1 Syntonix Pharmaceuticals, Inc., 9 Fourth Avenue,Waltham, MA 02451, USA, 2 Current address: Protein Structure Group/Discovery Technologies, Novartis Institutes for Biomedical Research Inc., 250 Massachusetts Avenue, Cambridge, MA 02139, USA
3 To whom correspondence should be addressed: Email: slow{at}syntnx.com
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Abstract |
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Key words: FcRn/FSH/lung/pulmonary delivery
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Introduction |
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One candidate transport pathway involves the neonatal Fc receptor (FcRn) that was first described in newborn rodents as being responsible for the transport of IgG from maternal milk into the neonatal bloodstream (Simister et al., 1997). Human FcRn was first described in the placenta where it transports IgG from mother to fetus (Story et al., 1994
). Although rodent FcRn levels decrease in epithelial tissues after weaning, human FcRn was recently detected in epithelial cells of adult human lung and intestine (Israel et al., 1997
), and has been shown to transport IgG across human epithelial cells in vitro (Dickinson et al., 1999
). Various proteins have been fused to the Fc domain of IgG1 and shown to retain biological activity (Ashkenazi et al., 1993
; Chamow and Ashkenazi, 1996
; Economides et al., 2003
; Bitonti et al., 2004
). Conjugation to Fc also increases the serum half-life of the fusion partner (Kim et al., 1998
) due to the ability to bind FcRn which is the major regulator of IgG half-life (Kim et al., 1994
; Junghans and Anderson, 1996
; Simister et al., 1997
). We therefore propose that FcRn may be used to transport therapeutic proteins, including FSH, across epithelial barriers and concomitantly increase the circulating half-life of the molecule.
FSH is a non-covalently linked dimeric protein consisting of and
subunits (Pierce and Parsons, 1981
). Subunit assembly is essential for bioactivity of FSH (Jia and Hseuh, 1986
) as well as for the stability of the
subunit (Keene et al., 1989
). However, it has been shown that a single chain fusion of the
and
subunits of FSH is fully active (Sugahara et al., 1996
) and has an increased serum half-life when fused with the carboxy-terminal peptide of hCG (Bouloux et al., 2001
; Duijkers et al., 2002
; Klein et al., 2003
).
In this study we have investigated the effect of linking the Fc domain of immunoglobulin G1 (IgG1) to FSH, either as a single chain or with the and
subunits linked to separate arms of the Fc fragment as a heterodimer FSH-Fc molecule. These fusion proteins were used to examine carrier-medicated transport via FcRn across intestinal epithelia in neonatal rats and lung epithelia in cynomolgus monkeys.
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Materials and methods |
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FSH was isolated from the same human pituitary mRNA library but without the signal sequence using the following primers: 5'-TACTTTCCCGGGGCTCCTGATGTGCAGGATT-3' and 5'-GCATCCGAATTCAGATTTGTGATAATAACAAGTAC-3'.
The area of each primer predicted to anneal to the corresponding template is underlined. The two FSH subunits were ligated to form a contiguous FSHFSH
fusion without a 3' termination codon. The Fc fragment of human IgG1 (hinge, CH2 and CH3 domains; amino acids 221447, EU numbering) was isolated from a human leukocyte cDNA library (Clontech) using the following primers: 5'-AAGAATTCGCCGGCGCCGCTGCGGTCGACAAAACTC-3' and 5'-TTCAATTGTCATTTACCCGGAGACAGGG-3'.
The area of each primer predicted to anneal to the Fc region of IgG1 is underlined. The 5' primer also contains sequence shown in bold that will create an eight amino acid linker sequence (EFAGAAAV) on the amino terminus of the Fc fragment. The Fc PCR product was cloned into the mammalian expression vector, pED.dC (Wyeth, USA) that contains an adenovirus major late promoter and a mouse dihydrofolate reductase gene as a selectable marker. The single chain FSH molecule was then cloned into pED.dC containing the human Fc sequence, thus creating a fusion molecule of FSHFSH
Fc with an eight amino acid linker between the FSH
and the Fc moieties (Figure 1a). Single chain FSHFc protein generated from this DNA construct will be
128 kDa in size.
