The inventors have designed novel FSH mutants with increased glycosylation and longer half-lives for use in inducing folliculogenesis in human patients. The use of a FSH mutant preparation of the invention permits the use of lower cumulative doses of FSH to achieve the same or better clinical result.

Description of the Invention

SUMMARY OF THE INVENTION

The inventors have designed novel FSH mutants with increased glycosylation and longer half-lives for use in treating reproductive disorders in human patients. The use of a FSH mutant preparation of the invention permits the use of lower cumulative doses of FSH to achieve the same or better clinical result.

Accordingly, the invention provides a mutant FSH showing FSH activity, the mutant FSH having at least one additional glycosylation site, as compared with wild type FSH, which glycosylation site bears a glycan. The invention further provides nucleic acid molecules encoding the mutant FSH. Also included in the invention is a vector containing any one of the nucleic acids of the present invention.

The present invention is also directed to recombinant host cells expressing a mutant FSH of the invention, particularly a host cell transformed with a vector comprising a FSH nucleic acid molecule encoding the mutant FSH. In further aspect, the invention provides for a composition that includes a mutant FSH and a pharmaceutically acceptable carrier or excipient.

Still further, the invention provides a method for producing a mutant FSH. The method includes providing a cell containing a FSH mutant nucleic acid, e.g., a vector that includes a FSH mutant nucleic acid, and culturing the cell under conditions sufficient to express the FSH mutant. The expressed FSH mutant is then recovered from the cell.

The present invention also provides for methods of treating male and female infertility, as well as reproductive disorders in mammals, including stimulating folliculogenesis, inducing ovulation and ovarian hyperstimulation.

In another aspect, the invention provides a use of mutant FSH in ovulation induction, stimulation of folliculogenesis and controlled ovarian hyperstimulation, particularly in conjuction with ART.

DETAILED DESCRIPTION OF THE INVENTION

Based on information derived from the crystal structure of FSH, the inventors have designed mutants with increased glycosylation by substituting specific amino acids for asparagine or threonine, thereby creating additional glycosylation recognition sites.

The specific substitutions were as follows: In the beta-subunit: A70N, V78N, G100N and D41N/A43T; In the alpha-subunit: F17T and E56N.

It is known that potential in vivo N-glycosylation sites are specific to the consensus pattern Asn-Xaa-Ser/Thr, and less specifically to the pattern Asn-Xaa-Cys, where Xaa can be any amino acid residue. The probability of N-glycosylation for the Asn-Pro-Ser/Thr sequence is about 50%, which is statistically much lower than the other consensus pattern of Asn-Xaa-Ser/Thr. The presence of the consensus tripeptide, however, is not sufficient to conclude that an asparagine residue will be glycosylated.

Protein folding plays an important role in the regulation of N-glycosylation. If consensus residues are buried in the interior of the folded protein structure, it is unlikely glycosylation will occur at the particular site. Furthermore, if a glycosylation site is introduced by mutation, the mutated residues should not disrupt the three-dimensional structure of the protein or substantially detract from the desired function of the protein, such as receptor binding or activation. For these reasons, structural knowledge of the protein molecule is critical to the success of the design of glycosylation mutants.

Two methods that have been used to gain insight into the 3-dimensional structure of proteins are modeling and X-ray crystal structure. Although hCG and FSH have essentially identical folding patterns, the two structures are significantly different (Fox et al, 2001), and the detailed structure for individual amino acid residues for the FSH molecule cannot be properly modeled from the previously determined hCG structures (Wu et al, 1994; Lapthorn et al, 1994). The inventors have designed mutants based solely on the crystal structures of the human FSH molecule (Fox, et al, 2001).

There are two FSH molecules (four subunits) in each asymmetric unit in the crystal structure. These two molecules were superimposed and compared, and each residue was visually inspected to identify potential N-glycosylation sites that would not disrupt the structure of the protein molecule.

