Abstract:
Recombinant Fc fusion proteins of human follicle-stimulating hormone (hFSH) with in vivo biological activities comparable to those of human follicle-stimulating hormone are disclosed. A recombinant hFSH-Fc fusion protein comprises β subunit of hFSH (hFSH β), CTP, α subunit of hFSH (hFSH α), a flexible peptide linker, and human IgG2 Fe variant (vIgG2Fc). A method is also disclosed to make such fusion proteins at good expression levels. These recombinant hFSH-Fc fusion proteins of the present disclosure exhibit sufficient biological activities and prolonged plasma half-lives, leading to improved pharmacokinetics and pharmacodynamics. Thus, a lower dosage may be used and/or better or different therapeutic efficacies with less side effects may be achieved. A method for the application of the recombinant hFSH-Fc fusion proteins in the treatment and/or prevention of human infertility is also disclosed.

Description:
TECHNICAL FIELD 
       [0001]    The present disclosure relates to molecular biology and medicine. More specifically, the present disclosure relates to a long-acting recombinant human follicle-stimulating hormone fusion protein, and preparation method and use thereof. The fusion protein has significantly extended in vito half-life, and has therapeutic efficacy better than that of existing human follicle-stimulating hormone. 
       BACKGROUND 
       [0002]    Infertility affects one in seven couples worldwide, becoming a severe disease right after cancer and cardiovascular disease in terms of its harmful effect on human health. Follicle stimulating hormone (FSH), either extracted from urine or produced by genetic engineering, has been widely used by specialists to increase fertility. 
         [0003]    The use of human FSH (hFSH) extracted from human urine is limited by its high cost, labor intensive collection and difficulty in tracing urine source, as well as potential risk of virus contamination. Recombinant hFSH is a better option as it can avoid the above problems. hFSH is a glycoprotein with a molecular weight of about 43 kilodalton (kD). As a therapeutic drug, it is necessary to possess the correct 3-dimensional structure and glycosylation to maintain its bioactivity. The ability to perform complex post-translational modifications is a major reason that most therapeutic biologicals are manufactured in mammalian cell lines. Among them, Chinese Hamster Ovary (CHO) cell is the most common host cell system for eukaryotic gene expression. Therapeutic recombinant proteins such as EPO, G-CSF have been successfully expressed in CHO cells. These proteins not only can properly be folded and glycosylated, but also they can be secreted which is favorable for subsequent downstream process, such as purification. 
         [0004]    Although recombinant hFSH produced by CHO cells is in market for years, several defects still need to be solved. First, current recombinant FSH has a short plasma half-life, requiring patients to be administrated repeatedly to achieve optimal therapeutic efficacy. For example, hFSH must be administered intramuscularly or subcutaneously as a daily injection, routinely for 8-12 days or more when used for ovulation induction, resulting in poor compliance. In addition, this treatment regimen is often accompanied with severe cytotoxic effects to nervous, endocrine and immune system, causing a number of frequently-occurring complications such as ovarian hyperstimulation syndrome, clinical manifestations associated with ovarian enlargement, increased vascular permeability and ascites formation, severe cases can be life-threatening. Furthermore, the production cost of recombinant hFSH is very high due to the low level of cell expression and extensive production process. Last but not least, hFSH is a glycosylated protein comprising an a subunit and a 0 subunit, connected via non-covalent bond, and its bioactivity depends on the correct assembly between the two subunits. It remains a challenging to maintain the right assembly of the two subunits during the protein expression and purification process which result in a biologically active molecule useful for therapeutic purpose until the instant disclosure. To resolve the defects and insufficiency of the existing hFSH products, the present disclosure makes a new molecule of recombinant hFSH with prolonged plasma half-life, sufficient biological activities and high level of protein expression, leading to improved pharmacokinetics and/or pharmacodynamics. Thus, a lower dosage may be used and/or better or different therapeutic efficacies with less side-effects may be achieved. 
       SUMMARY 
       [0005]    The present disclosure relates to a human follicle-stimulating hormone-Fc fusion protein (denoted by hFSH-Fc). The present disclosure also provides methods for preparation of the fusion protein and its use/application, aimed at solving the defects of current FSH, such as low expression level, extensive purification process and short plasma half-life. 
         [0006]    One aspect of the present disclosure relates to an hFSH-Fc fusion protein. This fusion protein comprises hFSH β, CTP, hFSH α, a flexible peptide linker (denoted by L), and human IgG2 Fc variant (vIgG2Fc), as shown in SEQ ID NO: 2 (hFSH β-CTP-hFSH α-L-vIgG2Fc amino acid sequence), wherein hFSH β is a beta subunit of FSH, hFSH α is an alpha subunit of FSH, L is a flexible peptide linker. The fusion protein of the present disclosure is abbreviated to hFSH-Fc. 
         [0007]    It is another aspect of the present disclosure that the amino acid sequence of hFSH β is the regular hFSH β subunit where its N-terminal 1-18 amino acid residues is deleted, as shown in SEQ ID NO: 5; The amino acid sequence of hFSH α is the regular hFSH α subunit where its N-terminal 1-24 amino acid residues is deleted, as shown in SEQ ID NO: 3. 
         [0008]    It is another embodiment of the present disclosure that the sequence of the CTP (carboxy-terminal peptide) refers to the 28-34 amino acid residues of C terminal of HCG β subunit, preferably, CTP is from 33 amino acid residues of C terminal of HCG β subunit, as demonstrated by SEQ ID NO: 4. 
         [0009]    In yet another embodiment of the present disclosure, provides a flexible peptide linker of about 20 or fewer, more preferably from about 2 to 20 amino acids in length and the flexible peptide linker contains or comprises of two or more of amino acids selected from the group consisting of glycine, serine, alanine, and threonine, preferably, the amino acid sequence of a flexible peptide linker:
       GlySerGlyGlyGlySerGlyGlyGyGlyGySerGlyGlyGlyGlySer.       
 
