analogs can be used as antagonists. In specific embodiments, heterodimers of TSH or TSH analogs capable of stimulating differentiated thyroid functions, even prophylactically, are administered therapeutically. Diseases and conditions that can be treated or prevented include, but are not limited to, hypothyroidism, hyperthyroidism, thyroid development, thyroid cancer, benign drops, enlarged thyroid, protection of thyroid cells from apoptosis, etc. . The absence of a lower level in the protein or the function of the TSH, or the protein and the function of the TSHR can be detected easily, for example, by obtaining a sample of the patient's tissue (for example, with biopsy tissue) and testing it in vitro in terms of RNA or protein levels, structure or activity of the expressed RNA or protein of TSH or TSHR. Thus, many normal methods in the art may be applied, including but not limited to, in immunological tests to detect or visualize the TSH protein or the TSHR (eg Northern blots, dot blot, in situ hybridization, etc.), etc. In particular embodiments, the Therapeutics of the invention are used to treat thyroid cancer. The mutant and analogue heterodimers of TSH are useful for the stimulation of thyroid and metastatic tissue before therapeutic ablation with radioactive iodine. For example, the heterodimer of the TSH of the invention can be administered to a patient suffering from thyroid cancer before administration of the iodine with radioactive labeling for radio ablation. The Therapeutics of the invention can also be used to stimulate the absorption of iodine by benign multinodular drops before radio ablation for the treatment of multinodular drops., or to stimulate the absorption of thyroid tissue before radio ablation for the treatment of thyroid enlargement. In particular, the radio ablation therapy is carried out with the Therapeutic of the invention, preferably administered intramuscularly, in a regimen of one to three doses, for example, but not limited to, one dose per day for two days, or one dose the first, fourth and seventh days of a regime of several days. The dosage is an appropriate dose, as described in Section 5.10 below, for example, but not limited to a dose of about 10 μg to 1 mg, preferably a dose of 10 μg to 100 μg. One day after the last dose of the regimen, the subject is administered iodine with radioactive labeling, preferably 131 I, in an amount sufficient to treat the cancer, the non-cancerous gout and the enlarged thyroid. The amount of iodine with radioactive labeling to be administered will depend on the type and severity of the disease. In general, 30 to 300 mCi of 131I are given to treat thyroid carcinoma. In other specific embodiments, the heterodimers of the mutant TSH may be used for the targeted delivery of Thyroid Therapeutics or thyroid cancer cells, for example, for the targeted delivery of nucleic acids for gene therapy (eg. example, targeted delivery of tumor suppressor genes to thyroid cancer cells) or for the targeted delivery of toxins, for example, but not limited to, ricin, diphtheria toxin, etc. The invention further comprises methods for diagnosis, prognosis, screening for thyroid cancer, preferably thyroid carcinoma, and for monitoring the treatment of thyroid cancer, for example, by the invention of the TSH heterodimers of the invention. In particular embodiments, the Therapeutics of the invention are administered to a subject to stimulate the absorption of iodine (preferably iodine with radioactive labeling, for example, but not limited to, 131 I or 125 I) by thyroid cells (including thyroid cancer cells) or to stimulate thyroglobulin secretion from thyroid cells. Upon subsequent administration of the Therapeutics, iodine with radioactive labeling may be administered to the patient, and then, the presence and location of iodine with radioactive labeling (ie, thyroid cells) may be detected in the subject (eg, but not by way of limitation). , whole body scan) or the thyroglobulin levels in the subject can be measured or detected, where the highest levels of radioactive iodine absorption or thyroglobulin secretion, compared to levels in a subject not suffering from cancer or Thyroid disease or with a normal level, indicates that the subject has thyroid cancer. Certain subjects who have undergone thyroidectomy or thyroid tissue ablation therapy may have little or no residual thyroid tissue. In these subjects, or any other subject lacking non-cancerous thyroid cells, the detection of any iodine uptake or thyroglobulin secretion (over any residual level remaining after thyroidectomy or ablation therapy or after loss of thyroid tissue by any other reason) indicates the presence of thyroid cancer cells. The location of the iodine with radioactive labeling in the subject can be used to detect an expansion or metastasis of the disease or malignant tumor. In addition, the diagnostic methods of the invention can be applied to monitor the treatment of thyroid cancer by measuring the change of iodine with radioactive labeling or thyroglobulin levels in response to a course of treatment or detecting regression or growth or metastasis of the thyroid. thyroid tumor. In particular, the diagnostic methods are carried out by administering the Therapeutics of the invention, preferably intramuscularly, in a regimen of one to three doses, for example, but not limited to, one dose per day for two days, or one dose the first, fourth and seventh days of a regimen of several days. The dosage is an appropriate dose, as described in Section 5.10 below, for example, but not limited to a dose of about 10 μg to 1 mg, preferably a dose of 10 μg to 100 μg. One day after the last dose of the regimen, the subject is administered iodine with radioactive labeling, preferably 131I, in an amount sufficient for the detection of thyroid cells (including cancer cells), in general 1-5 mCi of 131I is administered to diagnose Thyroid carcinoma. Two days after the administration of the iodine with radioactive labeling, the absorption of the iodine with radioactive labeling in the patient is detected or localized, for example, but not limited to, radioactive iodine exploration of the whole body. Alternatively, in cases where all or most of the thyroid tissue has been removed (for example, in patients with