Structural knowledge was also analysed to uncover potential functional important regions of the glycoprotein hormone molecules, including FSH, hCG and TSH. Comparative study of the crystal structures of hCG molecule alone (Wu et al, 1994; Lapthorn et al, 1994) and its complex with the Fv fragments of neutralizing and non-neutralizing antibodies (Tegoni, et al, 1999) provided clues of the important regions for receptor binding and activation. One face of the "waist-like" region of the hCG molecule is very likely to bind to the extracellular domain of the receptor, and this type of interaction was inferred for the FSH-FSHR system.

Using structural knowledge, the FSH ligand (Fox et al, 2001) was fine tuned to its receptor model (Jiang et al, 1995). The following six site mutations were selected, all of which avoid the ligand-receptor interface on one side of the "waist-region" of the FSH molecule: In the beta-subunit: A70N, V78N, G100N and D41N/A43T; In the alpha-subunit: F17T and E56N; wherein A is alanine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, N is asparagine, T is threonine, V is valine, and the notation "E4N" represents a replacement of a glutamic acid (E) at position 4 with an asparagine (N). For sequence numbering, the amino acid sequence of human FSH alpha is numbered according to the mature sequence shown in FIG. 6 (see Original Patent) or SEQ ID NO: 1. The amino acid sequence of human FSH beta is numbered according to the mature sequence shown in FIG. 7 (see Original Patent) or SEQ ID NO: 2.

Thus, the present invention relates to FSH preparations having increased half-life resulting from an increased glycosylation by adding one or more additional glycosylation sites on the protein. Such sites were introduced by substitution of residues in the FSH protein backbone with serine, threonine, lysine or asparagine residues, using, for example, mutagenesis. For in vivo glycosylation, the site introduced should be such as to form an "N-glycosylation site", of the following sequence: N-X'-S/T/C-X'', wherein X' is any amino acid residue except proline, X'' is any amino acid residue which may or may not be identical to X' and which preferably is different from proline, N is asparagine, and S/T/C represents a residue that may be serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine.

Methods for Generating FSH Mutants:

The FSH mutants of the present invention may be produced by any suitable method known in the art. These methods include the construction of nucleotide sequences encoding the respective FSH mutants and expressing the amino acid sequence in a suitable transfected host. FSH mutants of the present invention may also be produced by chemical synthesis or by a combination of chemical synthesis and recombinant DNA technology.

FSH mutants of the present invention may comprise the FSH alpha and FSH beta subunits in the form of two separate polypeptide chains, where the two chains become dimerized in vivo so as to form a dimeric polypeptide, or it may comprise a single chain construct comprising the two subunits covalently linked by a peptide bond or a peptide linker. The amino acid residues of the linker peptide should exhibit properties that do not interfere significantly with the activity of the FSH mutant.

The nucleotide sequence encoding the alpha or beta subunits of the FSH mutants of the invention may be constructed by isolating or synthesizing a nucleotide sequence encoding the parent FSH subunit, such as the hFSH-alpha or hFSH-beta with the amino acid sequences shown in FIGS. 6 and 7 (see Original Patent), respectively or SEQ ID Nos 1 and 2, respectively. The nucleotide sequence is then changed so as to effect the substitution of the relevant amino acid residues. The nucleotide sequence can be modified by site directed mutagenesis as in Example 1 of the present specification. In the alternative, the nucleotide sequence may be prepared by chemical synthesis, wherein oligonucleotides are designed based on the specific amino acid sequence of the FSH mutant.

The nucleotide sequence encoding the polypeptide is inserted into a recombinant vector and operably linked to control sequences necessary for expression of the polypeptide in the desired transfected host cell. One of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation.

The recombinant vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector is one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the nucleotide sequence encoding the polypeptide of the invention is operably linked to additional segments required for transcription of the nucleotide sequence. The vector is typically derived from plasmid or viral DNA. A number of suitable 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 enabling 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 replication.

The vector may also comprise a selectable marker, e.g. a gene whose product complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. 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 flanking sequences, including the mutant FSH DNA, can be selected for on appropriate media.