         [0011]    A further embodiment of the present disclosure provides the human IgG2 Fc variant of the present disclosure comprises a hinge with Pro331Ser mutant, CH2 and CH3 domains of human IgG2. 
         [0012]    The following chapters are detailed descriptions of IgG Fc variant, peptide linker and CTP: 
         [0013]    IgG Fc Variant 
         [0014]    Human immunoglobulins are the most abundant proteins in the blood, their plasma half-lives can reach as long as 21 days. The main reason for these is that every immunoglobulin comprises an Fe fragment, which has a unique function in stabilization of proteins. 
         [0015]    The Fc region of human immunoglobulins plays a significant role in immune defense system for the elimination of pathogens. Effector functions of IgG are mediated by the Fc region through two major mechanisms: (1) binding to Fc receptors (Fcγ Rs) on the cell surface can lead to ingestion of pathogens by phagocytosis or lysis by killer cells via the antibody-dependent cellular cytotoxicity (ADCC) pathway, or (2) binding to the C1q part of the first complement component C1 initiates the complement-dependent cytotoxicity (CDC) pathway, resulting in the lysis of pathogens. Among the four human IgG isotypes (IgG1, IgG2, IgG3, IgG4), the binding affinity of IgG2 to Fcγ R is the lowest and almost below detection. In addition, human IgG2 appears quite weak in binding to C1q and activating the complement cascade. For therapeutic use, when hFSH-Fc binds to various target cells, it may be determinative or important that the Fc region of the fusion protein will not mediate effector functions, leading to the lysis or removal of the target cells. Accordingly, it will become evident from the presently disclosed disclosure that the Fc region of hFSH-Fc should be Fc variant of a non-lytic nature. As discussed above, natural IgG Fc mediates various levels of effector functions. In contrast, Fc of a non-lytic nature is inert in terms of binding to Fcγ Rs and C1q for the triggering of effector functions. To obtain a non-lytic Fc, certain amino acids of the natural Fc region have to be mutated for the attenuation of the effector functions. 
         [0016]    By comparing amino acid sequences of human IgG isotypes, a portion of Fc near the N-terminal end of the CH2 domain is implicated to play an important role in the binding of IgG Fc to Fcγ Rs, and a portion of Fc near the carboxyl-terminal end of the CH2 domain is critical in binding of IgG to C1q. IgG2 does not bind to Fcγ Rs, but binds weakly to C1q. To minimize the binding of Fe to C1q and hence the CDC activity, IgG2 has been altered in this motif with Pro33 Ser mutation (As shown in  FIG. 1 ). This substitution resulted in an IgG2 variant with minimal CDC activity, which is more suitable for the production of recombinant hFSH-Fc fusion protein. 
         [0017]    Peptide Linker 
         [0018]    The length of the peptide linker plays an important role in the bioactivity of the recombinant dimeric protein. It has been reported that homodimeric EPO with two complete EPO subunits separated by a peptide linker of 3-7 amino acids showed decreased bioactivity comparing to the normal EPO (see, for example, Qiu H et al. J Biol Chem, 273:11173-11176, 1998). However, when the length of the peptide linker between the two EPO molecules reached 17 amino acids, the in vitro and in vivo bioactivity of the homodimeric EPO increased significantly (see, for example, Sytkowski A J et al. J Biol Chem, 274:24773-24778, 1999; U.S. Pat. No. 6,187,564). A possible explanation is as the length of the peptide linker between the functional molecules increases, steric hindrance decreases, the two functional molecules will not interfere with each other any more (see, for example, Ashkenazi A et al. Curr Opin in Immunol, 9:195-200, 1997). 
         [0019]    The instant disclosure for the first time provides a unique peptide linker at the hinge region to minimize the steric hindrance, a method to produce homodimeric hFSH-Fc fusion protein with the C terminal of hFSH α binding to the Fc mutant via a soft peptide linker. Instead of diminishing the bioactivity of FSH, this homodimeric hFSH-Fc fusion protein can maintain or even increase the bioactivity of FSH. Preferably, the amino acid sequence of the flexible peptide linker is:
       GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.       
 
         [0021]    CTP 
         [0022]    Glycosylation is very important to the bioactivity and plasma half-life of proteins. There are two types of glycosylation sites on a glycosylated protein, one is N-Glycosylation site and the other is O-Glycosylation site. CTP is a 28-34 amino acid-long peptide derived from the C terminal of HCG β subunit, and it has been reported that HCG has a much longer plasma half-life than the hFSH, mainly due to the glycosylation of CTP peptide. The CTP peptide possesses sites of O-Glycosylation, which will increase the glycosylation level of a protein, leading to improved pharmacokinetics and pharmacodynamics, such as increase of bioactivity and in vivo half-life. 
         [0023]    The instant disclosure provides a recombinant hFSH-Fc fusion protein with following characteristics. The hFSH-Fc fusion protein is a homodimeric protein which comprises β subunit of hFSH (hFSH β), CTP, α subunit of hFSH (hFSH α), a flexible peptide linker, and human IgG2 Fc variant (vIgG2Fc). The Fc variant of human IgG2 can prolong the in vivo half-life of fusion protein and can stabilize the protein. The non-lytic Fc variant can minimize the effector function when binding to Fc γ Rs and/or C1q, repress the ADCC and CDC pathway effectively, causing reduced cell cytotoxicity. CTP can increase the bioactivity and prolong the in vivo half-life of the protein without immunogenicity. CTP serves as a bridge between an α subunit and a β subunit of hFSH, leading to decrease the steric hindrance and contributing to the protein assembly and function. A soft peptide linker is inserted between the C terminal of hFSH α and an Fc variant, resulting in maintaining or even increasing the bioactivity of hFSH-Fc fusion protein. 
         [0024]    For the first time until the present disclosure, the CTP, peptide linker and IgG2 Fc variant are linked together in sequence with a hFSH molecule, leading to an innovative recombinant hFSH fusion protein. The elaborately designed array of CTP, peptide linker and IgG2 Fc variant can prolong the plasma half-life significantly without affecting the spatial configurations and bioactivity of hFSH, leading to minimized injection times and side effects. 
         [0025]    In another embodiment of the present disclosure, a method is disclosed for making or producing such recombinant hFSH-Fc fusion proteins from a mammalian cell line such as a CHO-derived cell line. A method for making the recombinant fusion protein includes the following procedures.
       (1) Construct an expression vector containing DNA encoding hFSH-Fc fusion protein;   (2) Generate stable mammalian cell lines expressing the fusion protein;   (3) Culture cells to high density;   (4) Purify the recombinant hFSH-Fc fusion protein.       
 