thyroidectomy or previous thyroid tissue ablation), thyroglobulin levels can be measured 2 to 7 days after administration of the last dose of the Therapeutics of the invention. Thyroglobulin can be measured by any method known in the art, including the application of a specific immunological assay for thyroglobulin, which is well known in the art. The heterodimer of the mutant TSH of the invention can also be used in TSH binding inhibition assays for the TSH receptor autoantibodies, for example, as described in Kakinuma et al. (1997, J. Clin. Endo. Met. 82: 2129-2134). Antibodies against the TSH receptor are present in certain thyroid diseases, for example, but not limited to, Graves' disease and Hashimoto's thyroiditis; therefore, these inhibition assays can be applied for the diagnosis of thyroid diseases such as Graves' disease and Hashimoto's thyroiditis. Briefly, the cells or the membrane containing the TSH receptor are contacted with the sample to be tested for TSHR antibodies and with the mutant TSH with radioactive labeling of the invention, the inhibition of the TSH binding with radioactive labeling of the invention in connection with the binding to the cells or membranes contacted with the mutant TSH with radioactive labeling but not with the sample to be tested, indicates that the sample to be tested has antibodies that bind to the receptor of TSH. The binding inhibition assay using heterodimers of the mutant TSH of the invention, which have higher biological activity than wild-type TSH, is more sensitive to antibodies to the TSH receptor than the link inhibition assay used by the TSH of wild type. Accordingly, an embodiment of the invention provides methods for diagnosing or selecting a disease or condition characterized by the presence of TSHR antibodies, preferably Graves' disease, comprising contacting cultured cells or isolated membranes containing recipients of TSH with a sample that putatively contains the antibodies of a subject with an amount effective from the diagnostic point of view of a heterodimer of the mutant TSH with radioactive labeling with the cultured cells or the isolated membrane, wherein a decrease in the binding of the TSH with radioactive labeling in relation to the binding in the absence of the sample or in the presence of an analogous sample without such a disease or condition indicates the presence of the disease or condition. Heterodimers and analogs can also be used in diagnostic methods to detect suppressed, but functional thyroid tissue in patients with thyroid nodules with hyper-function or exogenous thyroid hormone therapy. The heterodimers and analogs of the mutant TSH may have other applications, for example, but not limited to those of the diagnosis of combined central and primary and central hypothyroidism, thyroid hemiatrophy and resistance to the action of TSH. . 10 PHARMACEUTICAL COMPOSITIONS The invention provides diagnostic methods and methods of treatment by administering to a subject an effective amount of a Therapeutic of the invention. In a preferred aspect, the Therapeutics is considerably purified. The subject is preferably an animal, even, but is not limited to animals such as cows, pigs, horses, chickens, cats, dogs and is preferably a mammal. In a particular embodiment, the subject is a non-human mammal. The heterodimers of the mutant TSH and the TSH analogs of the invention are preferably tested in vitro, and then in vivo as desired, before their application in humans. In several specific embodiments, in vitro assays can be performed with cells representative of the cell types (eg, thyroid cells) present in the patient's condition, to determine whether a heterodimer of the mutant TSH or TSH analog has a desired effect in said cell types, for example as described in Section 5.8, supra. The compounds that will be applied in therapy can be tested in the model systems with appropriate animals before being tested in humans, including but not limited to rats, mice, chickens, cows, monkeys, rabbits, etc. For in vivo testing, prior to administration to humans, any animal model system known in the art can be used. Various delivery systems for administering the heterodimer of the mutant TSH or the TSH analogue of the invention are known and can be used, for example, the encapsulation of liposomes, microparticles, microcapsules, recombinant cells capable of expressing the heterodimer of the mutant TSH or the TSH analog, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 2 ^ 62: 4429-4432), etc. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal,. intravenous, subcutaneous, intranasal, epidural and oral. The compounds may be administered by any appropriate route, for example, by infusion or bolus injection, by absorption through epithelial or mucotutinal coatings (eg, oral mucosal, rectal and intestinal mucosa, etc.) and they can be administered together with other active agents from the biological point of view. The administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system through any appropriate route, including intraventricular and intrathecal injection; Intraventricular injection can be facilitated with an intraventricular catheter, for example, connected to a reservoir, for example an Ommaya reservoir. Pulmonary administration can also be applied, for example, by the use of an inhaler or nebulizer and the formulation with an aerosol agent. In a particular embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally in the area requiring treatment; this can be achieved for example, and not by way of limitation, with local infusion during surgery, by means of a catheter, by means of a suppository, or by means of an implant, the implant being porous, non-porous or gelatinous material , including membranes, for example sialastic membranes, or fibers. In another embodiment, the heterodimer of the mutant TSH or the TSH analog can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in The Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989), Lopez-Berestein, ibid., pp. 317-327, see ibid. ). In yet another embodiment, the heterodimer of the mutant TSH or the TSH analog can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra, Sefton, CRC Crit Ref Biomed Eng 14: 201 (1987), Buchwald et al .. Surgery 88: 507 (1980), Saudek et al. , N. Engl. J.

