MX2008009199A - Novel fsh glycosylation variant d3n - Google Patents

Abstract

FSH mutant with increased glycosylation and longer half-life is described. The use of this FSH mutant for inducing folliculogenesis in human patients is also described.

Description

D3N VARIANT OF GLYCOSILATION OF THE STIMULATING HORMONE OF THE NOVEDOSE FOLLICLE BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to human reproduction. More specifically, the present invention relates to fertilization therapies.

DESCRIPTION OF THE RELATED TECHNIQUE to. Gonadotrophins The follicle stimulating hormone (FSH) is a member of the gonadotropin family that plays the key roles in human fertility. Gonadotrophins, which also include luteinizing hormone (LH) and chorionic gonadotropin (CG), are heterodimers, each consisting of a subunit-a (92 amino acids) and a single common subunit-β (111 amino acids in FSH) . The amino acid sequences of the mature forms of the α and β subunits of FSH are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Human FSH has been isolated from the pituitary glands and from post-menopausal urine (EP 322,438) and has been produced recombinantly in mammary cells (U.S. Patent No. 5,639,640; U.S. Patent No. 5,156,957; of the United States of America No. 4,923,805; United States Patent No. 4,840,896; United States Patent No. 5,767,251; Patent EP 211,894; and EP 521,586. The latter references also disclose a modified human gene of the β-subunit of FSH U.S. Patent No. 5,405,945 discloses a modified human gene of the α-subunit of FSH comprising only one intron Liu et al, Biol J Chem 1993.15; 268 (2): 21613-7, Grossmann et al., Mol Endocrinol 1996 10 (6): 769-79, Roth and Dias (Mol Cell Endocrinol 1995 1; 109 (2): 143-9, Valove et al., Endocrinology 1994; 135 (6): 2657-61.1 Yoo et al., JBiol Chem 1993 25; 268 (18): 13034-42), U.S. Patent No. 5,508,261 and Chappel et al., 1998, Human Reproduction, 13 (3): 1835 disclose several studies of the relationship of the structure-function and identify residues of the amino acids involved in the binding of the receptor and activation and in the dimerization of FSH. b. The use of Gonadotrophins in Assisted Reproduction Techniques. Gonadotrophins play crucial roles in the reproductive cycle, and their use in exogenous therapies is essential for assisted reproduction techniques (ART), such as in vitro fertilization (IVF), IVF in conjunction with intracytoplasmic sperm injection (IVF) / ICSI) and embryo transfer (ET), as well as for the induction of ovulation (Ol) in anovulatory patients undergoing in vivo fertilization either naturally or through intrauterine insemination (IUI). The patent of the United States of North America No. 4. 589,402 and U.S. Patent No. 4,845,077 disclose purified human FSH which is free of LH and the use thereof for in vitro fertilization. Patent EP 322,438 discloses a protein with at least 6200 U / mg of FSH activity which is substantially free of LH activity, and wherein the -subunit and β-subunit of FSH, respectively, can be wild-type or specific truncated forms thereof. Prolonged therapy is necessary to achieve a therapeutic effect, typically 8-10 consecutive days and sometimes up to 21 days to stimulate folliculogenesis in women, and up to 18 months in hypogonadotropic males to induce spermatogenesis. The recombinant hFSH is typically administered as an i.m. or s.c. daily, with the consequent annoyance and the potential for the reaction of the site of the local injection. Decreasing the frequency of administration would facilitate therapy and would provide a more adequate, more tolerable and more appropriate gonadotrophin administration to the patient. c. Glycosylation of FSH Gonadotropins are glycoproteins, which have the side chains of otigosaccharide (N-enlance) attached to asparagine that are important for activity and function in vivo. The addition of the carbohydrate (glycosylation) to the polypeptides is a post-translational event that results in the addition of sugar chains to the specific asparagine (N-bond) or the serine / threonine amino acids (O-bond). In contrast to the invariable amino acid sequence of the protein portion of the glycoproteins, the carbohydrate structures are variable, a characteristic referred to as micro-heterogeneity. For example, N-glycosylation sites in the same protein may contain structures other than the carbohydrate. In addition, even in the same glycosylation site in a given glycoprotein, different carbohydrate structures can be found. This heterogeneity is a consequence of the non-directed synthesis of carbohydrate template. N-glycosylation of proteins occurs specifically in the Asn-Xaa / Ser / Thr consensus pattern, and to a lesser degree in the Asn-Xaa-Cys consensus pattern, where Xaa can be any amino acid residue. However, the presence of a consensus tripeptide is not enough to ensure that an asparagine residue is glycosylated. For example, N-glycosylation of the Asn-Pro-Ser / Thr sequence occurs at a rate 50 times lower than the other Asn-Xaa-Ser / Thr consensus standards. Human FSH contains four glycosylation sites: two in the common subunit-a at positions 52 and 78 and two in the β-subunit at positions 7 and 24. The carbohydrates attached to the subunit-a of FSH are critical for the assembly of the dimer, for the integrity, for the transduction of secretion and signal, while the carbohydrates of the β-subunit are important for the assembly of the dimer, for the secretion and clearance of the heterodimer from the circulation. Galway et al., Endocrinology 1990; 127 (1): 93-100 demonstrates that FSH variants produced in a CHO cell line of transferase-1 N-acetylglucosamine or a CHO cell line defective in sialic acid transport, is as active as FSH secreted by wild-type cells or purified pituitary FSH in vitro, but lacks in vivo activity, presumably due to the rapid clearance of inappropriately glycosylated variants in serum. D'Antonio and collaborators, Reprod. Humana 1999; 14 (5): 1160-7 describe several isoforms of FSH circulating in the bloodstream. The isoforms have identical amino acid sequences, but they differ in the extent of post-translational modification. It was found that the less acid groups of isoforms had a faster clearance in vivo, compared to the acid isoform group, possibly due to differences in acid content sialic between the isoforms. In addition, Bishop et al., Endocrinology 1995; 136 (6): 2635-40 concludes that the circulatory half-life seems to be the primary determinant of in vivo activity. These observations led to the hypothesis that the half-life of FSH could be increased by the introduction of additional glycosylation sites to increase the sialic acid content of the polypeptide. d. Variants of the FSH. FSH agonists with increased half-life have been developed by fusion of hCG carboxy-termite peptide (CTP) to recombinant native human FSH (rhFSH). The CTP fraction consists of amino acids 112-118 to 145 with four O-linked glycosylation sites located at positions 121, 127, 132 and 138. The United States Patent U.S. Patent No. 5,338,835 and U.S. Patent No. 5,585,345 disclose a modified β-subunit of FSH extended to Terminal-C Glu with the CTP fraction of hCG. The resulting modified analog is indicated to have the biological activity of native FSH, but a prolonged half-life of circulation. The patent of the United States of North America No. 5,405,945 discloses that the terminal portion of the carboxy subunit-β hCG or a variant thereof, has significant effects on the clearance of CG, FSH, and LH. U.S. Patent No. 5,883,073 discloses the simple-chain proteins comprising two subunits-a with an agonist or antagonist activity for CG, TSH, LH and FSH. U.S. Patent No. 5,508,261 discloses heterodimeric polypeptides that have LH-binding affinity and FSH receptors that comprise a glycoprotein