         [0030]    According to this instant disclosure, a method of constructing expression vector containing DNA encoding hFSH-Fc fusion protein is disclosed. The codon of nucleotide sequence of the hFSH-Fe gene is optimized and then synthesized (as shown in SEQ ID NO: 1). The fusion gene sequence is then inserted into a mammalian cell expression vector, leading to the plasmid pCDNA3-hFSH-Fc containing hFSH-Fc gene ( FIG. 4 ). Nucleotide sequence optimization is based on codon preference of a mammalian host cells. 
         [0031]    The expression vector of mammalian cells may be commercially available but not limited to vectors suitable for eukaryotic expression such as pCDNA3, pCMV/ZEO, pIRES, pDR, pBK, pSPORT etc., preferably, pCDNA3. 
         [0032]    For the instant disclosure, a method is disclosed for generating stable mammalian cell lines expressing the recombinant hFSH-Fc fusion protein. The expression plasmid containing hFSH-Fc gene is transfected into suitable mammalian host cells, and the cell lines are then screened for stable and high expression level of target protein. 
         [0033]    The mammalian host cells includes CHO, HEK293, COS, BHK, NS0 and Sp2/0, preferably, CHO; More preferably, Dihydrofolate Reductase (DHFR) deficient CHO cell, which has been adapted to suspension culture in serum free medium (CHO DHFR-). 
         [0034]    The transfection methods include phosphate calcium method, electroporation and liposome transfection, preferably, electroporation. 
         [0035]    A method for screening and obtaining cell lines of stable producers of FSH-Fc fusion protein is disclosed. The cells expressing fusion protein are initially screened by the screening markers, and the stable cell lines of high producers are made by amplification selectable markers. A screening marker is known in the art to be any suitable selective markers of resistance, for example, ZEO (Zeocin), G418 (amino glycosides antibiotics), PUR (puromycin) or HYP (Hygromycin), preferably, ZEO; A screening marker is also well known in the art to be any fluorescent labeling gene, including GFP (green fluorescent protein), RFP (red fluorescent protein), preferably. GFP. Amplification selectable marker is known in the art to be DHFR (DHFR) sequence or GS (Glutaminesynthetase) sequence, preferably, DHFR. Due to the cells of CHO-DHFR- are lack of dihydrofolate reductase, they can not synthesize tetrahydrofolate by their own, in order to survive, the addition of hypoxanthine, thymidine and glycine in the culture medium becomes essential. However, when the target gene is co-transfected with the DHFR gene, not only those cells can grow in the culture medium without the additives mentioned above, but also MTX resistant cell lines can be obtained because DHFR can be inhibited by MTX (methotrexate, folic acid analogue), under the selective pressure of MTX, DHFR gene must amplify to a certain large copy number in order to survive; And as the target gene together with the co-transfected DHFR gene are prone to integrate into the same domain of the cell chromosome, they are amplified simultaneously, leading to the expression of large amounts of exogenous target protein. 
         [0036]    Also according to this disclosure, a method of high-density cell culture is disclosed for producing recombinant hFSH-Fc fusion protein. The above-mentioned stable cell line is transferred to a shake bottle or bioreactor to culture in a larger scale, especially, through the optimization process of culture conditions, the instant disclosure obtains a high level of expression of the recombinant hFSH-Fc fusion protein in the culture medium. The method can accomplish a high density culture of cells, increase the quality and yield of recombinant proteins, and also improve the degree of glycosylation and content of sialic acid. 
         [0037]    The optimization of cell culture conditions includes the cooling culture method, specifically, when the cell density reaches 1×10 7 /mL, the culture temperature is reduced from 37° C. to 33° C., and then the cells are cultured at 33° C. until the cumulative protein production level no longer increases. This method can increase the bioactivity and cumulative yield of the target protein. 
         [0038]    The optimization of cell culture conditions also includes supplement of special additives in the culture medium, preferably, add 100 μM Cu 2+  to the basic medium and 2 mm ManNAc (N-acetyl-D-amino mannose) to the feeding medium. This method of additive supplement can increase the degree of glycosylation and the content of sialic acid by 20%. 
         [0039]    For the instant disclosure, a method of purification of the recombinant hFSH-Fc fusion protein is disclosed using the following procedures.
       1) Protein A affinity chromatography: centrifuge culture medium and collect the supernatant, according to the characteristic of the fusion protein coupled to an Fc fragment, use Protein A affinity chromatography to capture the target hFSH-Fc fusion protein.   2) Hydrophobic chromatography: Based on the hydrophobic characteristic of the recombinant hFSH-Fc fusion protein, use hydrophobic chromatography to further remove the impurities from the eluent of Protein A chromatography.       
 
