1EMD-Lexigen Research Center, Bedford Campus, 45A Middlesex Turnpike, Billerica, MA 01821, USA and 3Merck-Santé S.A., 4 Avenue du President François Mitterand, 91380 Chilly-Mazarin, France
2 To whom correspondence should be addressed. E-mail: klo{at}emdlexigen.com
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Abstract |
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Keywords: diabetes/Fc/immunoglobulin/leptin/obesity
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Introduction |
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Leptin, a mammalian cytokine of 167 amino acids, plays an essential role in mediating adaptive responses to environmental variations in the availability of energy, regulating the storage of excess energy as triglycerides in adipose cells when energy supplies are plentiful and the subsequent utilization of this stored energy during prolonged periods of nutritional deprivation (Spiegelman and Flier, 1996). Through studies of profoundly obese and diabetic phenotypes in mice and humans associated with mutations in the ob gene, the ob gene product leptin was identified as the endocrine link between the brain and the storage of fat in adipose tissue (Zhang et al., 1994
; Green et al., 1995
). Similar investigations of the genetic loci associated with obesity and diabetes subsequently led to the discovery of a leptin receptor (OB-R) (Tartaglia et al., 1995
). To date, six differently spliced isoforms of OB-R have been identified (Lee et al., 1996
). The longer Ob-Rb isoform, predominantly expressed in the hypothalamus, seems to be the form of OB-R uniquely responsible for the neuroendocrine effects of leptin in energy homeostasis (Chen,H. et al., 1996
; Ghilardi and Skoda, 1997
), via its ability to activate jak/stat signaling pathways (Baumann et al., 1996
; Ghilardi et al., 1996
; Vaisse et al., 1996
; Ghilardi and Skoda, 1997
).
Leptin exerts a regulatory effect on lipid levels as a result of its expression and secretion by adipocytes in proportion to their levels of stored triglycerides. Indeed, circulating levels of leptin in the blood are known to be closely correlated with the degree of obesity (Considine et al., 1996). The key physiological effects of leptin that make it a potentially useful therapeutic agent for the treatment of obesity-related disorders, reducing appetite, facilitating weight loss and reversing obesity-related insulin resistance have been most clearly demonstrated in studies involving the administration of recombinant leptin to mice with the ob/ob phenotype, a leptin-deficient mouse strain (Weigle et al., 1995
; Seufert et al., 1999
). Although the most obvious biological effects of leptin are associated with adiposity, energy homeostasis in mammals involves many other physiological systems in addition to the regulation of fat storage and consumption. As a primary modulator of the multi-factorial metabolic systems involved in energy homeostasis, leptin has systemic and far-reaching behavioral and physiological effects that influence not only adiposity (Friedman and Halaas, 1998
), but also many other functional areas such as food intake (Schwartz et al., 2000
), glucose homeostasis (Seufert et al., 1999
; Schwartz et al., 2000
), fatty acid homeostasis in non-adipocytes (Unger et al., 1999
), reproduction and sexual development (Brann et al., 2002
; Moschos et al., 2002
), immune response (Lord et al., 1998
; Marti et al., 2001
), angiogenesis (Sierra-Honigmann et al., 1998
), wound healing (Ring et al., 2000
) and bone remodeling (Ducy et al., 2000
).
Two of the major problems that must be addressed for the use of leptin as a therapeutic agent are its short circulating half-life and poor solubility. In an early clinical trial, the use of leptin to reduce body weight in humans met with only limited success (Friedman and Halaas, 1998). To achieve clinical benefit, high doses had to be injected three times daily for 6 months, which caused local reactions in the skin. Furthermore, clinical benefit was observed in only a small fraction of patients in this 6-month trial. More recently, an extensive, randomized and controlled clinical trial to observe the effects of exogenous leptin in groups of obese and lean adults also yielded disappointing results (Heymsfield et al., 1999
). Although a small but statistically significant weight loss was observed in some of the study participants, it was only amongst the most obese subjects who were given the highest doses of leptin. In addition, inflammatory responses at the sites of injection produced redness and swelling that were severe enough to cause some of the subjects to drop out of the study prematurely.