hormone-a subunit and a β-subunit polypeptide which occurs non-naturally, wherein the β-subunit polypeptide is a chain of amino acids comprising four linked sub-sequences, each of which is chosen from a list of specific sequences. Klein et al. (2003) disclose a simple chain analog of FSH with an increase in half-life, where the-a and -β subunits are linked by an oligopeptide containing two glycosylation sites of -N bonds. WO 01/58493 discloses 77 mutations that can be made in the α-subunit of FSH and 51 mutations that can be made in the β-subunit of FSH in an attempt to improve the in vivo half-life of FSH. In addition, WO 01/58493 discloses that one or more glycosylation sites can be added to the N-terminus of FSH to improve its half-life or be inserted at several sites within the FSH polypeptide. Patent WO 01/58493, while describing that glycosylation sites can be inserted into the FSH polypeptide, does not provide a guide as to the specific site (the sites), where one could insert a glycosylation site and maintain the activity of the FSH. WO 01/58493 further discloses that the mutant-a and subunits can be used individually: (1 additional glycosylation site) or in a combination (2) additional glycosylation sites). The 128 candidate mutants were identified using 50 3D structure models of FSH that were generated based solely on the structure of hCG and a sequence alignment of FSH and hCG despite only 32 percent identity between the β-subunits of hCG and FSH. WO 01/58493 does not disclose the production or testing of any of the a-subunits or β-FSH where a glycosylation site was introduced by site-directed mutagenesis. Patent WO 05/020934 discloses GM1, with the mutations in the α-subunits and β-FSH, including a double mutation in β E55N / V57T, that is, the residue E at the position of amino acid 55 mutated to N and residue V at the position of amino acid 57 mutated to T. The amino acid sequence of ß E55N / V57T is shown in SEQ ID NO: 3. There is a clinical need for a product that provides some or all of the therapeutically relevant effects of the FSH, and that they can be administered with fewer frequency intervals compared to the FSH products currently available, and that they preferably provide a more stable level of circulating FSH activity compared to those that can be achieved by the treatments currents The present invention is directed to such products as well as to the means for manufacturing such products.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to mutant FSH molecules, wherein the a-subunit of FSH comprises SEQ ID NO: 4 and wherein the β-subunit of FSH comprises SEQ ID NO: 3. FSH can to be N-glycosylated in 0, 1, 2, 3, 4, 5 or 6 asparagine residues of said mutant FSH. In an N3 modality of the mutant subunit-a of SEQ ID NO: 4 it can be glycosylated. The present invention also relates to isolated DNA molecules that encode a mutant of the FSH subunit that comprises the sequence of SEQ ID NO: 4. The present invention also relates to an isolated DNA encoding a β-subunit of FSH comprising the sequence of SEQ ID NO: 3. The present invention also relates to a vector comprising a DNA encoding a mutant of the a-subunit of FSH comprising the sequence of SEQ ID NO: 4. The vector can be an expression vector. The present invention also relates to a vector comprising a DNA encoding a mutant of the β-subunit of FSH comprising the sequence of SEQ ID NO: 3. The vector can be an expression vector. The present invention also relates to a vector comprising a first DNA and a second DNA in which the first DNA encodes a mutant of the FSH subunit of the sequence of SEQ ID NO: 4 and wherein the second DNA encodes a mutant of the β subunit of FSH comprising the sequence of SEQ ID NO: 3. The vector can be an expression vector. The present invention also relates to a cell comprising a vector comprising the coding of the DNA of a mutant of the FSH subunit which comprises the sequence of SEQ ID NO: 4. The vector can be an expression vector . The cell can be a mammalian cell, for example, a CHO cell. The present invention also relates to a cell comprising a vector comprising a DNA encoding a mutant of the β-subunit of FSH comprising the sequence of SEQ ID NO: 3. The vector can be an expression vector. The cell can be a mammalian cell, for example, a CHO cell. The present invention also relates to a cell comprising a vector comprising a first DNA and a second DNA, wherein the first DNA encodes a mutant of the FSH subunit which comprises the sequence of SEQ ID NO: 4 and wherein the second DNA encodes a mutant of the β-subunit of the FSH comprising the sequence of SEQ ID NO: 3. The vector can be an expression vector. The cell can be a mammalian cell, for example, a CHO cell. The present invention also relates to a cell comprising a first and a second vector, wherein the first vector comprises a DNA encoding a mutant of the FSH subunit consisting of SEQ ID NO: 4 and the second vector comprising a DNA encoding a mutant of the β subunit of the FSH comprising the sequence of the SEC ID NO: 3. The vector (vectors) can be an expression vector. The cell can be a mammalian cell, for example, a CHO cell. The present invention also relates to a method for producing an FSH mutant comprising the culture of mammalian cells capable of glycosylating proteins, wherein said cells comprise a first expression vector comprising a DNA encoding a mutant of the subunit -a of the FSH comprising the sequence of SEQ ID NO: 4 and a second expression vector comprising a DNA encoding a mutant of the β-subunit of the FSH comprising the sequence of SEQ ID NO: 3. In another embodiment of the present invention said cells comprise a single vector comprising a DNA encoding a mutant of the a-subunit of the FSH comprising the sequence of SEQ ID NO: 4 and further comprising a DNA encoding a mutant of the β-subunit of FSH comprising the sequence of SEQ ID NO: 3. The present invention also relates to a composition comprising a mutant of FSH and a pharmaceutically acceptable carrier or excipient. ptable, wherein the FSH subunit comprises the sequence of SEQ ID NO: 4 and wherein the β-subunit of FSH comprises SEQ ID NO: 3. The present invention also relates to a method for try an infertile mammal, comprising administering to a mammal in need thereof, an effective amount of a mutant FSH mutant, wherein the a-subunit of FSH comprises the sequence of SEQ ID NO: 4 and wherein the β-subunit of FSH comprises SEQ ID NO: 3. The present invention also relates to a method of stimulating follicle-genesis in a mammal, comprising administering to a mammal in need thereof an effective amount of an FSH mutant, wherein the a-subunit of FSH comprises the sequence of SEQ ID NO: 4 and wherein the β-subunit of FSH comprises SEQ ID NO: 3. The present invention also relates to a method for inducing ovarian hyper-stimulation in a mammal, which comprises administering to the mammal in need thereof an effective amount of a mutant FSH, wherein the a-subunit of the FSH comprises the sequence of SEQ ID NO: 4 and wherein the subunit -ß of the FSH comprises the SEC ID NO : 3.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows an alignment of the mutants of the D3N subunit (SEQ ID NO: 4) to the human subunit of the FSH (SEQ ID NO: 1). The residue numbers refer to the human subunit-a of FSH (SEQ ID NO: 1) with 1 being the first amino acid of the polypeptide mature. The exposures of Figure 2 show a dose response curve for the mutant FSH, which comprises a D3N mutant subunit-a and a GM-1 subunit comprising SEQ ID NO: 3, as compared to a curve of response to the dose of wild-type FSH. The dose response curve indicates the activity of FSH in several dilutions of both the FSH mutant and the wild type. Clone 15C refers to the D3N mutant. Clone 14C is an unrelated mutant of FSH.