         [0042]    The suitable resins for hydrophobic chromatography can be selected from the following: Butyl Sepharose 4 Fast Flow, Octyl Sepharose 4 Fast Flow, Phenyl Sepharose 6 Fast Flow, Butyl-S Sepharose 6 Fast Flow, Butyl Sepharose 4B, Octyl Sepharose CL-4B and Phenyl Sepharose CL-4B, preferably, Phenyl Sepharose 6 Fast Flow. 
         [0043]    The instant disclosure discloses a preparation method of the recombinant hFSH-Fc fusion protein with high expression yield, and due to its coupling to an IgG2 Fc variant, a convenient and efficient purification process can be achieved by the protein A affinity chromatography. The purity of the fusion protein reached 98% or more after the subsequent hydrophobic chromatography. In addition, the α and β chain of the recombinant hFSH-Fc fusion protein can be correctly folded together, avoiding the unwanted formations of α-α dimer and β-β dimer, greatly simplifies the purification process and significantly reduces the production cost. 
         [0044]    Another embodiment of the present disclosure provides a pharmaceutical composition comprising the recombinant hFSH-Fc fusion protein, wherein a pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient or diluent, and an effective amount of the recombinant hFSH-Fc fusion protein of present disclosure. 
         [0045]    Specifically, the pharmaceutical composition contains an effective dose (such as 0.000001-90 wt %; preferably, 0.1-50 wt %; more preferably, 5-40 wt %) of the recombinant hFSH-Fc fusion protein and a pharmaceutically acceptable carrier. Typically, an effective amount of the fusion protein is formulated into a non-toxic, inert and pharmaceutically acceptable aqueous carrier, the pH of formulation is usually about 5-8, preferably, 6-8. 
         [0046]    According to this disclosure, the pharmaceutical acceptable carrier includes but not limited to sucrose, mannitol, Tween 20, methionine, saline, buffer, glucose, water, glycerol, and their compositions thereof. Typically the pharmaceutical preparation of compositions and administration route should be matched, wherein the pharmaceutical preparation of compositions in the present disclosure can be made into injection form, such preparation using saline or water solution containing glucose and other excipient agent by conventional methods. The pharmaceutical composition is manufactured under sterile conditions. The amount of active ingredients is of the effective treatment dose. The pharmaceutical preparation of the present disclosure can also be made into a sustained-release form. 
         [0047]    The effective amount of the fusion protein of the instant disclosure can be varied according to the mode of administration and the severity of diseases. The preferred effective amount of the fusion protein can be determined by those of skill in the art according to a variety of factors such as by clinical trials. The factors include but are not limited to the pharmacokinetic parameters of the fusion protein such as bioavailability, metabolic rate and half-life, the severity of disease, a patient&#39;s weight, a patient&#39;s immune status and route of drug administration etc. 
         [0048]    A further embodiment of the present disclosure provides a method for the application of the recombinant hFSH-Fc fusion protein in the treatment and/or prevention of human infertility. 
         [0049]    The recombinant hFSH-Fc fusion protein of this instant disclosure prolongs the in vivo half-life significantly, leading to improved pharmacokinetics and pharmacodynamics, reduces not only the number of injections and side effects, but also the pain and economic burden of patients comparing with the existing hFSH in clinical application. 
         [0050]    There are many advantages with the present disclosure stated below for the recombinant hFSH-Fc fusion protein and the preparation method.
       1) The recombinant hFSH-Fc fusion protein is a novel fusion protein comprising the CTP, a peptide linker, human IgG2 Fc variants (vIgG2Fc) and hFSH linked in right sequence order. The hFSH-Fc fusion protein maintains the correct spatial configuration of hFSH, significantly extends the in vivo half-life and greatly improves the expression level in CHO cells. Moreover, the in vitro and in vivo bioactivity of the recombinant hFSH-Fc fusion protein is similar to those of the existing hFSH.   2) The α and β chain of the homodimeric hFSH-Fc fusion protein is coupled correctly by covalent bonds, avoiding the formation of α-α dimer and β-β dimers, greatly simplifying the purification process and reducing the production cost.   3) The in vivo half-life of the recombinant hFSH-Fc fusion protein is prolonged significantly, and its plasma half-life is four times longer than the existing hFSH, leading to the significant reduction of the injection times and side effects caused by the existing treatment regimen in clinical application.       
 
     
    
     
       BRIEF DESCRIPTIONS OF THE DRAWINGS 
         [0054]      FIG. 1  shows the amino acid sequence alignment from the binge and CH2 regions of human IgG2 and its variants. Three portions are compared: amino acid position 228, 234-237, and 330-331. Amino acid mutations of the variants are indicated in bold italics. The EU numbering system is used for the amino acid residues. 
           [0055]      FIG. 2  shows the schematic diagram of single stranded and dimerized recombinant hFSH-Fc protein. a) single stranded hFSH-Fc; b) dimerized hFSH-Fc. 
           [0056]      FIG. 3  shows the nucleotide sequence and deduced amino acid sequence of hFSH-Fc fragment between HindIII and EcoRI fragment in pCDNA3 expression vector. The nucleotide sequence of the recombinant hFSH-Fc comprises a leading peptide (amino acid residues 1-18), hFSH β chain, CTP, mature hFSH α chain, peptide linker, and IgG2Fc variant (vIgG2Fc). Mature recombinant hFSH-Fc fusion protein contains mature hFSH β chain (amino acid residues 19-129), CTP (amino acid residues 130-162), mature a chain (amino acid residues 163-254), peptide linker (amino acid residues 255-270) and IgG2Fc variant (vIgG2Fc) (amino acid residues 271-493). 
           [0057]      FIG. 4  shows the schematic representation of eukaryotic expression plasmid pCDNA3-hFSH-Fc. The full-length of the plasmid is 9063 bp, comprising 10 major gene fragments: 1. CMV promoter; 2. Target gene hFSH-Fc; 3. IRES: 4. The zeocin resistance gene; 5. BGH terminator; 6. SV40 promoter; 7. DHFR amplification gene; 8. SV40 terminator; 9. Ampicillin resistance gene (ampicillin); 10. The ColE1 origin of replication (Ori). 
           [0058]      FIG. 5  shows cumulative recombinant hFSH-Fc protein level (mg/L) secreted from cells cultured in a 7 L bioreactor. 
           [0059]      FIG. 6  shows successful expression of recombinant hFSH-Fc fusion protein in CHO cells by Western blotting analysis in non-reduced SDS-PAGE: Lane 1, human urinary hFSH (about 43 kDa); Lane 2, recombinant hFSH-Fc fusion protein of the present disclosure (about 140 kDa). 
           [0060]      FIG. 7  shows the map of 10% SDS-PAGE for single stranded and dimerized hFSH-Fc under reduced condition and non-reduced condition. a) non-reduced, dimerized hFSH-Fc (about 140 kDa); b) reduced, single stranded hFSH-Fc (about 70 kDa). 
           [0061]      FIG. 8  shows the metabolic curve in rats for the recombinant hFSH-Fc fusion protein, recombinant hFSH and human urinary hFSH. 
       