Med. 321: 574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Relay, Langer and Wise (eds.), CRC Pres., Boca Mouse, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev.

Macromol Chem. 23:61 (1983); see also Levy et al., Science 228: 190 (1985); During et al., Ann. Neurol. 25: 351 (1989); Howard et al., J. Neurosurg. 71: 105 (1989)). In yet another embodiment, a controlled release system can be placed close to the therapeutic target, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Reread, supra, vol 2, pp. 115-138 (1984)).

Langer discusses other controlled release systems in his review (Science 249: 1527-1533 (1990)). In a particular embodiment, a heterodimer of the mutant TSH encoding nucleic acid or a TSH analog can be administered in vivo to promote the expression of its encoded protein, constructing it as part of a nucleic acid expression vector and administering it for it to become intracellular, for example, by the use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by the use of bombardment of mircroparticles (e.g., a gene gun) , Biolistic, Dupont), or lipid coating or cell surface receptors or transfection agents, or by administering at the junction of a homeobox-like peptide that is known to enter the nucleus (see, for example, Joliot et al. ., 1991, Proc. Nati, Acad. Sci. USA 88: 1864-1868), etc. Alternatively, a heterodimer of the mutant TSH encoding nucleic acid or a TSH analog can be introduced intracellularly and incorporated into the host cell DNA for expression, by homologous recombination. The present invention also provides pharmaceutical compositions. Said compositions comprise a therapeutic effective amount of a heterodimer of the mutant TSH or the TSH analog, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory unit of the federal or state government or that appears in the U.S. Pharmacopoeia or other pharmacopoeia generally accepted for use in animals and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered. Said pharmaceutical carriers can be sterile liquids, such as water and oils, even those of petroleum, of animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Among the appropriate pharmaceutical excipients are starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycol. of propylene, water, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting agents or emulsifiers, or pH buffering agents. The composition can be formulated as suppositories, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulation and the like. The composition can be formulated as a suppository,. with traditional binders and transporters such as triglycerides. The oral formulation may include normal carriers, as pharmaceutical qualities of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. In "Remington's Pharmaceutical Sciences" by E.W. Martin describes examples of suitable pharmaceutical carriers. The compositions will include a therapeutic effective amount of the heterodimer of mutant TSH or TSH analog, preferably n purified form, together with a suitable amount of carrier, such that the form for appropriate administration is provided to the patient. patient. The formulation must be adapted to the administration modality. In a preferred embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration in humans. In general, compositions for intravenous administration are solutions that are found in a sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to relieve pain at the site of injection. Generally, the ingredients are supplied already singly or mixed in dosage unit, for example, as a dry or water free concentrate lyophilized powder in a hermetically sealed as an ampoule or sachet indicating the quantity of agent active. In cases in which the composition is to be administered by infusion, it can be distributed with an infusion bottle containing water or sterile saline of pharmaceutical quality. In cases where the composition is administered by injection, a sterile water vial for injection or saline may be provided so that the ingredients can be mixed prior to administration. The heterodimers of the mutant TSH or the TSH analogs of the invention can be formulated as neutral or salt forms. Prominent among the acceptable salts pharmaceutically those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric, etc., and those formed with free carboxyl groups such as those derived of sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. The amount of the mutant TSH heterodimer or TSH analogue of the invention that will be effective in the treatment of a particular condition or condition will depend on the nature of the condition or condition, and can be determined by normal clinical techniques. . In addition, in vitro assays can optionally be applied in animal models to help identify optimal dosage ranges. The precise dose to be applied in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be decided according to the judgment of the doctor and the circumstances of each patient. In specific embodiments, the Therapeutics of the invention are administered intramuscularly. Appropriate dosage ranges for intramuscular administration are generally about 10 μg to 1 μg per dose, preferably about 10 μg to 100 μg per dose. In general, for diagnostic and therapeutic methods in which the mutant TSH heterodimer is administered to stimulate iodine uptake, the heterodimers of the mutant TSH can be administered in a regimen of 1-3 injections. In one embodiment, the Therapeutic is administered in two doses, wherein the second dose is administered 24 hours after the first dose; In another embodiment, the Therapeutic is administered in three doses, one of these doses being administered on days 1, 4 and 7 of a 7-day regimen. Effective doses can be extrapolated from the dose-response curves derived from the test systems of in vitro or animal models. Suppositories generally contain the active ingredient in the range of 0.5% to 10% by weight; Oral formulations preferably contain 10% to 95% active ingredient. The invention also provides a package or kit for therapeutic or diagnostic use comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally, a notification in the form established by a government agency that regulates the manufacture, use or sale of pharmacists or diagnostic products may be related to the recipient (s), mentioning the approval of the dependence of the manufacture, use or sale for administration in humans. 