A therapeutically effective form of leptin suitable for the clinical treatment of obesity may be possible if leptin can be produced as a highly soluble protein with a long circulating half-life. In this paper, we describe a series of novel leptin immunofusins (immunoglobulin fusion proteins) that have several desirable properties with the potential to enhance the clinical efficacy of leptin. These fusion proteins consist of the Fc fragment of the immunoglobulin gamma chain as the N-terminal fusion partner, followed by leptin. Whereas the recombinant leptin used in previous clinical trials was produced in the form of insoluble inclusion bodies in bacteria and has solubility problems following the renaturation process (Fawzi et al., 1996), soluble Fcleptin fusion proteins were produced in mammalian cells and secreted into the media, from which they could be readily purified to homogeneity by protein A affinity chromatography. Purified Fcleptin is highly soluble and possesses a very consistent and potent biological activity that is many-fold greater than that of its bacterially produced counterpart. More importantly, Fcleptin exhibits a much longer serum half-life, which obviates the need for daily injections and potentially makes it a much more acceptable pharmacological agent for the treatment of obesity.
The use of a fused N-terminal immunoglobulin Fc domain (FcX) has been shown to enhance significantly the production and secretion of proteins expressed in mammalian cells in addition to providing an easy route to their purification (Lo et al., 1998). The protein of interest is expressed as a fusion to a signal peptide and an immunoglobulin Fc domain, which lead to high levels of cellular expression and secretion. This FcX approach should be superior to an XFc expression system since X may not always be a protein that is normally targeted to the endoplasmic reticulum or expressed at a high level. Furthermore, as a relatively large and highly soluble protein vehicle, the Fc domain can potentially extend the circulating half-life of pharmacological proteins in vivo protecting them from degradation and preventing their clearance by renal filtration. The use of Fc domains, however, may produce some unwanted properties in pharmacological fusion proteins, since the Fc domain itself may be recognized by the Fc receptors of the immune system, triggering the cellular activation responses associated with the immune effector functions of Fc (Ravetch, 1997
) and reducing the circulating half-life of the fusion protein (Gillies et al., 1999
). This can be largely circumvented by the use of immunoglobulin Fc isotypes such as Fc
2 that have a lower affinity for the Fc receptor (Cole et al., 1997
; Gillies et al., 1999
). Site-directed mutagenesis can also be used to abrogate further Fc receptor binding by making selective changes to the receptor binding regions in the Fc domain itself (Gillies et al., 1999
).
In the first part of the study described here, the feasibility of the Fc fusion approach for leptin treatment is demonstrated in leptin-deficient and normal, lean mice, using fusion constructs combining murine Fc (muFc) and murine leptin (muLeptin). The initial use of murine constructs in mice allowed us to establish a proof of principle for the clinical use of Fcleptin in the absence of the experimental artifacts that arise as a result of the immunogenicity of the injected agent. The expression properties and in vivo activities of Fcleptin and leptinFc constructs are compared, along with an additional leptinlinkerFc construct that we designed to try to circumvent some of the expression problems that we encountered with the leptinFc constructs.
Having established the proof of principle for the use of Fcleptin for controlling obesity, the second part of this study describes similar experiments with constructs combining human Fc and human leptin. The huFc2hhuLeptin construct, a potential clinical candidate for the treatment of obesity and its related disorders in humans, consists of a modified human immunoglobulin Fc chain of the
2 isotype (huFc
2h) fused with human leptin (huLeptin). Its expression properties and activity are compared with those of human Fc
1 and Fc
2 fusion constructs. The Fc
2 domain has a greatly reduced affinity for the Fc receptor compared with Fc
1, but in trying to produce the recombinant protein we observed that it is much more prone to post-translational misfolding and aggregation. This appears to be because Fc
2 has four disulfide bridges instead of the two that are present in Fc
1, therefore post-translational misfolding can more readily lead to cross-linking and aggregation. The Fc
2h chain is a modified form of Fc
2 that we engineered to have only two disulfide bridges as described in Materials and methods, thereby making it a more amenable fusion partner for recombinant protein expression.
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Materials and methods |
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RNA was prepared from the fat cells of a normal C57/BL6 mouse and reverse transcribed with reverse transcriptase. The resultant cDNA was used as a template for polymerase chain reactions (PCRs) to clone and adapt the murine leptin cDNA for expression as an muFcmuLeptin fusion protein (where muFc is the Fc region of murine immunoglobulin-2a and muLeptin is murine leptin). The forward primer was 5'-CCCGGGTAAAGTGCCTATCCAGAAAGTCC, where the sequence CCCGGG (XmaI restriction site) TAAA encodes the carboxyl terminus of the immunoglobulin heavy chain, followed by a sequence encoding the N-terminus of leptin. The reverse primer was 5'-CTCGAGTCAGCATTCAGGGCTAACATC, which encodes the anti-sense strand of the C-terminal sequence of leptin with its translation STOP codon, followed by a XhoI restriction site. The 450 base-pair PCR product was cloned and sequenced. The expression vector pdCsmuFcmuLeptin was constructed by ligating the XmaIXhoI fragment encoding muLeptin into the pdCsmuFc vector (Lo et al., 1998
). The resultant vector pdCsmuFcmuLeptin was used to transfect human 293 and mouse NS/0 cells for protein expression as described previously (Lo et al., 1998
).