    
    
     DETAILED DESCRIPTION 
       [0062]    The present disclosure will further be elaborated with illustrative embodiments below. The methods are intended to illustrate and not to limit this disclosure. Specific experimental conditions not stated in the following embodiments can be operated according to the conventional conditions such as Sambrook etc. Molecular cloning: a laboratory manual (New York: Cold Spring Harbor Laboratory Press press, 1989), or manufacturer&#39;s recommendations. 
       Example 1. Preparation of the Gene Encoding the Recombinant hFSH-Fc Fusion Protein 
       [0063]    The design of gene sequence was optimized on the basis of preferred codons of CHO cells. The gene encoding the leader peptide and mature protein of hFSH β chain, CTP and mature protein of hFSH α chain were synthesized de novo. The resulting DNA fragment of 756 bp in length was inserted into a holding vector such as pUCS7 at the EcoRV restriction enzyme site to give the phFSH plasmid. The sequence of the hFSH gene was confirmed by DNA sequencing. 
         [0064]    The gene encoding the linker peptide (L) and human IgG2Fc variant (vIgG2Fc) with restriction sites of BamHI (5′) and EcoRI (3′) was synthesized de novo. Resulting DNA fragment of L-vIgG2Fc was inserted into a holding vector such as pUC19 between the BamHI and EcoRI sites to give the pL-vIgG2Fc plasmid. The sequence of the pL-vIgG2Fc gene was confirmed by DNA sequencing. To prepare the hFSH-L-Fc fusion gene, the hFSH fragment containing the leader peptide sequence, hFSH β, CTP and hFSH α was excised from the phFSH plasmid with SpeI and BamHI, and then purified by agarose gel electrophoresis. The purified fragment was then inserted to the 5′-end of the peptide linker in the pL-vIgG2Fc plasmid, linked by T4 ligase to give the phFSH-L-vIgG2Fc plasmid. The resulting fusion gene of phFSH-L-vIgG2Fc plasmid comprised hFSH 0, CTP, hFSH α, peptide linker, and Fc variant gene. The single stranded structure is shown in  FIG. 2 a    and dimeric structure is shown in  FIG. 2   b.    
         [0065]    To construct the expression vector for hFSH-Fc, the hFSH-L-vIgG2Fc fragment was excised from the phFSH-L-vIgG2Fc plasmid with restriction enzyme SpeI and EcoRI and was purified by agarose gel electrophoresis. The purified fragment was then inserted into the corresponding restriction sites of the mammalian expression plasmid, such as pcDNA3 (Invitrogen), to give the pCDNA3-hFSH-L-vIgG2Fc plasmid (pCDNA3-hFSH-Fc), as shown in  FIG. 4 . The plasmid comprised a cytomegalovirus (CMV) early gene promoter-enhancer which was required for high level expression of exogenous protein, two kinds of selective marker gene, leading to ampicillin resistance in bacteria and zeocin resistance in mammalian cells. In addition, this expression vector comprised the dihydrofolate reductase (DHFR) gene which was in a position to enable the co-amplification of the hFSH-L-vIgG2Fc fusion gene together with the DHFR gene in the presence of methotrexate (MTX) when the host cells were deficient in the DHFR gene expression. 
         [0066]    The presence of CTP between the hFSH β and hFSH α moieties (and chemically bound to both) is favorable to the right assembly of the two chains. The presence of a peptide linker, preferably a flexible linker, between the hFSH and Fc moieties (and chemically bound to both) increases the flexibility of the hFSH domain and enhances its biological activity. For the present disclosure, a peptide linker of about 20 or fewer amino acids in length is preferred. While a single amino acid is within the scope of the present disclosure, it is preferred to have a flexible peptide linker of about 20 to about 2 amino acids in length. Peptide linker containing or comprising of two or more of amino acids selected from the group consisting of glycine, serine, alanine, and threonine can be used preferably. One embodiment of the present disclosure has the peptide linker containing a Gly-Ser peptide component, and its amino acid sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer. 
       Example 2. Stable Expression of the Recombinant hFSH-Fc Fusion Protein in Mammalian Cells 
       [0067]    The pCDNA3-hFSH-L-vIgG2Fc expression vector plasmid made from example 1 was transfected into a mammalian host cell line to achieve the expression of the fusion protein. For stable high levels of expression, a preferred host cell line was CHO cells deficient in the DHFR enzyme (CHO DHFR-).  FIG. 2 b    shows the schematic diagram of the recombinant dimerized hFSH-Fc fusion protein. A preferred method of transfection was electroporation. Ten μg of plasmid DNA linearized with PvuI was added to 2 to 5×10 7  cells in a cuvette using Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, Calif.) set at an electric field of 250 V and a capacitance of 960 μFd. Two days following the transfection, the media was replaced with growth media containing resistant marker gene of 100 μg/mL Zeocin. Transifectants resistant to the selection chemical were analyzed for the expression level of hFSH-Fc protein by Western blotting using anti hFSH antibody. To achieve higher levels of the fusion protein expression, co-amplification was carried out by utilizing the gene of DHFR that could be inhibited by the MTX drug. In growth media containing increasing concentrations of MTX, the transfected fusion protein gene was co-amplified with the DHFR gene. Transfectants capable of growing in media with up to 10 μM/mL of MTX were then subcloned by limiting dilutions. The subcloned cells were further analyzed by measuring the secretion rates. To obtain stable cell lines of high expression of recombinant hFSH-Fc fusion protein, cell clones yielding secretion rate levels over 10 μg/million cells/24 h (preferably about 20 μg/million cells/24 h) were adapted to suspension culture using serum-free growth media. 
       Example 3. Purification and Characterization of the Fusion Protein 
       [0068]    The high expression cell line from Example 2 was first undergone a domestication process using serum-free medium in the culture dish, and then transferred to the shake flask for suspension culture. During the above serum-free medium culture process, medium optimization was also carried out for testing different ingredients to detect various parameters, such as the growth state, growth trend, bioactivity and sialic acid etc. The following conditions of cell culture were preferred: basic medium comprising 100 μM Cu 2+ , feeding medium comprising 2 mM ManNAc (N-acetyl amino mannose). This culture condition can increase the glycosylation content of the recombinant hFSH-Fc fusion protein, such as about 20% increase of the content of sialic acid. For cell growth at a 7 L bioreactor, when the cell density reached 1×10 7 /mL at 37° C., the culture temperature was adjusted to 33° C. to allow longer accumulation and more stable of secreted fusion protein than those at 37° C. The optimum culture period for one batch of cell production was approximately 20 days. Small amount of the recombinant fusion protein was initially purified by chromatography of 1 ml Protein A column to determine the expression level, as shown in  FIG. 5 , the cumulative yield of recombinant hFSH-L-vIgG2Fc cell line was 1.87 g/L. 
         [0069]    The purification of the recombinant hFSH-Fc fusion protein included the following steps:
       1) Protein A affinity chromatography: The culture media containing the hFSH-Fc fusion protein were centrifugated, and the supernatant was collected for subsequent loading onto a Protein A column pre-equilibrated in phosphate-buffered saline (PBS). After binding of the fusion protein to Protein A resin, the flow-through fractions were discarded. The column was then washed with PBS until OD at 280 nm was below 0.01. The bounded fusion protein was eluted with 20 mM sodium acetate buffer (pH 4.0), and the elution was neutralized with 1M Tris-HCl buffer (pH10.0). The purity of the hFSH-Fc protein after this step could reach 95% or more.   2) Hydrophobic chromatography: The elution from above Protein A chromatography was buffer changed to 20 mM Tris-HCl-1.5 M NaCl (pH8.0) with ultrafiltration method, and loaded onto a phenyl-6 Fast Flow column equilibrated in 20 mM Tris-HCl-1.5 M NaCl (pH8.0) buffer. The column was washed with the same equilibration buffer, and then washed with 20 mM Tris-HCl-1.35M NaCl (pH8.0) before its elution with 2 mM Tris-HCl-0.5M NaCl (pH8.0).       
 