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In the same way, the Fc fragment of IgG1 was isolated from a human leukocyte cDNA library (Clontech) using the following primers: 5'-GGAGGCGGATCCGGTGGAGGCGGGTCCGGCGGTGGAGGGAGCGACAAAACTCACACGTGCC-3' and 5'-GTTAGGCGGCCGCTCATTTACCCGGAGACAGGG-3'.
In each case, the sequence of the primers predicted to anneal to the corresponding template is underlined. A 15 amino acid linker sequence (GGGGSGGGGSGGGGS) was generated between the carboxy terminus of FSH and the amino terminus of Fc. The sequence of each primer corresponding to this linker sequence is shown in bold. The adenovirus major late promoter in pED.dC was replaced by a cytomegalovirus (CMV) promoter using standard PCR techniques. The mammalian expression vector pcDNA6 (Invitrogen, USA) that contains a CMV promoter was the template for this PCR reaction. The FSH
and Fc PCR fragments were then cloned into this CMVpED.dC vector (Figure 1b).
For FSHFc, FSH
was isolated from a human pituitary mRNA library (Clontech) using the following primers: 5'-GGCTAGCCTGCAGGCCACCATGAAGACACTCCAGTTTTTCT-3' and 5'-TCCACCGGATCCGCCTCCACCTTCTTTCATTTCACCAAAGGAG-3'.
In the same way, the Fc fragment of IgG1 was isolated from a human leukocyte cDNA library (Clontech) using the following primers: 5'-GGAGGCGGATCCGGTGGAGGCGGGTCCGGCGGTGGAGGGAGCGACAAAACTCACACGTGCC-3' and 5'-GTTAGGCGGCCGCTCAGTGGTGATGGTGATGATGATGTTTACCCGGAGACAGGG-3'.
The sequence of each FSH and Fc primer predicted to anneal to the corresponding template is underlined. A 15 amino acid linker sequence (GGGGSGGGGSGGGGS) was generated between the carboxy terminus of FSH
and the amino terminus of Fc. The sequence of each primer corresponding to this linker sequence is shown in bold. To facilitate protein purification, a carboxy terminal 6Histidine (6His) tag sequence was created on the Fc fragment. The sequence of the Fc primer corresponding to the 6His tag is shown in italics. The FSH
and Fc6His fragments were then cloned into the mammalian expression vector pcDNA6 (Invitrogen) that contains a CMV promoter and blasticidin gene for selection purposes (Figure 1b). Heterodimer FSHFc protein generated from co-transfection of FSH
Fc and FSH
Fc constructs will be
94.5 kDa in size.
Expression and purification of single chain and heterodimer FSHFc
Single chain FSHFc was transfected into CHO DG44 cells lacking a dihydrofolate reductase gene (obtained from Dr L.Chasin, Columbia University, NY, USA; Urlaub et al., 1986) using standard Superfect transfection protocols (Qiagen, USA). After 48 h, transfected cells were selected in minimum essential medium (MEM)
without ribonucleosides and deoxyribonucleosides containing 5% dialysed fetal bovine serum (FBS). To obtain higher protein expression levels, cells were treated with methotrexate at concentrations
200 nmol/l. For protein production, cells were seeded into roller bottles in Dulbecco's modified Eagle's medium (DMEM): F-12 (Invitrogen) +10% FBS and incubated for 3 days before changing the medium to DMEM: F-12+5 µg/ml human insulin (Invitrogen). Conditioned medium was collected daily for 10 days, then filtered through 0.2 µm filters, and stored at 4°C until purification. Single chain FSHFc was purified from culture medium using protein A affinity chromatography. After medium containing single chain FSHFc was loaded, protein A columns were washed with 510 column volumes of PBS (10 mmol/l phosphate pH 7.4, 2.7 mmol/l KCl and 137 mmol/l NaCl) and bound protein eluted with 0.1 mol/l glycine, pH 3.0. Eluted single chain FSHFc was then dialysed into PBS and stored at 80°C in aliquots containing 10% glycerol. Single chain FSHFc was run on Trisglycine gels (Invitrogen) and stained with GelCode Blue (Pierce Chemical Company, USA) under reducing and non-reducing conditions to determine the purity of the protein. Single chain FSHFc was
90% pure following a single protein A chromatography step (Figure 1d).