6. EXAMPLES Two examples of the novel mutant TSH heterodimers are given below. The data show that the heterodimers of the mutant TSH have higher biological activity than the wild-type TSH. 6. 1 MATERIALS Restriction enzymes, DHNA markers and other molecular biological reagents were purchased either at Gibco BRL (Gaithersburg, MD) or Boehringer-Mannheim (Indianapolis, IN). Means for cell culture, fetal bovine serum and LIPOFECTAMINE were purchased from New England Biolabs (Beverly, MA). The full length cDNA (840 bp) subcloned in BamHI / Xhol sites of the pcDNA i / Neo vector (Invitrogen, San Diego, CA) and the hCG-β gene were obtained from T.H. Ji (University of Wyoming, Laramie, WY). The inventors constructed the minigene hTSH-β without the first intron, with the first untranslated exon and authentic translation initiation site. The human TSH norm was from Genzyme (Framingham, MA). Chinese hamster ovary cells (CHO) with a stably expressed hTSH receptor (CHO-hTSHR clone JP09 and clone JP26) were provided by G. Vassart (University of Brussels, Brussels, Belgium). cAMP 125I human TSH 125 I-, and bovine TSH 125 I - with radioactive labeling with respect to a particular activity of 40-60 μCi / μg were obtained from Hazleton Biologicals (Vienna, VA). 6.2 METHODS 6.2.1 MUTAGENESIS DIRECTED TO A SITE Mutagenesis of a-cDNA was achieved with the PCR-based mega-initiator method (Sarkar et al., 1990, Biotechniques, 8: 404-407). Amplification was optimized using DNA Vent® polymerase (New England Biolabs). After digestion with BamHI and Xhol, the PCR product was ligated with pcDNA I / Neo (Invitrogen) with the excised BamHI / Xhol fragment. MC1061 / p3 cells of Escherichia coli were transformed using the Ultracomp E. coli Transformation Kit (Invitrogen). The QIAprep 8 Plasmid Kit (Qiagen) was used for DNA preparations with multiple plasmids. The Qiagen Mega and Maxi Purification were followed Protocols for purifying larger quantities of a-cDNA containing plasmids with single or multiple mutations as a matrix for further mutagenesis. The mutations were confirmed by the double-helical sequence applying the chain termination method of Sanger dideoxynucleotide. The construction of fusion of the β subunit of TSH with CTEP is described in Joshi et al. (1995, Endocrinology, 136: 3839-3848). 6. 2.2 EXPRESSION OF RECOMBINANT HORMONES CH0-K1 cells (ATCC, Rockville, MD) were maintained in HAM F-12 medium with glutamine and 10% FBS, penicillin (50 units / ml), and streptomycin (50 μg / ml). . Cell plates (100-mm culture flasks) were cotransfected with a-DNA in the pcDNA I / INEO and the minigene of the mutant hTSHβ inserted into the vector p (LB) CMV vector, using a LIPOFECTAMINE reagent (Gibco BRL). After 24 hours the transfected cells were transferred to serum free CHO serum (CHO-SFM-II, Gibco BRL). Culture media that included control medium for simulated transfections using the expression plasmids without gene inserts were grown 72 hours after transfection, concentrated and centrifuged; aliquots were stored at -20 ° C and thawed only once before each test. The wild-type and mutant hTSH were measured and verified by applying various activity assays and immunological assays. 6.2.3 STIMULATION OF cAMP STIMULATION IN MAMMALIAN CELLS EXPRESSING THE HUMAN TSH RECEPTOR CHO-Kl cells stably transfected with the hTSH receptor cDNA (JP09 or JP26) were cultured and incubated with serial TSH-type solutions wild and mutant described. The cAMP released in the medium was determined by radioactive immunological assay. Equivalent amounts of the protein from the total media were used as a sham control and as the samples containing the hTSH from the transfected cells. 6. 2.4 IN VIVO BIOLOGICAL ACTIVITY TESTS The thyrotropic activity of hTSH was evaluated for its ability to induce the production of cAMP in cells in CHO that expressed hTSH receptors (clones JP09 and JP26) and in FRLT-5 cells that expressed the receptor of the endogenous TSH of the rat. FRTL-5 cells were also used to test cell growth induced by hTSH. For that purpose, CHO cells stably expressing the hTSH receptor (JP09 or JP26) were cultured to coalesce in a HAM F-12 medium in 96-well tissue culture plates. Subsequently, the cells were incubated either under salt-free conditions or with a physiological concentration of NaCl (0.9%) for 2 hours at 37 ° C, 5% CO; with solutions in wild-type and mutant hTSH series as well as control means for simulated transfections. The amount of cAMP released in the medium was determined by radioactive immunological assay. The in vivo activity of the TSH analogues was tested by determining the total levels of thyroxine (total T) after intraperitoneal injections in suppressed T3 mice, as described by Szkudlinski et al., In Nat. Biotechnol. 14: 1257 (1996). 6. 3 RESULTS The results presented in Figures 4-8 support the conclusion that the heterodimers of the TSH mutated according to the invention showed higher biological activity when compared to the wild-type TSH. More particularly, the results indicated that single or multiple mutations within the TSH subunits of the above described methods could be incorporated into the heterodimers with higher biological activity in vitro and in vivo. This was true for several different mutations and combinations of these, and illustrates the basic principle of the present invention. In one example illustrating the mutation in the structure of the higher hormonal activity of the common human subunit, a mutant was created in which the glycine residue usually present at position 22 of subunit a was replaced with a residue of arginine (aG22R). SE produced a heterodimer of the mutant TSH comprising this mutation in combination with a wild-type β subunit, temporarily expressing the subunits of CH0-K1 cells together. The produced mutant heterodimer was tested in a biological activity assay using CHO-JP26 cells expressing the TSH receptor. As indicated by the results presented in Figure 4, the mutant hormone bound TSHR and induced a higher level of cyclic AMP production than wild-type TSH.