         [0072]    As shown in  FIG. 6 , Western blotting analysis indicated that the recombinant hFSH-Fc fusion protein in CHO cells was successfully expressed; Non-reduced SDS-PAGE showed hybridization bands of target protein respectively: 1) 43 kDa band, human urine hFSH (commercial product); 2) 140 kDa band, recombinant hFSH-Fc fusion protein (the present disclosure), proving that the recombinant hFSH-Fc fusion protein comprised hFSH component.  FIG. 7  indicated SDS-PAGE of the hFSH-Fc fusion protein under reduced and non-reduced conditions. The result demonstrated that the purity of the hFSH-Fc protein could reach 98% or more, and the molecular weight of the hFSH-Fc protein under reduced condition was half of that under non-reduced condition. 
       Example 4. In Vitro and In Vivo Bioactivity Assay 
       [0073]    In vitro bioactivity of the recombinant hFSH-Fc fusion protein (Immunological activity) was assayed by ELISA kit from BIOCHECK (USA) Company. Experimental procedure was conducted according to specifications of the kit. In vivo activity was assayed by measuring ovarian weight gain based on the 2010 edition of the British Pharmacopoeia. Protein quantitation was determined using the traditional LOWRY method. According to British Pharmacopoeia, small amount of background HCG (701 U/ml) was used to increase assay sensitivity for ovarian weight gain in rats. Sample diluent comprising 701 U/ml HCG was prepared by adding 0.1% albumin phosphate buffer (pH7.2±0.2). For in vivo assay, each of sample diluent (pH7.2±0.2) was prepared to equally contain 1.67 IU/ml FSH based on ELISA activity of test samples, that includes the FSH standard sample (positive control), recombinant hFSH, human urine FSH and recombinant hFSH-Fc fusion protein. Female Wistar rats of 19 to 28 days old could be used, however, one experiment was required for those rats of age differences no more than 3 days and weight differences no more than 10 grams to minimize experimental variation. The 24 rats were equally divided into four groups for the following samples: FSH standard sample (positive control), recombinant hFSH (commercial product), human urine FSH and recombinant hFSH-Fc fusion protein. Each group were injected with corresponding above sample subcutaneously at the same time every day, two times a day, 0.2 ml each time for 3 consecutive days. Twenty-four hours after the last injection, animals were killed in accordance with the sequence of administration by cervical vertebra dislocation, and ovaries were drawed and weighed after drying the surfaces. The in vivo activities of recombinant hFSH, human urine hFSH and recombinant hFSH-Fc were assayed by the quantity response parallel line method based on the ovarian weight gain of the standard group (positive control). The data showed In vitro bioactivity of recombinant hFSH-Fc, recombinant hFSH and human urine FSH were 10105, 9928 and 9321 IU/ml, respectively; and in vivo bioactivity were 10230, 8190 and 9051 IU/ml, respectively. These results indicated that, the recombinant hFSH-Fc fusion protein of present disclosure has both in vitro and in vivo biological activity. 
       Example 5. Pharmacokinetics of Recombinant hFSH-Fc Fusion Protein 
       [0074]    Fifteen Male Wistar rats with weight between 200-250 g were randomly and equally divided into 3 groups for recombinant hFSH-Fc, human urine FSH and recombinant hFSH. All groups were subcutaneously injected with single dose corresponding sample protein of 15 IU/kg. The blood samples were collected at different time intervals, i.e., at 1, 2, 3, 4, 6, 8, 12, 36, 56 h for human urine FSH and recombinant hFSH groups, at 1, 2, 4, 8, 12, 24, 56, 120, 176, 200, 264, 340 h for recombinant hFSH-Fc group. The above samples were centrifuged at 3000 rpm for 5 min and plasma supernatants were stored at −20° C. until assays were performed. The immunological activities of FSH in plasma at each time point were tested by ELISA kit (BIOCHECK, USA). The pharmacokinetic parameters were calculated by statistical method using PKSolver 2 software. The pharmacokinetic curves of each group were shown in  FIG. 8 , and the data of half-life were shown in Table 1. The results indicated that the half-life of recombinant hFSH and human urine FSH in rats were 11.35±1.0 h and 12.7±2.8 h, respectively. The half-life of recombinant hFSH-Fc fusion protein of present disclosure was 47.24±13.92 h, which was at least 4 times of that of recombinant hFSH and human urine FSH. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 T½ Comparation 
               
             
          
           
               
                   
                 Group 
                 T 1/2  (h) 
               
               
                   
                   
               
               
                   
                 Recombinant hFSH-Fc 
                 47.24 ± 13.92 
               
               
                   
                 Recombinant hFSH 
                 11.35 ± 1.0  
               
               
                   
                 Human urine FSH 
                 12.7 ± 2.8  
               
               
                   