Heterodimer FSHFc was expressed by co-transfection of FSHFc and FSH
Fc6His expression vectors in CHO DG44 (Urlaub et al., 1986
) cells using standard Superfect transfection methods. Forty-eight hours after transfection, cells were incubated in MEM
without ribonucleosides and deoxyribonucleosides containing 5% dialysed FBS and 10 µg/ml blasticidin (Invitrogen) to select only for cells containing both FSH
Fc and FSH
Fc expression vectors. To obtain higher expression levels, cells were treated with methotrexate
50 nmol/l. For protein production, cells were seeded into roller bottles and culture medium containing secreted protein collected in the same way as for single chain FSHFc. Because FSH
Fc and FSH
Fc are co-transfected, culture medium contains not only the desired heterodimer FSHFc, but also FSH
Fc homodimer and FSH
Fc homodimer that require separation. An initial purification using protein A affinity chromatography was performed in the same way as described above for single chain FSHFc. Proteins were further purified using nickel affinity chromatography. FSH
Fc homodimers and heterodimer FSHFc bound to nickel affinity columns due to the presence of the 6His tag on FSH
Fc. Heterodimer FSHFc was separated from FSH
Fc homodimer by elution with an imidazole gradient (0500 mmol/l). The imidazole concentration was then increased to 1 mol/l to remove any remaining protein. Heterodimer FSHFc eluted at
30 to 90 mmol/l imidazole. Heterodimer FSHFc was run on Trisglycine gels (Invitrogen) and stained with GelCode Blue (Pierce Chemical Company) under reducing and non-reducing conditions to determine the purity of the protein. Heterodimer FSHFc was
90% pure following protein A and nickel affinity chromatography steps (Figure 1d).
In vivo bioactivity: ovarian weight gain assay
In vivo activity assays were carried out according to the protocol of Steelman and Pohley (1953) with minor modifications. Briefly, 21 day old female rats (10 rats per group) were given a single s.c. dose (1 nmol/kg) of recombinant (r)FSH (Follistim; Organon, The Netherlands), single chain FSHFc or heterodimer FSHFc in PBS. Seventy-two hours after dosing, ovarian weight was measured in each rat. Statistics were analysed using SigmaStat version 2.0 (RockWare Inc., USA).
Pharmacokinetic studies after oral dosing in neonatal rats
Ten day old neonatal rats (four rats/group) were dosed orally with 0.3 mg/kg single chain FSHFc or heterodimer FSHFc in PBS containing 5 mg/ml soybean trypsin inhibitor. At various times after dosing, blood was collected by cardiac puncture, allowed to clot, then serum prepared and stored at 20°C. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed using an FSH coating antibody (Fitzgerald Industries, USA) and a horseradish peroxidase conjugated Fc detection antibody (Pierce Chemical Company, USA). The standard curve for the ELISA (2000.78 ng/ml in 2-fold dilutions, limit of detection 3 ng/ml) was created with the same lot of protein used to dose the rats. Samples were analysed in triplicate. Pharmacokinetic parameters were estimated using WinNonlin version 4.1 (Pharsight, USA).
Role of FcRn in oral delivery of single chain FSHFc and heterodimer FSHFc in neonatal rats
Single chain FSHFc and heterodimer FSHFc were iodinated with [125I]sodium iodide (Perkin Elmer, USA) using iodobeads (Pierce Chemical Company) according to manufacturer's protocols. Free iodine was separated from iodinated protein on a PD-10 desalting column. Ten day old rats were dosed orally with iodinated single chain or heterodimer FSHFc (25 pmol) or a mixture of iodinated single chain or heterodimer FSHFc (25 pmol) and a 300-fold molar excess of unlabelled human IgG (7.5 nmol; ICN, USA) in PBS with 5 mg/ml soybean trypsin inhibitor. Three hours after dosing, blood was collected by cardiac puncture and serum prepared. A 100 µl aliquot of serum was incubated with protein A Trisacrylamide beads (Pierce Chemical Company) at 4°C for 1 h. Protein A beads were then washed twice with PBS and eluted with sodium dodecyl sulphate (SDS) sample buffer containing 20% -mercaptoethanol. Samples were boiled and analysed on 420% Trisglycine gels, dried and quantification was performed on a Storm Phosphorimager (Molecular Dynamics, USA).