DETAILED DESCRIPTION OF THE INVENTION While it has been shown that increasing the carbohydrate content of FSH can lead to increased half-life in vivo, improving the half-life of FSH is more complicated than simply adding additional glycosylation sites. While a glycosylation consensus sequence is necessary for the addition of the carbohydrate, it is not sufficient to ensure that a carbohydrate addition site has been used. Other factors, such as folding and conformation of the local protein during biosynthesis, determine whether an oligosaccharide is attached to a given site in the consensus sequence. Furthermore, as a function of an additional glycosylation leading to an increase in half-life in vivo, the consensus sequence must be in such a position that the glycosylation of the site does not interfere with the binding of the receptor, nor that it compromises the folding, the conformation nor the stability of the glycoprotein. At this point, FSH analogs with increased half-lives have been limited primarily to fusion proteins wherein fused portions of the polypeptide included additional glycosylation sites. 1. Mutant FSH A mutant of FSH is provided with the proviso that it has been modified to create additional glycosylation recognition sites. The a-subunit of the FSH mutant may have one of the following mutations, compared to the wild-type subunit-a: D3N and Q5T. A mutant FSH may comprise the above mutant subunit in combination with the mutant β subunit, ie β GM1 containing the following mutation: E55NA / 57T. One or more of the additional glycosylation sites of the recombinant FSH can be glycosylated. One or more additional glycosylation sites of the mutant FSH can be glycosylated in vitro or in vivo. The FSH mutant can be produced by some suitable method known in the art. These methods include the construction of the nucleotide sequences encoding the respective mutants of the FSH and expressing the amino acid sequence in a suitable transfected host. The mutant of FSH can also be produced by chemical synthesis or by a combination of chemical synthesis and recombinant DNA technology.