                   
               
             
          
         
       
     
       Example 6. Effect of Recombinant hFSH-Fc Fusion Protein on Promoting Ovulation in Female Rats 
       [0075]    Ninety female SD rats with weight between 200-250 g were fed and observed for more than 7 days in the experimental environment to ensure their healthy conditions. Sixty of the healthy female SD rats were selected, randomly and equally divided into 4 groups: negative control, recombinant hFSH-Fc fusion protein and recombinant hFSH, human urine FSH. In addition, the weight distribution of each group rats was similar. Firstly, one estrous cycle of these rats was observed and monitored by the method of vagina smear. The group of recombinant hFSH-Fc fusion protein was subcutaneously injected with corresponding drug (fusion protein) only one time on the first day among diestrous with a dose of 45 IU/kg per rat; The recombinant hFSH group and human urine FSH group were subcutaneously injected with corresponding drug on the first day and consecutive two days among diestrous (15 IU/kg/day) to give a total dose of 45 IU/kg per rat; Negative control group was subcutaneously injected with same volume of normal saline. All groups were injected with HCG (10 IU/each rat) subcutaneously on the fourth day (proestrus). On the fifth day (estrus), all rats were anesthetized, ovaries were drawn and weighed. The separated ovaries were fixed by bouin&#39;s fluid and embedded in paraffin. Consecutive sections of fixed ovary at 6 m intervals and microscopic organizational observation were performed, and the number of follicles was also recorded. 
         [0076]    The number of follicles each group was shown in Table 2. The result indicated that, compared with the negative control group, recombinant hFSH-Fc fusion protein, recombinant hFSH and human urine FSH can all promote superovulation significantly (P&lt;0.01) on female rats. Importantly, for achieving equivalent efficacy, recombinant hFSH-Fc fusion protein of present disclosure only needs one time administration whereas both the recombinant hFSH and human urine FSH need three times administration. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Effect of Recombinant hFSH-Fc Fusion Protein 
               
               
                 on Promoting Ovulation in Female Rats 
               
             
          
           
               
                   
                   
                 Number of large 
                 Number of small 
               
               
                 Group 
                 n 
                 follicles/section 
                 follicles/section 
               
               
                   
               
               
                 Negative control 
                 15 
                     8.2 ± 5.1 
                 119.0 ± 36.1  
               
               
                 Recombinant hFSH-Fc 
                 15 
                 24.3 ± 8.4 a   
                 233.1 ± 65.9 a   
               
               
                 Recombinant hFSH 
                 15 
                 21.1 ± 5.9 a   
                 227.4 ± 55.2 a   
               
               
                 Human urine FSH 
                 15 
                 20.4 ± 4.1 a   
                 225.4 ± 31.6 a   
               
               
                   
               
               
                 Notes: 
               
               
                 t test, Compared with the negative control group, 
               
               
                   a p &lt; 0.01. 
               
             
          
         
       
     
       Example 7. The Therapeutic Effect of Recombinant hFSH-Fc Fusion Protein on Androgen Induced Anovulation in Female Rats 
       [0077]    Seventy-five juvenile SD female rats of 9 days old were randomly divided into two groups: normal control group (15 rats) and animal model group (60 rats). The normal control group was subcutaneously injected once with 0.05 ml neutral tea-seed oil on the nape of neck, the animal model group was subcutaneously injected once with 1.25 mg testosterone propionate on the nape of neck. Vagina was opened at 70th day, and two cycles of consecutive vaginal smear (total 10 days as one cycle lasting for 5 days) were performed. The normal control group should have typical estrous cycle (proestrus, estrus, metestrus, diestrus) with consecutive vaginal smear, and any rats with abnormal estrous cycle was eliminated from the normal control group. The animal model group&#39;s vaginal epithelial cells should be free of estrous cycle with sustained keratinization, indicating that the anovulatory rat model was successful, and any rats with abnormal performance were eliminated from the model group. At 81th day and later on, 13 rats from the normal control group were injected subcutaneously with distilled water of 10 ml/kg/day. Fifty-two rats from the anovulatory model mentioned above were randomly and equally divided into 4 groups: negative control (animal model), recombinant hFSH-Fc, recombinant hFSH and human urine FSH. Rats of the negative control group were injected subcutaneously with distilled water of 10 ml/kg/day; Rats of the recombinant hFSH-Fc group were injected subcutaneously with hFSH-Fc of 45 IU/kg/time, once every three days lasting for 15 days to give a total of 5 injections; Rats from the recombinant hFSH group and human urine FSH group were injected subcutaneously with corresponding drugs of 7.5 IU/kg/time, twice a day lasting for 15 days to give a total of 30 injections. After administration, rats in each group were given a continuous vaginal smear for two estrous cycles to examine their ovulation condition. Rats in each group were weighed and anesthetized by intraperitoneal injection with Urethan before the estrous cycle. The left ovary and uterus tissue specimens were fixed in 10% neutral formaldehyde for pathorphological observation of ovary and uterus. Quantitative indexes of ovarian observation include the number of follicles at different stages, the number of corpus luteums; and index of uterus observation was measured by endometrial thickness. 
         [0078]    Table 3 showed results of each group&#39;s estrous cycles, table 4 showed results of each group&#39;s pathological changes of uterus and ovaries. Compared with the negative control group of animal model, the recombinant hFSH-Fc fusion protein of present disclosure, recombinant hFSH and human urine FSH had a significant promoting effect on inducing estrus of anovulatory female rats; the number of large follicles and corpus luteums of these later three groups were significantly higher (P&lt;0.01) than those of the negative control group within anovulatory model. However, the recombinant hFSH and human urine FSH both were required for total 30 times administration and the recombinant hFSH-Fc fusion protein was only required for 5 times to achieve the same potency. Results showed that the recombinant hFSH-Fc fusion protein of present disclosure, recombinant hFSH and human urine FSH all had a significant promoting effect on inducing estrus of anovulatory female rats, but the dosing frequency of the fusion protein from present disclosure was significantly less than that of the recombinant hFSH and human urine FSH. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Promoting effect of recombinant hFSH-Fc fusion protein 
               
               
                 on inducing estrus with anovulatory female rats 
               
             
          
           
               
                   
                 Number of 
                 Animals with 
                   
               
               
                 Group 
                 animals 
                 estrous cycles 
                 Ovulation rate 
               
               
                   
               
             
          
           
               
                 Normal control 
                 13 
                 13      
                 100 
               
               
                 Negative control 
                 13 
                 0     
                 0 
               
               
                 Recombinant hFSH-Fc 
                 13 
                 10 a   
                 76.9% a   
               
               
                 Recombinant hFSH 
                 13 
                 8 a   
                 71.5% a   
               
               
                 Human urine FSH 
                 13 
                 6 a   
                 70.2% a   
               
               
                   
               
               
                 Notes: 
               
               
                 X 2  test, Compared with the negative control group, 
               
               
                   a p &lt; 0.01. 
               