Oral bioactivity: testis weight gain assay
Oral bioactivity in male rats was determined according to a published protocol (Meachem et al., 1996) that demonstrated an increase in Sertoli cell number, resulting in an increase in testis weight, in neonatal rats treated with FSH daily for 1020 days. Briefly, 2 day old male rats (10 rats per group) were orally dosed with 1 nmol/kg human rFSH (Follistim; Organon), single chain FSHFc or heterodimer FSHFc in PBS with 5 mg/ml soybean trypsin inhibitor. Rats were dosed daily for 14 days before measuring testis weight in each rat. Statistics were analysed using SigmaStat version 2.0 (RockWare, Inc.).
Pharmacokinetic/pharmacodynamic studies in cynomolgus monkeys
All studies with cynomolgus monkeys were conducted using approved protocols, following NIH guidelines for the care and use of research animals. Prior to pulmonary administration, animals were anesthetized with a combination of ketamine and valium and intubated with endotracheal tubes. Aerosols of single chain FSHFc (in PBS, pH7.4) or heterodimer FSHFc (in PBS, pH 7.4 with 0.1% HSA) were created with an Aeroneb ProTM nebulizer (Aerogen, USA) and administered to cynomolgus monkeys (deposited dose 45 µg/kg) through the endotracheal tubes. A Bird Mark 7A respirator (Bird Products, USA) regulated the depth (2040% vital capacity) and rate of respiration (2830 breaths per minute) of each monkey such that the delivery of single chain FSHFc or heterodimer FSHFc was targeted to the central airways where epithelial expression of FcRn is predominantly located (Bitonti et al., 2004
). Aerosol particle size was
45 µm. Blood samples were collected at various times after pulmonary dosing and serum prepared. Single chain FSHFc and heterodimer FSHFc levels were quantified using commercially available FSH ELISA kits (DRG International, USA) according to manufacturer's directions. The standard curve for each assay (60 ng/ml to 2.5 ng/ml, limit of detection
10 ng/ml) was created with the same lot of single chain FSHFc or heterodimer FSHFc used to dose the monkeys. Pharmacokinetic parameters were estimated using WinNonlin version 4.1 (Pharsight). Serum samples obtained after pulmonary dosing of single chain FSHFc or heterodimer FSHFc were also used to determine inhibin levels using commercially available ELISA kits (Diagnostic Systems Laboratories, USA) following manufacturer's instructions.
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Results |
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In vivo bioactivity: ovarian weight gain assay
We compared the activity of single chain FSHFc and heterodimer FSHFc fusion proteins to that of recombinant human FSH in 21 day old female rats. Rats were given a single s.c. dose of 1 nmol/kg of either rFSH, single chain FSHFc or heterodimer FSHFc. Seventy-two hours after dosing, the right ovary from each animal was weighed (Figure 2). Ovarian weight was significantly increased in female rats treated with a single dose of rFSH compared to vehicle (14.3±1.7 mg compared to 12.1±1.0 mg, P=0.003). Single chain FSHFc and heterodimer FSHFc produced a greater increase in ovarian weight compared to vehicle- and FSH-treated groups (20.8±3.9 mg and 26.9±6.1 mg respectively; P<0.001). Heterodimer FSHFc was significantly more active than single chain FSHFc in this experiment (P=0.016).