The mutant FSH may comprise the α- and β-subunits of FSH in the form of two separate polypeptide chains, where the two chains are dimerized in vivo to form a dimeric polypeptide, or may comprise a single chain construct comprising both subunits covalently linked by a peptide linkage or a peptide linker. The amino acid residues of the linker peptide may exhibit properties that do not appreciably interfere with the activity of the FSH mutant. The FSH mutant may have an increased half-life compared to wild-type FSH. The FSH mutant may also have a stability compared to wild-type FSH. The FSH mutant may comprise the oligosaccharides at 0, 1, 2, 3, 4, 5 or 6 of the N-linked glycosylation sites. A population of the FSH mutants are also provided., which may comprise one or more mutant isoforms of FSH, where each isoform comprises the oligosaccharides at 0, 1, 2, 3, 4, 5 or 6 of the N-linked glycosylation sites. The nucleotide sequence encoding the subunit-a- or β of the FSH mutant can be constructed by isolating or synthesizing a nucleotide sequence that encodes the mother subunit of FSH, such as hFSH-alpha or hFSH-beta with the amino acid sequences shown in the SEC ID Nos .: 1 and 2, respectively. The nucleotide sequence can then be changed to perform the reinsertion or replacement of the relevant amino acid residues. The nucleotide sequence can be modified by the site of directed mutagenesis. In the alternative, the nucleotide sequence can be prepared by chemical synthesis, wherein the oligonucleotides are designed based on the specific amino acid sequence of the FSH mutant. The nucleotide sequence encoding the polypeptide can be inserted into a recombinant vector and operably linked to a control sequence necessary for the expression of the polypeptide in the desired transfected host cell. The control sequences can be any component that is necessary or advantageous for the expression of a polypeptide. Examples of suitable control sequences for directing transcription in mammalian cells include the early and late promoters of SV40 and the adenovirus, for example the major late promoter of adenovirus 2, the MT -1 promoter (metallothionein gene) and the promoter of the immediate early gene of human cytomegalovirus (CMV). A person skilled in the art can make a selection between these vectors, the sequences of expression control and the hosts without undue experimentation. The recombinant vector can be a self-replicating vector, ie, a vector that exists as an extrachromosomal entity, the replica of which is independent of chromosomal replication, for example a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome of the host cell and replicates together with the chromosome (the chromosomes) in which it has been integrated.