Human forms of Fcleptin were produced in a similar manner. A human leptin cDNA was obtained by PCR cloning from Human Fat Cell Quick-Clone cDNA (Clontech, Palo Alto, CA) and ligated as an XmaI to XhoI fragment into the pdCshuFc1 expression vector (Lo et al., 1998
) encoding the Fc fragment of human immunoglobulin-
1 (huFc
1). The PCR primers used were 5'-CCCGGGTAAAGTGCCCATCCAAAAAGTCCA and 5'-CTCGAGTCAGCACCCAGGGCTGAGGTC for the forward and reverse primers, respectively. For the construction of huFc
2 hhuLeptin, the genomic DNA encoding Fc
2 was obtained by PCR on cellular DNA isolated from human peripheral blood mononuclear cells (PBMC). The forward primer has the sequence 5'-CCTTAAGCGAGCGCAAATGTTGTGTCGAG, where CTTAAGC (containing an AflII restriction site) was introduced just upstream of the
2 hinge coding region. The reverse primer has the sequence 5'-CCTCGAGTCATTTACCCGGGGACAGGGAG, where an XhoI restriction site CTCGAG was introduced immediately after the translation stop codon (anticodon TCA). In addition, the reverse primer also introduced an SmaI CC CGGG by silent mutation (A to G substitution underlined). The 910 bp PCR fragment was cloned into TOPO TA Cloning Vector (Invitrogen, Carlsbad, CA) for sequence verification. The natural SmaI restriction site in the DNA sequence encoding the upper CH3 region was deleted by a silent mutation introduced by an overlapping PCR technique. The forward primer has the sequence 5'-CTGCCCCCATCACGGGAGGAGATGACCAAG, where the C to A substitution is underlined; and the reverse primer has the sequence 5'-GGTCATCTCCTCCCGTGATGGGGGCAGGGTGTAC, where the G to T substitution is underlined. After sequence verification, the resultant AflIIXhoI restriction fragment encoding the Fc of
2 contains a unique SmaI site upstream of the translation stop codon, followed by the XhoI site. The AflIISmaI fragment encoding Fc
2 was then used to replace the corresponding restriction fragment encoding Fc
1 in pdCshuFc
1 to produce pdCshuFc
2.
The immunoglobulin-2 hinge region contains four cystine disulfide bonds. The AflIIStuI fragment 5'-CTTAAGCGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAG containing the native
2 hinge exon in pdCshuFc
2Leptin was replaced by the corresponding AflIIStuI fragment 5'-CTTAAGCGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAG containing the modified
1 hinge exon from pdCshuFc
1Leptin. The
1 hinge sequence in pdCshuFc
1 contains a Cys to Ser mutation (underlined) that eliminates the Cys residue which forms a disulfide bond with the light chain in Ig
1 (Lo et al., 1998
). Since the StuI sites in both the
1 and
2 exons are C-methylated and the StuI restriction endonuclease is methylation sensitive, both plasmids had to be isolated from a DNA cytosine methylase (DCM) negative strain of bacteria before they could be digested with the StuI enzyme. The resultant pdCshuFc
2Leptin with the hinge region from pdCshuFc
1 was designated pdCshuFc
2 hLeptin (
2h: gamma-2 hinge mutant).
Cloning and expression of leptinFc fusion proteins
The murine leptin cDNA was adapted for expression as a muLeptinmuFc fusion protein by PCR. The forward primer, 5'-CTTAAGCGTGCCTATCCAGAAAGTCCA, introduced an AflII (CTTAAG) restriction site for ligating the cDNA encoding the mature N-terminus of murine leptin to the DNA encoding a signal peptide (Lo et al., 1998). The reverse primer, 5'-GATATCGCATTCAGGGCTAACATC, introduced an EcoRV restriction site into the sequence encoding the C-terminus of murine leptin, without the STOP codon. The EcoRV site served as a linkeradaptor for an in-frame fusion of the murine leptin to the murine Fc, which was engineered to contain a unique EcoRV site at the 5' end of the hinge region with the following sequence GATATCTTAAGCGAGCCCAGA, where the CTTAAG is the AflII site preceding the DNA sequence encoding the hinge region (Lo et al., 1998
). The reconstructed pdCsmuLeptinmuFc expression vector contains a DNA fragment encoding a signal peptide and the mature murine leptin, followed by muFc. A different expression plasmid, designated pdCsmuLeptinlinkermuFc, was engineered in the same fashion except that it contained an insertion at the EcoRV site, of a linker encoding GlyAlaGly2SerGly2Ser.