The plasma half-life and stability of the common subunits of wild type or mutant of the superactive heterodimers of TSH comprising four mutations, Q13K + E14K + P16K + Q20K, this is a4K, increased by the joint expression of the subunit a4K and the ß subunit of wild type human TSH or the fusion of the β subunit of human TSH with the cTEP of hCG (β-CTEP) in CHO-K1 cells. The heterodimer of wild-type and mutant TSH were quantified by applying an immunological chemical luminescence assay. (Nichols Institute). The results are shown in Table 1 (100% expression is 47 ng wild-type TSH per ml).

TABLE 1 hTSH type 100 6 salva e hTSH-a4K 26 5 hTSH-a4K + CTEP 20 3 The presence of CTEP did not reduce the expression of the heterodimer comprising the fusion protein a4K and β-CTEP compared to the heterodimer comprising the a4K and the TSHß of wild type. The capacity of the heterodimers of the TSH of wild-type and mutants to bind to the TSHR by stimulating the production of cyclic AMP in CHO-JP09 which are stably expressed as transfected TSHR. As indicated by the results presented in Figure 5, the a4K / β-CTEP heterodimer showed a 200-fold increase in power and an increase of 1.5 in Vmax compared to wild-type TSH. It was surprising that the inclusion of CTEP, which is expected to prolong the in vivo half-life of the a4K / ß-CTEP heterodimer, will also increase its in vitro activity by 3-4 times over that of a ß a4K / wild-type heterodimer. The results show that the mutations that increase the The biological activity of a mutant TSH can be combined with a mutation or modification that prolongs the circulatory half-life to create mutant hormones that exhibit superior in vitro and in vivo characteristics.

In other examples, mutations in the ßL3 hairpin structure of the human common subunit also increased hormonal activity. One of the mutations was the substitution of the alanine residue at position 81 with a lysine residue (Aa81k). The other mutation was the substitution of the asparagine residue at position 66 with a lysine residue (An66k). Each of the mutant subunits was temporarily expressed in CHO-Kl cells in combination with wild-type human TSH-β subunits to produce mutant TSH hormones. Each of these heterodimers of the mutant TSH was tested in a biological activity assay using CHO-JP09 cells that expressed the human TSH receptor. The results of these procedures indicated that both mutant hormones stimulated higher levels of cAMP production than the wild-type hormone. The substitution of alanine 81 by lysine (Aadlk) in subunit a represents the first demonstration of the introduction of a lysine residue, which is not present in other homologous sequences, within a β-hairpin structure. In yet another example, a mutation near the ßL1 hairpin structure of the β subunit of human TSH increased the hormonal activity of a heterodimer that included this mutant subunit. The mutation was a substitution of the glutamate residue at position 6 with an asparagine residue (BE6N) that removes the negatively charged residue at the periphery of the BL1 hairpin structure. The β subunit of the human mutant TSH was temporarily expressed in CHO-K1 cells in a combination with a common human wild-type subunit to produce a heterodimer of the mutant TSH. The heterodimer of the mutant TSH was then tested in a biological activity assay using CHO-JP09 cells that expressed the TSH receptor. Again, the results indicate that this mutant TSH hormone bound to the receptor and induced higher levels of cAMP production than wild-type TSH. The results presented in Figures 6 and 7 further confirm that the ßL1 structure can be used to produce mutant heterodimers that advantageously possess higher biological activity when tested using in vitro assays. More particularly, the results presented in Figure 6 indicate that the individual mutants ßFIR, ßE6N and ßAl7R could be combined with a wild-type subunit to form mutant heterodimers that possess higher hormonal activity. Figure 7 shows that ß subunits that included combinations of either ßFIR and ßE6N, or ßFIR, ßE6N and ßA17R also possessed higher hormonal activity. According to the in vitro findings described above, the analogs of human hTSH showed parallel increases in their in vivo activities. Indeed, in a demonstration of the in vivo activity of the TSH mutants prepared according to the procedure described above, a ß-subunit of TSH (ß3R) was created with mutations at three points: ßI58R, ßE63R and ßL69R. The a4K mutant subunit and the ß3R mutant β subunit were co-expressed intracellularly, collected from the conditioned medium and the resulting heterodimer was tested for measurable biological activity in the form of total T4. Mutagenesis did not significantly influence the release of the analogs from the circulation. The results shown in Figure 8a indicate that two distinct mutant heterodimers showed markedly greater biological activity in vivo. The results shown in Figure 8B indicate that the magnitude of higher biological activity of the a4K / ß3R heterodimer in relation to the wild-type control was at least 100 times higher. Moreover, these results confirmed that combinations of mutant TSH subunits could significantly improve hormonal activity in vivo. In light of these results, it can reasonably be expected that the TSH mutants and analogs described herein are superior to conventional recombinant hTSH for the diagnostic management of thyroid cancer.