The vector can be an expression vector in which the nucleotide sequence encoding the polypeptide of the invention is operably linked to the additional segments required for transcription of the nucleotide sequence. The vector can be derived from a plasmid or viral DNA. A number of expression vectors for expression in the host cells mentioned herein are commercially available or described in the literature. The recombinant vector may further comprise a DNA sequence that enables the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 origin of the replication. The vector may also comprise a selectable marker, for example a gene whose product complements a defect in the host cell, such as the coding of the gene for dihydrofolate reductase (DHFR) or one that confers resistance to a drug, eg, ampicillin. , kanamycin, tetracycline chloramphenicol, neomycin, hygromycin or methotrexate. The vector may also comprise an amplifiable gene, such as DHFR, such that cells having multiple copies of the amplifiable gene and the flanking sequences, including the mutant FSH DNA, may be chosen for an appropriate medium. Also provided is a DNA encoding a subunit-a of the FSH mutant. The nucleotide sequence encoding the alpha beta subunits of the FSH mutant, prepared by site-directed mutagenesis, synthesis, PCR or other methods, optionally may also include a nucleotide sequence encoding a signal peptide. The signal peptide may be present when the polypeptide is to be secreted by the cells in which it is expressed. Such a signal peptide, if present, can be one recognized by the cell chosen for the expression of the polypeptide. The signal peptide can be homologous (for example), which is normally associated with a subunit of hFSH) or can be heterologous (ie originating from another source than hFSH) to the polypeptide or can be homologous or heterologous to the host cell, i.e., is a signal peptide normally expressed from the host cell or from one that is not normally expressed from the host cell. Any suitable host can be used to produce the polypeptides, including bacteria, fungi (including yeasts), plants, insects, mammals, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. Examples of mammalian host cells include the CHO cell lines of the Chinese hamster ovary, e.g., CHO-KL; ATCC CCL-61), Mono Green cell lines (Green Monkey), (COS) (eg, COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (for example NSIO), cell lines (BI-EK) from Baby Hamster Kidney (for example ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g. BEK 293 (ATCC CRL-1573)), as well as plant cells in tissue cultures. Additional suitable cell lines are known in the technique and are available from public depositaries such as the American Type Culture Collection, (American Type Culture Collection) United States of North America. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, electroporation, dextran-mediated transfection, liposome-mediated transfection, and viral vectors. The cells can be cultured in a nutrient medium suitable for the production of the polypeptide using methods known in the art. For example, the cell can be cultured by cultivation in a shaker, fermentation or large-scale or small scale flask (including continuous fermentation, feed batch or solid state) in the laboratory or in industrial fermenters carried out in a medium suitable and under conditions that allow the polypeptide to be expressed and / or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and organic salts, using methods known in the art. Suitable media are available from commercial suppliers or can be prepared according to the published compositions (for example in the Catalog of the American Type Culture Collection). If the polypeptide is secreted in the nutrient medium, it can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from those used in the cell. A high-throughput production method of the FSH mutants of the invention is by amplification by the use of dihydrofolate reductase (DHFR) in DHFR-deficient CHO cells, by the use of successive levels of methotrexate as described in U.S. Patent No. 4,889,803. The resultant mutant FSH polypeptide may be covered by methods known in the art. For example, it may be coated with the nutrient medium by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. The mutant FSH polypeptides can be purified by a variety of methods known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromate-focusing, and size exclusion). ), electrophoretic procedures (eg, preparative isoelectric focusing), differential solubility (eg, precipitation of ammonium sulfate), SDS-PAGE, or extraction. Also provided is a pharmaceutical composition comprising the FSH mutant. Such a pharmaceutical composition can be used to stimulate follicle-genesis, for example, in conjunction with assisted ovulation or reproductive induction (ART) techniques. Because the FSH mutant of the present invention can be effective in inducing multiple follicles to develop and mature, it can be particularly suitable for use in ART, where it is desired to collect multiple oocytes. The mutant of FSH can be used to induce mono- follicle-genesis for Ol, or paucifollic-genesis (up to approximately three follicles) for IUI, for in vivo fertilization. Mono-folliculogenesis can also be achieved with a reduced dose of the FSH mutant, or less frequent dosing compared to conventional preparations of FSH. For example, in Ol a preparation of the FSH of the invention can be administered at 225-400 IU every three days, or lower doses, depending on the response of the patient. The patient's response can be followed by sonography. The mutant of the FSH of the invention can be used in a controlled hyper-stimulation regime COH. Standard regimens for CHO include a down-regulation phase in which the endogenous luteinizing hormone (LH) is down-regulated by the administration of a gonadotropin-releasing agonist hormone (GnRH) followed by a stimulation phase in which the development follicular (follicle-genesis) is induced by the daily administration of follicle-stimulating hormone (FSH), generally at approximately 150-225 lU / day. Alternatively stimulation can be initiated with FSH after spontaneous or induced menstruation, followed by the administration of a Gnrh-Antagonist (typically starting around day six of the stimulation phase). When there are at least 3 follicles >At 16 mm (one of 18 mm), a single hCG bolus (IU 5-10,000) can be administered to mimic the natural surge of LH and induce ovulation. Oocyte retrieval can be timed for 36-38 hours after of hCG injection. The mutant of the FSH of the invention can also be used for 01 or for IUI, the FSH stimulation can be started after the menstruation either spontaneous or induced at a daily dose of 75-150 IU. When 1 or 3 follicles have reached a diameter of at least 16 mm, a single hCG food bolus can be administered to induce ovulation. The insemination can be done in vivo, through a regular sexual relationship or through IUI. Because the FSH mutant may have an increase in half-life with respect to the wild-type FSH preparations, regimens such as the one described above may employ lower doses of IU of the FSH, and / or may be modified. by decreasing the period of stimulation of FSH, while achieving the same or a better response in terms of quantity and viability of follicles. For example, an adequate follicle-genesis can be achieved with daily doses of approximately 50-150, 50-100 or 50-75 IU of FSH. The dosage of FSH can be on a daily or semi-daily basis. The dosing period may be less than or approximately 14, 12, 11 or 10 days. For Ol, the preparation of the FSH mutant can be administered in doses of 25-150 or 50-125 IU of FSH / day. For the treatment of male infertility, the preparation of the FSH mutant can be administered at 3 X 150 up to 300 IU / week until the spermatogenesis reaches adequate levels for insemination, either by regular intercourse techniques or by ART. Due to the longer half-life of the FSH mutant, it can be administered as a long-acting preparation, which can be administered less frequently than every two days. Conventional FSH can be administered at or about 300 IU on every other day, while results similar to administration are achieved every day at or about 150 IU. The FSH mutant can be administered every 3, 4, 5, 6 or 7 days, achieving similar or better results than the daily administration of conventional FSH. The mutant of FSH can be used for the manufacture of a drug for the treatment of diseases, conditions or disorders. In another aspect the polypeptide of the pharmaceutical composition according to the invention is used in a method for treating a mammal, in particular a human, which comprises administering to the mammal in need of a polypeptide or a pharmaceutical composition. It is obvious to those skilled in the art that an effective amount of a polypeptide, preparation or composition depends, inter alia, on the disease, the dose, the administration schedule, whether the polypeptide or the preparation or composition is administered alone or in conjunction with other therapeutic agents, the serum half-life of the compositions, and the general health of the patient. Typically, an effective dose of the preparation or composition is sufficient to ensure a therapeutic effect.