Western blotting analyses
For transient transfection analysis, 293 cells (1.5 x 106 cells on a 100 mm plate) were transfected with plasmid DNA using LipofectAmine Plus Reagent (Life Technologies, Gaithersburg, MD). Three days after transfection, cell culture medium was harvested and after two washes with PBS, cells were lysed in a triple-detergent lysis buffer (50 mM TrisHCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 100 mg/ml phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml aprotinin, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate). Total cell lysate or cell culture media samples were incubated with protein A Sepharose beads at 4°C overnight. Beads were then washed several times with PBS containing 1% Triton X-100. Bound proteins were eluted in SDS gel buffer and separated on a 420% polyacrylamide gradient gel. After electrophoresis, the proteins were blotted on to PVDF membranes (Millipore, Bedford, MA) and visualized by reaction with either horseradish peroxidase (HRP)-conjugated anti-mouse Fc antisera (Jackson ImmunoResearch, West Grove, PA) or biotinylated anti-mouse leptin antibody (R&D Systems, Minneapolis, MN), followed by incubation with HRP-conjugated avidin. For characterizing the huFchuLeptin in the pharmacokinetics study, 3 µl of mouse serum samples per lane were loaded on a 420% polyacrylamide gradient gel. Western blot analysis was performed with HRP-conjugated anti-human IgG (Jackson ImmunoResearch).
Specific activity analyses
The specific activities of the huFchuLeptin constructs were measured using a standard proliferation assay using the murine pro-B cell line BAF-3 (a gift from R&D Systems) transfected with the gene for the human OB receptor (Gainsford et al., 1996). This cell line is dependent on leptin for growth and is under G418 selection for optimal expression of the human OB receptor. Proliferation was measured using the uptake of [3H]thymidine. Washed BAF-3 (OB-R) cells in the log phase (10 000 cells/well) were incubated with leptin for 32 h. [3H]thymidine (Dupont-NEN-027) was then added and the incubation continued for an additional 16 h. The cells were then lysed with water and harvested from the wells on to glass microfiber filter plates and radioactivity was measured by liquid scintillation counting.
Treatment of mice with leptin fusion proteins
C57BL/6 J mice, AKR/J, wild-type and C57BL/6J ob1J/ob1J mice, which were homozygous for the obese gene mutation (ob/ob mice), were purchased from Jackson Research Laboratories (Bar Harbor, ME). All mice were allowed ad libitum access to food and water and their body weights were measured daily and before any injections. The leptin fusion proteins were suspended in phosphate-buffered saline (PBS) and administered following the dose schedules indicated in the figure captions. The amounts of leptin injected were normalized to a given weight of leptin per kilogram of mouse body weight.
Pharmacokinetics of leptin fusion proteins
The pharmacokinetics of muFcmuLeptin and murine leptin (R&D Systems) were compared. Ob/ob mice were injected in the tail vein (six mice per group) with control leptin or fusion proteins in PBS. The amounts of leptin injected were normalized to 1 mg of leptin per kilogram of mouse body weight. Blood samples were obtained by retro-orbital bleeding immediately after injection (0 min) and at 0.1, 0.5, 1, 2, 4, 8, 24 and 48 h post-injection. Blood samples were collected in tubes containing heparin to prevent clotting and placed on ice. Cells were removed by centrifugation in an Eppendorf high-speed microcentrifuge for 4 min. The concentration of leptin in the plasma was measured using a commercial mouse leptin immunoassay kit (R&D Systems). The pharmacokinetics of huFchuLeptin in ob/+ mice were determined in a similar manner, except that the concentrations and integrity of huFchuLeptin in the serum samples were determined by huFc ELISA and western blot analysis, respectively.