             
          
         
       
     
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                 TABLE 4 
               
             
             
               
                   
               
               
                 Pathological changes of uterus and ovaries 
               
               
                 with anovulatory female rats 
               
             
          
           
               
                   
                 Number of 
                 Number of 
                 Endometrial 
               
               
                 Group 
                 large follicles 
                 corpus luteums 
                 thickness (mm) 
               
               
                   
               
               
                 Normal control 
                 8.9 ± 3.3  
                 6.1 ± 1.2  
                 0.44 ± 0.15 
               
               
                 Negative control 
                 2.1 ± 0.6  
                 0.4 ± 0.2  
                 0.39 ± 0.14 
               
               
                 Recombinant hFSH-Fc 
                 7.7 ± 2.6 a   
                 6.2 ± 2.3 a   
                 0.42 ± 0.11 
               
               
                 Recombinant hFSH 
                 6.8 ± 1.8 a   
                 5.8 ± 2.1 a   
                 0.42 ± 0.08 
               
               
                 Human urine FSH 
                 6.7 ± 1.1 a   
                 5.2 ± 1.4 a   
                  0.4 ± 0.15 
               
               
                   
               
               
                 Notes: 
               
               
                 t test, Compared with the negative control group, 
               
               
                   a p &lt; 0.01. 
               
             
          
         
       
     
       Example 8. Effect of Recombinant hFSH-Fc Fusion Protein on Ovarian Stimulation in Rats 
       [0079]    Forty female Wistar rats of 22-day old were randomly and equally divided into 4 groups: saline buffer (negative control), recombinant hFSH-Fc fusion protein, recombinant hFSH (positive control), human urine FSH. The last three groups of rats were injected subcutaneously for consecutive four days daily with corresponding protein of 10 IU/day/rat of the recombinant hFSH-Fc fusion protein, recombinant hFSH and human urine FSH. At 26-day, these three groups were injected subcutaneously with 30 IU HCG; For the negative control group, the 22-day to 26-day age of rats was daily injected subcutaneously with the same volume of saline buffer. At 28-day age, all four groups of rats were injected with 0.1 ml of 1% Blue Evans (EB, Sigma) for 30 minutes staining. All rats were then killed by cervical vertebra luxation and abdominal cavities were cut open to observe whether there was ascites. The rats with little or no ascites were injected intraperitoneally with 5 ml of saline, and the perfusate was collected and diluted to 10 ml in a test tube. Then, 0.05 ml of 0.1 M NaOH solution was added to the test tube, centrifuging for 10 minutes at 3000 rpm at room temperature. Spectrophotometer was used to determine the OD at 600 nm. Finally, the ovaries of both sides were detached and weighed with electronic balance immediately. Observation indexes include: (1). Permeability of abdominal capillary: the EB content of peritoneal lavage fluid was calculated according to the standard curve ploted thereby. (2). Ascites grading: grade 1: no ascites; grade 2: a small amount of ascites; grade 3: medium amount of ascites; grade 4: large amount of ascites; grade 5: massive ascites or ascites overflows from abdominal incision. (3). Ovarian weight: According to Golan&#39;s criteria for the diagnosis of ovarian hyperstimulation syndrome, only all three criteria of ovarian volume increasing, abdominal capillary permeability increasing and ascites emerging were met together that ovarian hyperstimulation syndrome can be diagnosed. 
         [0080]    After 4 days administration of same dose of protein sample, as shown in Table 5, the EB content and ascites score of the recombinant hFSH-Fc group had no significant difference from that of the saline control group. However, in comparison with the saline control group, the ovarian weight of the recombinant hFSH-Fc group was significantly increased but no ovarian hyperstimulation syndrome was observed. In contrast, the EB content, ovarian weight and ascites score of recombinant hFSH group and human urine FSH group were significantly higher than thseat of the saline control group and recombinant hFSH-Fc group, indicating that the recombinant hFSH and human urine FSH could cause ovarian hyperstimulation syndrome. These results demonstrated that the side effect of recombinant hFSH-Fc was relatively smaller than that of recombinant hFSH and human urine FSH, indicating recombinant hFSH-Fc protein was more safe for clinical application. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Comparison of ascites grading, EB content 
               
               
                 in abdominal fluid and ovary weight 
               
             
          
           
               
                   
                   
                 EB content in 
                   
               
               
                   
                 Ascites 
                 abdominal fluid 
                 Ovary weight 
               
               
                 Group 
                 grading 
                 (μg/ml) 
                 (mg) 
               
               
                   
               
               
                 Saline control 
                 1.00 ± 0.00     
                 0.52 ± 0.01     
                 28 ± 3      
               
               
                 Recombinant 
                 1.17 ± 0.25     
                 0.63 ± 0.22     
                 117 ± 24 a   
               
               
                 hFSH-Fc 
               
               
                 Recombinant hFSH 
                 2.36 ± 0.31 ab   
                 3.24 ± 0.33 ab   
                 165 ± 23 ac   
               
               
                 Human urine FSH 
                 2.58 ± 0.46 ab   
                 3.78 ± 0.28 ab   
                 155 ± 24 ac   
               
               
                   
               
               
                 Notes: 
               
               
                 t test, Compared with the normal control group, 
               
               
                   a p &lt; 0.01; Compared with the hFSH-Fc group; 
               
               
                   b p &lt; 0.01, 
               
               
                   c p &lt; 0.05