The FSH mutant can be administered in a composition that includes one or more pharmaceutically acceptable carriers or excipients. By "pharmaceutically acceptable" is meant a vehicle or excipient that does not cause any adverse effect in the patients to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art and the polypeptide can be formulated into the pharmaceutical compositions by well-known methods (see for example, Remington's Pharmaceutical Sciences, edition 18, eighteenth edition, AR Gennaro, Ed. Mack Publishing Company ( 1990), Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor &Francis (2000), and Handbook of Pharmaceutical Excipients, A. Kibbe, Pharmaceutical Press (2000)). The pharmaceutically acceptable excipients that can be used in the compositions comprising the polypeptide include, for example, buffering agents, stabilizing agents, preservatives, iso-toning agents, non-ionic surfactants or detergents "wetting agents"), antioxidants, thickening agents or fillers, chelating agents and co-solvents. Pharmaceutical compositions comprising the FSH mutant can be formulated in a variety of ways, including liquids, for example ready-to-use solutions or suspensions, gels, freeze-dried, or any other suitable form, for example, powders or crystals suitable for prepare a solution The shape of the composition can depending on the particular indication that is being treated and will be evident to a person skilled in the art. Pharmaceutical compositions comprising the FSH mutant can be administered intravenously, intramuscularly, intraperitoneally, intradermally, subcutaneously, sublingually, buccally, intranasally, transdermally, by inhalation or in any other acceptable manner, for example, using PowderJect or ProLease technology. or a pen injection system. The mode of administration may depend on the particular indication to be treated and will be apparent to a person of skill in the art. The composition can be administered subcutaneously, which can allow the patient to self-administer. The pharmaceutical compositions can be administered in conjunction with other therapeutic agents. These agents can be incorporated as part of the same pharmaceutical composition or can be administered separately from the polypeptides, either concurrently or in accordance with any other acceptable treatment program.