The results presented above confirm that the mutation of the TSH subunits according to the teaching of the present invention can be used advantageously to manufacture and use TSH mutants with improved biological activities. The results presented in this document also indicate that the peripheral regions of the aLl and ßL3 structures of the hTSH represented "permissive modification" domains that can be created with genetic engineering for greater receptor binding and activity. The location of these permissive modification domains creates a bivalent ligand in which said symmetry of binding interfaces is a result of head-to-tail dimerization of the homodimers or heterodimers, and is believed to mediate the dimerization of the receptor induced by the ligand Although dimerization of the functionally relevant receptor has not been described for any protein-coupled G receptor, a putative interaction site has been located in the sixth transmembrane domain of the adrenergic receptors, and the corresponding region is a "point sensitive "to consecutively activate mutations of the TSH receptor. Thus, by applying a rational strategy based on evolutionary considerations and homology comparisons, by testing at least 20 mutants, we have identified the a4KI / ß3R analogue of hTSH with only seven mutations of a total of 204 amino acids of the molecule. TSH that has an increase of up to 50,000 times in the binding affinity and up to 1,300 times increase in hormonal potency. Not wishing to be bound by any particular theory of functioning, it is possible that the modulation of the peripheral fork structures during the evolution of the glycoprotein hormone may have modified the hormonal function in several phylogenetic stages. The synergy of the hairpin structures created by genetic engineering at the receptor link suggests that the combined modifications in such distant spatial domains, which were optimized at different stages of hormonal evolution, can provide a universal strategy for creating human protein analogues by genetic engineering. Recombinant analogs of the type described herein have a combination of basic residues that are not present in any known natural hormone at any stage of evolution, and they exceed the binding affinity of the receptor or the TSH activity of any species. It will be appreciated that certain variations to the present invention may be suggested to those skilled in the art. The above detailed description should be clearly understood as given by way of illustration, the spirit and scope of the present invention being interpreted with reference to the appended claims.

Claims (64)

1. CLAIMS 1. A mutant subunit comprising an amino acid substitution at position 22 of the amino acid sequence of subunit a as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1) 2 . The mutant subunit of claim 1 wherein the amino acid substitution at position 22 is arginine. 3 . The mutant subunit of claim 1 which is purified. Four . The mutant subunit of claim 1 further comprising one or more amino acid substitutions with no amino acid residues selected from positions 11-21 of the amino acid sequence of subunit a as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER) : 1) . 5 . The mutant subunit of claim 5 wherein the amino acid substitution or substitutions are at amino acid residues selected from positions 11, 13, 14, 16, 17, and 20 of the amino acid sequence of subunit a as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1). 6 The mutant subunit of claim 1 further comprising one or more amino acid substitutions in the ß Ll hairpin structure of subunit a. 7 The mutant subunit of claim 4 or 6 wherein the substitution or substitutions are amino acids selected from the group consisting of arginine and lysine. 8 The subunit a of claim 4 wherein the substitution or substitutions are selected from the group consisting of aQ13K, aE14K, aP16K, aF17R, and aQ20K. 9. A Qi mutant subunit wherein the single mutation is an amino acid substitution at position 22 of the amino acid sequence of subunit a as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1). 10 The mutant subunit of claim 9 wherein the substitution at position 22 is arginine. eleven . The mutant subunit of claim 9 which is purified. 12 A heterodimer of mutant TSH comprising subunit to mutant comprising an amino acid substitution at position 22 of the amino acid sequence of subunit a as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1) and subunit a β, wherein the biological activity of the heterodimer of the mutant TSH is greater than the biological activity of the wild-type TSH heterodimer. 13 The heterodimer of the mutant TSH of claim 12 wherein the amino acid substitution at position 22 is arginine. 14 The heterodimer of the mutant TSH of claim 12 which is purified. fifteen . The heterodimer of the mutant TSH of claim 12 wherein the β subunit is a subunit to human β. 16 The heterodimer of the mutant TSH of claim 12 further comprising one or more amino acid substitutions at amino acid residues selected from positions 11-21 of the amino acid sequence of subunit a as shown in Figure 1 (IDENTIFICATION NUMBER) OF SEQUENCE: 1). 17 The heterodimer of the mutant TSH of claim 12 wherein the amino acid substitution or substitutions are in amino acid residues selected from positions 11, 13, 14, 16, 17, and 20 of the amino acid sequence of subunit a. 18 The heterodimer of the mutant TSH of claim 12 further comprising one or more amino acid substitutions in the β1-cleave structure of subunit a. 19 The heterodimer of the mutant TSH of claim 16 or 18 wherein the substitution or substitutions are amino acids selected from the group consisting of arginine and lysine. twenty . The heterodimer of the mutant TSH of claim 16 wherein the substitution or substitutions are selected from the group consisting of aQ13K, aE14K, aP16K, aFl7R, and aQ20K 21. A heterodimer of the mutant TSH A wherein the single mutation is an amino acid substitution at position 22 of the amino acid sequence of subunit a as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1). 22 The heterodimer of the mutant TSH of claim 21 wherein the substitution at position 22 is arginine.
2. 2.