The term "control sequences" is defined herein to include all components which are necessary or advantageous for the expression of the polypeptide of the invention. Examples of suitable control sequences for directing transcription in mammalian cells include the early and late promoters of SV40 and adenovirus, e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter and the human cytomegalovirus immediate-early gene promoter (CMV).

The nucleotide sequences of the invention encoding the FSH mutants, whether prepared by site-directed mutagenesis, synthesis, PCR or other methods, may optionally also include a nucleotide sequence that encodes a signal peptide. The signal peptide is present when the polypeptide is to be secreted from the cells in which it is expressed. Such signal peptide, if present, should be one recognized by the cell chosen for expression of the polypeptide. The signal peptide may be homologous (e.g. be that normally associated with a hFSH subunit) or heterologous (i.e. originating from another source than hFSH) to the polypeptide or may be homologous or heterologous to the host cell, i.e. be a signal peptide normally expressed from the host cell or one which is not normally expressed from the host cell.

Any suitable host may be used to produce the polypeptide subunits of the invention, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. Examples of suitable mammalian host cells include Chinese hamster ovary (CHO) cell lines, (e.g. CHO-KL; ATCC CCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NSIO), Baby Hamster Kidney (BI-EK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g. BEK 293 (ATCC CRL-1573)), as well as plant cells in tissue culture. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, USA. Methods for introducing exogeneous DNA into mammalian host cells include calcium phosphate-mediated transfection, electroporation, DEAE-dextran mediated transfection, liposome-mediated transfection and viral vectors.

Cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g. in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, it can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

The resulting mutant FSH polypeptide may be recovered by methods known in the art. For example, it may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

The mutant FSH polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g. ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g. preparative isoelectric focusing), differential solubility (e.g. ammonium sulfate precipitation), SDS-PAGE, or extraction.

Use of FSH Mutants:

In one aspect, the invention provides a pharmaceutical composition comprising FSH mutants according to the invention. Such pharmaceutical compositions can be used to stimulate folliculogenesis, for example in conjunction with ovulation induction or assisted reproductive techniques (ART). Because the FSH mutants of the invention is particularly effective in inducing multiple follicles to develop and mature, it is particularly suitable for use in ART, in which it is desired to collect multiple oocytes.

Alternatively, with careful tailoring of the dose, FSH mutants of the invention may be used to induce mono-folliculogenesis for OI, or paucifolliculogenesis (up to about three follicles) for IUI, for in vivo fertilisation. Mono-folliculogenesis can also be attained with a reduced dose of an FSH mutant, or less frequent dosing as compared with conventional FSH preparations. For example, in OI an FSH preparation of the invention may be administered at 225-400 IU every three days, or lower doses, depending on the patient response. Patient response may be followed by sonography.

The FSH mutants of the invention may be used in a controlled ovarian hyperstimulation (COH) regimen. Standard regimens for COH include a down-regulation phase in which endogenous luteinising hormone (LH) is down-regulated by administration of a gonadotrophin releasing hormone (GnRH) agonist followed by a stimulatory phase in which follicular development (folliculogenesis) is induced by daily administration of follicle stimulating hormone (FSH), usually at about 150-225 IU/day. Alternatively stimulation is started with FSH after spontaneous or induced menstruation, followed by administration of a GnRH-antagonist (typically starting around day six of the stimulatory phase). When there are at least 3 follicles>16 mm (one of 18 mm), a single bolus of hCG (5-10,000 IU) is given to mimic the natural LH surge and induce ovulation. Oocyte recovery is timed for 36-38 hours after the hCG injection.

The FSH mutants of the invention may also be used for OI and IUI. For example, FSH stimulation with a preparation of the invention is started after spontaneous or induced menstruation, at a daily dose of 75-150 IU. When 1 or 3 follicles have reached a diameter of at least 16 mm, a single bolus of hCG is administered to induce ovulation. Insemination is performed in vivo, by regular intercourse or IUI.