Additionally the polypeptide, the preparation or the pharmaceutical composition can be used as an adjunct to other therapies. The present invention has multiple aspects, illustrated by the following non-limiting examples.

EXAMPLE 1 Mutants of the FSH The 3D crystal structure of human FSH was used to identify the candidate glycosylation sites in the FSH subunit or in the regions of the FSH molecule where a candidate glycosylation site can be inserted. The two molecules of FSH (4 subunits) are present in each asymmetric unit of the crystal structure. The two FSH molecules were superimposed and compared, each residue being visually inspected to identify potential N glycosylation sites. The crystallographic structure of FSH was combined with the knowledge of the FSH / FSHR receptor interaction to further assist in the selection of potential N-glycosylation sites. The main design criteria were the minimal disruption of the 3D structure, the minimum interruption of predicted binding and activation sites, and the predicted 3D structure compatible with glycosylation. Based on the criteria above, the following mutant was identified for the amino acid sequence of the FSH subunit: EXAMPLE 2 Morphological analysis of the FSH mutants The aliquots of the concentrated culture supernatants of the transient expression of the FSH subunit mutant were analyzed by SDS-PAGE under the non-reduction conditions allowing the resolution of the intact FSH heterodimers of the subunits. free-a and -ß units. By comparing the apparent molecular weights of each mutant heterodimer to that of wild-type FSH it can be determined whether the mutant FSH is hyperglycosylated in relation to wild-type FSH. Briefly, after electrophoresis, the proteins were electrophoretically transferred to PVDF and visualized using Serono 9-14 antibody directed against the α-subunit of FSH. As a control, wild-type human FSH, mutant GM1, FSH-CTP and Gonal F were also analyzed. Table 1 shows the apparent molecular weight of the heterodimer formed by the subunit-a mutant and the wild-type β subunit as calculated based on molecular weight standards.

TABLE 1 As shown in Table I, the FSH mutant, ie D3 mutation, showed increased glycosylation evidenced by a commutation of the apparent molecular weight distribution of the heterodimer compared to wild-type human FSH.

EXAMPLE 3 In Vitro Function of the FSH Mutants To determine the activity of the FSH mutants, the mutant was tested for its ability to stimulate cAMP production in a CHO cell line that recombinantly expresses the human FSH receptor. CHO-FSHR cells were maintained in the growth medium of FSHR [MEM to (-) (Gibco, cat # 12561 -056) + 10 percent FBS dialysate (Gibco, cat # 26300-020) + 600 μg / ml Geneticin (Gibco, cat # 10131-035) + 0.02μMTX M]. CHO-FSHR cells were seeded in 2x10"cells / source in 100 μl / source (2 x 106 cells / 10 ml = 1 plate) and incubated at 37 ° C for 24 hours before assay. assay if at least 70 percent confluent A serial 1: 3 dilution of point 12 was made starting with 67.5 nM for all samples and the internal standard (Gonal F was used as an internal standard). performed in the assay medium [DMEM / F12 (phenol-free, Gibco, cat # 1 1039-021) + 1 mg / ml BSA (Sigma, A-6003) + 0.1 mM IBMX (3-isobutyl-1-methylxanthone inhibitor) phosphod this rasa, Sigma, cat # l-7018)]. Growth media was removed from the assay plate, 25μl of assay media was added (supplied with a MA6000 cAMP MSD - Meso Scale Discovery, Gaithersburg, MD ), the plates were recovered and incubated at 37 ° C for 15 minutes, then the sources were dosed with 25 μl / source of the test sample, The plates were coated and incubated for 1 hour at 37 ° C. After 1 hour of incubation, samples and media were removed from the sources. 25 μl of standard lysis buffer (supplied with a set of MA 6000 Meso Scale Discovery) was then added for each source, plates were covered with plate sealer (Packard, cat # 60O5185) and shaken for 5 minutes. After 5-minute lysis incubation, 25 μl of the lysate cell material was transferred to the cAMP Meso Scale Discovery (supplied with an MA6000 MSD) and incubated with Mix gently at room temperature for 30 minutes. 25 μl of cAMP-AP conjugate was added to each source and mixed. Then 25 μl of anti-body cAMP was added to each source, the plates were covered with plate sealer and stirred for 30 minutes at room temperature. The plates were then washed six times with 350 μl / source of wash buffer in an automatic plate washer. Then, 100 μl of Sapphire II RTU substrate improver (Ready to use) was added to each source, the plates were covered with plate sealer and incubated for 30 minutes in the dark at 25 ° C. The plates were read in one second per source with low levels of cAMP showing a high signal and high levels of cAMP showing a low signal. The dose response curve of the FSH mutant is shown in Figure 2. The EC50 values were calculated and shown in Table 2. As shown in Figure 2 and Table 2, the FSH mutant has an in vitro activity comparable to that of wild-type FSH.