3. 3 . The mutant heterodimer of claim 21 which is purified. 24 A mutant heterodimer of TSH comprising (a) a β subunit of bound TSH via a peptide bond at its carboxyl terminus with the amino terminus of the carboxyl terminal extension peptide of human chorionic gonadotropin; and (b) a subunit a, wherein at least the β subunit of the TSH or subunit a of the TSH contains at least one amino acid substitution, and wherein the biological activity of the heterodimer of the mutant TSH is greater than that of the heterodimer of wild-type TSH. 25 The heterodimer of the mutant TSH of claim 24 wherein at least the substitution of an amino acid is in amino acid residues selected from positions 11-21 of the amino acid sequence of the human subunit as shown in Figure 1 ( SEQUENCE IDENTIFICATION NUMBER: 1). 26 The heterodimer of the human mutant TSH of claim 24 wherein at least one amino acid substitution is in amino acid residues selected from positions 58-69 of the amino acid sequence of the β subunit of the TSH as shown in the Figure 2 (SEQUENCE IDENTIFICATION NUMBER: 2). 27 The heterodimer of the human mutant TSH of claim 26 wherein the substitution of at least one amino acid is selected from the group consisting of ßI58R, ßE63R and ßL69R. 28 The heterodimer of the mutant TSH of claim 24 comprising a subunit to a human mutant and a β subunit of the human mutant TSH, wherein the human mutant subunit comprises at least one amino acid substitution at amino acid residues selected from the group consisting of positions 11-22 of the amino acid sequence of the human subunit as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1), and wherein the β subunit of the human mutant TSH comprises at least one amino acid substitution in amino acid residues selected from positions 58-69 of the amino acid sequence of the β subunit of human TSH as shown in Figure 2 (SEQUENCE IDENTIFICATION NUMBER: 2). 29 The heterodimer of the mutant TSH of claim 24 which is a mutant of the heterodimer of human TSH. 30 A TSH analog comprising a subunit a that is covalently linked to a β subunit of the TSH, wherein at least one of the subunits comprises at least one amino acid substitution in its amino acid sequence, and wherein the biological activity of the TSH analog is greater than the biological activity of the TSH analog comprising a wild type a subunit covalently linked to a β subunit of the wild-type TSH. 31 The analogue of the TSH of claim 30 wherein at least one amino acid substitution is in in amino acid residues selected from positions 11-22 of the amino acid sequence of subunit a as shown in Figure 1 (NUMBER OF IDENTIFICATION OF SEQUENCE: 1). 32 The TSH analog of claim 30 wherein the substitution of at least one amino acid is in amino acid residues selected from positions 58-69 of the amino acid sequence of the β subunit of human TSH as shown in the Figure 2 (SEQUENCE IDENTIFICATION NUMBER: 2). 33 The TSH analog of claim 30 wherein both the α subunit and the β subunit comprise one or more amino acid substitutions. 3.
4. 4 . The TSH analog of claim 33 wherein the a subunit has at least one amino acid substitution in amino acid residues selected from positions 11-22 of the amino acid sequence of the human subunit as shown in Figure 1 (SEQUENCE IDENTIFICATION NUMBER: 1), and the β subunit of human TSH has at least one amino acid substitution selected from positions 58-69 of the β subunit amino acid sequence of human TSH as shown in Figure 2 (SEQUENCE IDENTIFICATION NUMBER: 2) . 35 The TSH analog of claim 30 wherein the TSH subunit is bound by a peptide bond at its carboxyl terminus to the amino terminus of the carboxyl terminal extension peptide of human chorionic gonadotropin 36. The TSH analog of claim 30 wherein the TSH subunit is bound by a peptide bond at the carboxyl terminus to the amino terminus of the a subunit. 37 The heterodimer of the mutant TSH of claim 24 wherein the hormonal half-life in the in vivo circulation of the heterodimer of the mutant TSH is greater than that of the wild-type TSH. 38 The mutant analogue of the TSH of claim 30 wherein the hormonal half-life in the in vivo circulation of the mutant analogue of TSH is greater than that of the wild-type TSH. 39 A nucleic acid comprising a nucleotide sequence encoding the mutant subunit of claim 1. 40. A nucleic acid comprising a nucleotide sequence encoding the TSH analog of claim 30, wherein the a subunit is bound to the β subunit via a peptide bond. 41 A method for treating or preventing hypothyroidism comprising administering to a subject for whom said treatment or prevention is desired a quantity of TSH heterodimer of claim 24 sufficient to treat or prevent hypothyroidism. 42 A method for treating or preventing hypothyroidism comprising administering to a subject for whom said treatment or prevention is desired a quantity of TSH heterodimer of claim 30 sufficient to treat or prevent hypothyroidism. 43 A method for treating thyroid cancer which comprises administering to a subject for whom said treatment or prevention is desired an amount of the TSH heterodimer of claim 24 sufficient to stimulate the absorption of iodine and subsequently administer to the subject an amount of iodine sufficient radioactive labeling to treat thyroid cancer. 44 A method for treating thyroid cancer which comprises administering to a subject for whom said treatment or prevention is desired an amount of the TSH analogue TSH heterodimer of claim 30 sufficient to stimulate the absorption of iodine and subsequently administer to the subject an amount of iodine radioactive labeling sufficient to treat thyroid cancer. Four. Five . A method for diagnosing thyroid cancer which comprises administering to a subject an amount of the heterodimer of the mutant TSH of claim 24 sufficient to stimulate iodine uptake by thyroid cancer cells and an amount of sufficient radioactive labeling iodine to diagnose thyroid cancer; and detecting the iodine radioactive labeling, wherein a relative increase in a subject who does not have thyroid disease in the absorption of radioiodinated iodine indicates that the subject has thyroid cancer. 