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Discussion |
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Single chain and heterodimer FSHFc are active in both ovarian and testis weight gain assays in rats. This is somewhat surprising since it has been shown that the carboxy terminal of the subunit of FSH is critical for binding to the FSH receptor (Chen et al., 1992
; Arnold et al., 1998
). In addition, the recent publication of the crystal structure of human FSH complexed with its receptor clearly shows that the carboxy terminus of FSH
is in close contact with the FSH receptor (Fan and Hendrickson, 2005
), and comparison of this crystal structure with that of FSH alone (Fox et al., 2001
) indicates that the carboxy terminus of FSH
undergoes a conformational change when bound to the FSH receptor, causing the carboxy terminus of FSH
to become more rigid in structure. Since the carboxy terminus of FSH
is critical for receptor binding, it would seem unlikely that FSH would retain biological activity if the FSH
subunit is tethered at the carboxy terminus by Fc. In both single chain and heterodimer FSHFc fusion proteins, a linker sequence (of eight or 15 amino acids respectively) was used to connect the carboxy terminus of FSH
to the Fc moiety. It is possible that these linker sequences provide enough flexibility for FSH to bind to its receptor, thus retaining in vivo FSH activity for both fusion proteins.
Single chain and heterodimer FSHFc both retain bioactivity after oral dosing in male neonatal rats, leading to an increase in testis weight in treated animals compared to rFSH and vehicle controls. Since FSH binding in the testis is restricted to the Sertoli cell (Simoni et al., 1997), and since it has been shown that daily treatment of newborn male rats with FSH for 1020 days results in an increase in Sertoli cell number resulting in an increase in testis weight (Meachem et al., 1996
), it seems likely that single chain and heterodimer FSHFc treatment results in an increase in Sertoli cell number that is similarly reflected by an increase in testis weight. Although single chain and heterodimer FSHFc are active after oral dosing in neonatal rats, rFSH is not. Since we also show that transport of FSHFc fusion proteins is specifically mediated by FcRn binding and transport through the Fc moiety, it is likey that rFSH is not transported effectively after oral administration, thus explaining the lack of effect on testis weight.
Bioactivity of single chain and heterodimer FSHFc after s.c. dosing was also demonstrated in 21 day old female rats using the SteelmanPohley assay (Steelman and Pohley, 1953). FSH binds to specific receptors on granulosa cells of the ovary and is responsible for the selection and growth of ovarian follicles that under specific conditions results in an increase in ovarian weight. Rats treated with rFSH or either FSHFc fusion protein had increased ovarian weights compared to vehicle-treated controls, likely due to an increased number and size of ovarian follicles. However, the stimulation in ovarian weight was greater for the FSHFc fusion proteins compared to rFSH. Since it is unlikely that the FSHFc fusion proteins will inherently have more activity than rFSH, the increased activity is probably due to the long terminal half-life (6069 h) of these molecules in rodent circulation.
Ovarian and testis weight gain assays in rats indicated that both single chain and heterodimer FSHFc are significantly more active than rFSH. However, it is apparent that heterodimer FSHFc is also significantly more active than single chain FSHFc. Pulmonary delivery of single chain and heterodimer FSHFc in cynomolgus monkeys also supports this observation in that inhibin levels are increased to a higher level in response to heterodimer FSHFc compared to single chain FSHFc. Since single chain and heterodimer FSHFc have similar terminal half-lives in both rodents and monkeys, it is likely that the structure of heterodimer FSHFc may present the FSH and FSH
subunits in a more favourable conformation for bioactivity than if the FSH
and FSH
are fused directly together with Fc to form single chain FSHFc.
rFSH is a common therapy in the treatment of both male and female infertility that requires daily injections for varying lengths of time. For example, treatment of hypogonadotrophic men currently requires daily injections of FSH for up to several months in order to induce spermatogenesis (Schaison et al., 1993; Schoot et al., 1994
). The half-life of heterodimer FSHFc in cynomolgus monkeys in this study is 182219 h. This is significantly longer than the half-life of rFSH of
24 h in humans (le Cotonnec et al., 1994
) and in non-human primates (Porchet et al., 1993
; Weinbauer et al., 1994
). Thus an obvious advantage of using heterodimer FSHFc in infertility treatments is the potential for a greatly reduced dosing frequency. In addition, pulmonary or oral delivery of FSHFc fusion proteins using endogenous FcRn expressed in epithelial cells of the lung and intestine could significantly improve tolerability of current infertility treatments.
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Submitted on January 27, 2005; resubmitted on March 3, 2005; accepted on March 8, 2005.
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