TABLE 2 EXAMPLE 4 In vivo half life of FSH mutants Two different batches of D3N mutant were analyzed in separate pharmacokinetic studies (PK). The two studies were of the same design: 33 21-day-old immature SD female rats (approximately 40 g of body weight, Charles River Laboratories, Wilmington, MA) were randomly divided into 5 treatment groups (n = 6). ) and a baseline group (n = 3). The choice of immature female rats was based on the use of this age and sex for in vivo biological assessments of FSH activity. The animals in the treatment groups received subcutaneous (s.c.) injections of 4 ug of GM1 (control), mutant D3N or 8 ug of Gonal-F rhFSH (control). Blood was collected from the retro-orbital sinus at 0 hours from the baseline group and at 1, 2, 4, 6, 10, 24, 48 and 72 hours of the animals in the treatment groups (n = 3 / point of time, the rats alternated so that they were not bled at 2 subsequent sampling points). Approximately 0.1 ml of the blood was collected from each rat in each Bleeding and plasma was cultured and stored at -80 ° C until analyzed by ELISA. The assay used to measure the FSH proteins in the serum of both studies was from ELISA coated source FSH from DSL (Diagnostics Systems Laboratories, Webster, TX). Each of the serum samples was analyzed in triplicate. The half-lives of the D3N mutant was significantly longer than that of the wild-type FSH.

EXAMPLE 5 Biological Activity in vivo The in vivo model used to assess the biological activity of the D3N mutant is the ovarian test for weight gain in rats. The treatment of immature females rats of 21 days with FSH or molecules with the type activity of FSH, for example mutant D3N, causes the growth of follicles and the ovarian production of oocytes. This growth is easily detected by measuring the ovarian weight at the end of the treatment period. In the model, the substance to be tested is given by injection for three days and the ovaries are collected and weighed after the last dose. This trial was used for several decades as the basis for assigning the potency of FSH to clinical products for label purposes. It measures the relevant physiological action of the FSH and has a clear correlation for the performance of the products in the clinic. The in vivo activity of the D3N mutant was compared to that of the wild-type FSH. All doses were defined based on the predicted equivalence of FSH, taking into account the in vitro potency and the half-life in the rats. It was discovered that the D3N mutant possesses potent FSH activity with a magnitude similar to that of wild-type FSH.

Claims (17)

NOVELTY OF THE INVENTION CLAIMS

1. - A nucleic acid encoding a mutant subunit of FSH wherein the subunit-a comprises the sequence of SEQ ID NO: 4.

2. A vector comprising the nucleic acid of claim 1.

3.- The vector according to claim 2, further characterized in that the vector is an expression vector.

4. The vector according to claim 2, further characterized in that the vector further comprises a nucleic acid encoding the sequence of SEQ ID NO: 3.

5. A host cell comprising the vector of claim 2.

6. - The host cell according to claim 5, further characterized in that the cell is a cell of a mammal.

7. A mutant FSH, wherein the β-subunit comprises the NO NO: 3, and wherein the α-subunit comprises a sequence encoded by the nucleic acid of claim 1.

8.- The FSH mutant in accordance with the claim 1, further characterized in that any of the 0 to 6 residues of asparagine are glycosylated.

9. - The FSH mutant according to claim 7, further characterized in that the subunit-a comprises the sequence of SEQ ID NO: 4 and wherein N3 is glycosylated.

10. A method for producing an FSH mutant comprising: a) providing a cell comprising the nucleic acid of claim 1 and a second nucleic acid encoding SEQ ID NO: 3; b) culturing the cell under the conditions that allow the expression of the first and second nucleic acids.

11. The method according to claim 1, further characterized in that the cell is able to glycosylate a protein.

12. The method according to claim 11, further characterized in that the cell comprises a single vector comprising the nucleic acid of claim 1 and a nucleic acid encoding SEQ ID NO: 3.

13. The method of compliance with claim 11, further characterized in that the cell comprises a vector comprising the nucleic acid of claim 1 and further comprising a second vector comprising a nucleic acid encoding SEQ ID NO: 3.

14. A pharmaceutical composition comprising the FSH mutant of claim 1 and optionally a pharmaceutically acceptable carrier or excipient.

15. The use of the pharmaceutical composition of claim 14, for the manufacture of a medicament useful for treating an infertile mammal.

16. - The use of the pharmaceutical composition of claim 14, for the manufacture of a medicament useful for the stimulation of follicle-genesis in a mammal.

17. The use of the pharmaceutical composition of claim 14, for the manufacture of a medicament useful for inducing ovarian hyper-stimulation in a mammal.