46 A method for diagnosing thyroid cancer which comprises administering to a subject lacking non-thyroid cancer cells to a subject an amount of the heterodimer of the mutant TSH of claim 24 sufficient to stimulate iodine uptake by thyroid cancer cells and an amount of radioiodine iodine sufficient to diagnose thyroid cancer; and detect radioiodinated iodine; wherein an increase in radioiodine iodine uptake indicates that the subject has thyroid cancer. 47 A method for diagnosing thyroid cancer which comprises administering to a first subject an amount of the heterodimer of the mutant TSH of claim 24 sufficient to stimulate the release of thyroglobulin in vivo and measure the levels of thyroglobulin in the first subject, in which an increase in thyroglobulin levels relative to thyroglobulin levels in a sample from a second subject that does not have thyroid cancer indicates that the first subject has thyroid cancer. 48 A method for diagnosing thyroid cancer which comprises administering to a subject an amount of the TSH analog of claim 30 sufficient to stimulate the absorption of iodine by the thyroid cancer cells and an amount of radioiodine sufficient for radioiodination. diagnose thyroid cancer; and detecting the iodine radioactive labeling, wherein a relative increase in a subject who does not have thyroid disease in the absorption of the radioactive labeling iodine indicates that it has. 49 A method for diagnosing thyroid cancer which comprises administering to a subject lacking non-thyroid cancer cells to a subject an amount of the mutant TSH heterodimer of claim 30 sufficient to stimulate iodine uptake by Thyroid cancer cells and enough radioiodine iodine to diagnose thyroid cancer; and detect radioiodinated iodine; wherein an increase in radioiodine iodine uptake indicates that the subject has thyroid cancer. fifty . A method for diagnosing thyroid cancer which comprises administering to a first subject an amount of the heterodimer of the mutant TSH of claim 30 sufficient to stimulate the release of thyroglobulin in vivo and measure the levels of thyroglobulin in the first subject, wherein an increase in thyroglobulin levels relative to thyroglobulin levels in a sample from a second subject that does not have thyroid cancer indicates that the first subject has thyroid cancer. 51 A method of diagnosis and surveillance of a disease or condition characterized by the presence of antibodies against the TSH receptor comprising contacting cultured cells or isolated membrane receptors containing TSH with a mixture containing putatively antibodies of a. first subject with an effective amount from the diagnostic point of view of the radiolabelled mutant TSH heterodimer of claim 24; and measure the binding of radiolabelled mutant TSH to cultured cells or the isolated membrane, where a decrease in the binding of radiolabelled TSH in relation to the linkage in the absence of the sample or in the presence of a one second sample subject that does not have the disease or condition, indicates the presence of the disease or condition in the first subject 52 The method of claim 51 wherein the disease or condition is the Graves disease. 53 A method of diagnosis and surveillance of a disease or condition characterized by the presence of antibodies against the TSH receptor comprising contacting cultured cells or isolated membrane receptors containing TSH with a mixture containing putatively antibodies of a first subject with an effective amount from the diagnostic point of view of the heterodimer of the radiolabelled mutant • TSH of claim 30; and measure the binding of radiolabelled mutant TSH to cultured cells or the isolated membrane, where a decrease in the binding of radiolabelled TSH in relation to the linkage in the absence of the sample or in the presence of a one second sample subject that does not have the disease or condition, indicates the presence of the disease or condition in the first subject 54 The method of claim 53 wherein the disease or condition is Graves disease. 55 A pharmaceutical composition comprising a therapeutic effective amount of the human mutant TSH of claim 12; and a pharmaceutically acceptable carrier. 56 A pharmaceutical composition comprising a therapeutic effective amount of the human mutant TSH of claim 24; and a pharmaceutically acceptable carrier. 57 A pharmaceutical composition comprising a therapeutic analog amount of the TSH analog of claim 30; and a pharmaceutically acceptable carrier. 58 A diagnostic composition comprising an amount of the heterodimer of the mutant TSH of claim 12 sufficient to stimulate iodine uptake by the thyroid cancer cells; and a pharmaceutically acceptable carrier. 59 A diagnostic composition comprising an amount of the heterodimer of the mutant TSH of claim 24 sufficient to stimulate iodine uptake by the thyroid cancer cells; and a pharmaceutically acceptable carrier. 60 A diagnostic composition comprising an amount of the heterodimer of the mutant TSH of claim 30 sufficient to stimulate iodine uptake by the thyroid cancer cells; and a pharmaceutically acceptable carrier. 61 A kit comprising in one or more containers an effective amount from the heterodimer diagnosis point of view of mutant TSH a of claim 12 or 24 or the analogue of the TSH of claim 30. 62. A kit comprising in one or more containers an effective amount from the point of view of the heterodimer diagnosis of the mutant TSH a of claim 12 or 24 or the analog of the TSH of claim 30. 63 The nucleic acid of claim 38 or 40 which is isolated. 64 The composition of claim 55, 56, 58 or 59 wherein the TSH mutant TSH heterodimer is purified.