Because the FSH mutants of the invention has an increased half-life with respect to known FSH preparations, regimens such as that described above may employ lower IU doses of FSH, and/or may be modified by decreasing the FSH stimulation period, while achieving the same or better response, in terms of number and viability of follicles. For example, using an FSH preparation of the invention, adequate folliculogenesis may be achieved with daily doses of at or about 50-150 IU FSH, preferably at or about 50-100, more preferably at or about 50-75 IU FSH. Dosing of FSH is usually on a daily or semi-daily basis. The dosing period may be less than at or about 14 days, preferably less than at or about 12 days, more preferable less than at or about 11 or 10 days.

For OI, the FSH mutant preparations of the invention may be administered at doses from 25-150 IU FSH/day, preferably, 50-125 IU FSH/day.

For the treatment of male infertility, an FSH mutant preparation of the invention may be administered at 3.times.150 to 300 IU/week until spermatogenesis reaches levels adequate for insemination, either through regular intercourse or ART techniques.

Because of the longer half-life of the mutant FSH of the invention, it may be administered as a long-acting preparation. Conventional FSH may be administered at or about 300 IU on every second day, while achieving similar results to administration every day at or about 150 IU. The expression "long-acting" is meant to encompass FSH preparations that may be administered less frequently than every two days. Preferred mutant FSH of the invention may be administered every three days, every four days, every five days, every six days or every seven days, while achieving similar or better results than daily administration of conventional FSH.

Pharmaceutical Compositions of the Invention:

In one aspect the FSH mutants or their pharmaceutical compositions according are used for the manufacture of a medicament for treatment of diseases, disorders or conditions.

In another aspect the polypeptide or the pharmaceutical composition according to the invention is used in a method of treating a mammal, in particular a human, comprising administering to the mammal in need thereof such polypeptide or pharmaceutical composition.

It will be apparent to those of skill in the art that an effective amount of a conjugate, preparation or composition of the invention depends, inter alia, upon the disease, the dose, the administration schedule, whether the polypeptide or conjugate 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 of the invention is sufficient to ensure a therapeutic effect.

The FSH mutants of the present invention are normally administered in a composition including one or more pharmaceutically acceptable carriers or excipients. "Pharmaceutically acceptable" means a carrier or excipient that does not cause any untoward effects in patients to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art, and the polypeptide or conjugate of the invention can be formulated into pharmaceutical compositions by well-known methods (see e.g. Remington's Pharmaceutical Sciences, 18th edition, A. R. 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, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000)). Pharmaceutically acceptable excipients that may be used in compositions comprising the polypeptide or conjugate of the invention include, for example, buffering agents, stabilizing agents, preservatives, isotonifiers, nonionic surfactants or detergents ("wetting agents"), antioxidants, bulking agents or fillers, chelating agents and cosolvents.

The pharmaceutical composition of the FSH mutants of the invention may be formulated in a variety of forms, including liquids, e.g. ready-to-use solutions or suspensions, gels, lyophilized, or any other suitable form, e.g. powder or crystals suitable for preparing a solution. The preferred form will depend upon the particular indication being treated and will be apparent to one of skill in the art.

The pharmaceutical composition containing a FSH mutant of the invention may be administered intravenously, intramuscularly, intraperitoneally, intradermally, subcutaneously, sublingualy, buccally, intranasally, transdermally, by inhalation, or in any other acceptable manner, e.g. using PowderJect or ProLease technology or a pen injection system. The preferred mode of administration will depend upon the particular indication being treated and will be apparent to one of skill in the art. In particular, it is advantageous that the composition be administered subcutaneously, since this allows the patient to conduct self-administration.

The pharmaceutical compositions of the invention may be administered in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the polypeptide or conjugate of the invention, either concurrently or in accordance with any other acceptable treatment schedule. In addition, the polypeptide, conjugate or pharmaceutical composition of the invention may be used as an adjunct to other therapies.
 

Claim 1 of 23 Claims

1. An isolated mutant of human Follicle Stimulating Hormone (FSH) having FSH activity, wherein one N-glycosylation site has been introduced into the FSH alpha subunit by the mutation F17T.
 

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