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Results |
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The Fc portion of IgG has been extensively used as a fusion partner. In most cases the configuration of these molecules places the Fc at the C-terminus (Bush et al., 2002; Christadoss and Goluszko, 2002
). Leptin contains two cysteine residues that form a covalent linkage between the middle of the protein and the C-terminus (Zhang et al., 1997
). This could potentially make it more difficult for an N-terminal leptinFc fusion protein to form the correct secondary structure since the C-terminal cysteine of leptin would be peptide bonded to the hinge region of the Fc. Such misfolding could negatively affect the production and secretion rate of the protein and also its affinity for the leptin receptor. We compared the expression of both the traditional leptinFc configuration and the oppositely oriented fusion construct Fcleptin by transient expression analysis in 293 human kidney carcinoma cells. Both expression plasmids used identical promoters, signal sequences and other regulatory elements, so that any differences in expression would be the result of the properties of the proteins themselves. Transient expression analyses did indeed indicate that the level of leptin production and its ability to be secreted into the cell culture medium were greatly influenced by the orientation of the Fc fusion partner (Figure 1a). In fact, with murine leptin as the N-terminal domain of the fusion protein, the majority of the immunoreactive protein detected with either murine Fc- or murine leptin-specific antibodies was cell associated. The problem of secretion was not alleviated when a peptide linker of eight amino acids was added between leptin and the Fc domain, to allow more flexibility in protein folding. In contrast, muFcmuLeptin was readily secreted into the medium at a level estimated to be at least 20-fold higher than that of muLeptinmuFc. Such a difference was also observed in stable NS/0 clones. For muFcmuLeptin, the expression levels of the clones were about 30 µg/ml. By contrast, for muLeptinmuFc and muLeptinlinkermuFc the expression levels of the highest producing clones were only about 1 µg/ml.
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Specific activity analyses
An in vitro bioassay was used to demonstrate that the leptin domain in the recombinant huFchuLeptin constructs is recognized by the leptin receptor (OB-R) and can successfully signal through that receptor to cause cell proliferation. The specific activities of the huFchuLeptin constructs were measured using the proliferation of murine pro-B cell line BAF-3 transfected to express the human OB receptor. Figure 2 shows a representative experiment comparing the specific activities of the three huFchuLeptin constructs huFc1huLeptin, huFc
2huLeptin and huFc
2hhuLeptin relative to the WHO international standard (Robinson et al., 2001
). The leptin in these three different huFchuLeptin molecules range in average specific activity from about one-quarter to about half of the specific activity of the international standard for human leptin.
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Groups of 5- to 6-week-old ob/ob mice (three mice per group) received daily i.p. injections of either muFcmuLeptin (0.25 mg of leptin per kg body weight) or vehicle (PBS), over an approximately 3-month period. The control group exhibited a steady 40% increase in mean body weight (from 50 to 70 g). The treatment group had a 45% reduction in mean body weight (from 50.5 to 28 g) over the first month, after which the mean reduction in body weight stabilized at between 45 and 47% (Figure 3a). The mice did not receive treatment over the weekends, which caused their body weights to increase slightly at weekends, with the resumption of daily treatment leading to a steady decrease in their body weights during the week. As shown in Figure 3a, muFcmuLeptin was shown to be effective for over 3 months. During the first 2 weeks of this study, food intake for the treatment group was below detectable limits. After 34 weeks, when the mean body weight had decreased by about 41% and most excess adipose tissue was apparently depleted, the mice consumed a daily average of about 3 g of food per mouse. This is consistent with the results of an earlier study (Mounzih et al., 1997), which showed that the daily food consumption of ob/ob mice receiving leptin treatment at 20 mg/kg resumed at about 2.63.2 g at day 45. The remarkable weight loss represented by the data in Figure 3a, and also the general physiological effects of muFcmuLeptin on ob/ob mice, are graphically illustrated by the series of photographs of one of the treated ob/ob mice, shown in Figure 4.
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S.c. injection of muFcmuLeptin was found to be as effective as i.p. injection in reducing body weight in 5- to 6-week-old ob/ob mice (three mice per group). After 17 days, the mice receiving daily (Monday through Friday) s.c. injections of muFcmuLeptin at 0.1 and 0.25 mg of leptin per kg body weight had reductions of 14% and 22% in mean body weight, respectively, while the control group receiving PBS exhibited a 15% weight gain. The decrease in food intake in mice receiving s.c. injections was similar to that in mice receiving i.p. injections of equivalent doses; i.v. injection was also effective. Ob/ob mice (two mice per group) received daily i.v. injections of muFcmuLeptin at 0.25 or 1 mg/kg. Treatment was stopped after 5 days, but body weights continued to be recorded daily. As shown in Figure 3b, treatment with 0.25 and 1 mg/kg of muFcmuLeptin caused the mean body weight to decrease for the next 48 and 72 h, respectively. These results suggest that muFcmuLeptin has a much longer circulating half-life than murine leptin, based upon the high, frequent doses of murine leptin that have been shown to be necessary for reducing body weight (Friedman and Halaas, 1998).
Dosing schedules of three times weekly or once every 4 days were effective
To show that daily injections of muFcmuLeptin are not necessary, different dosing schedules were tested on ob/ob mice (three mice per group). The results shown in Figure 3c demonstrate that s.c. injections of 0.25 mg/kg of muFcmuLeptin three times weekly (Monday, Wednesday and Friday) were effective in stabilizing a mean reduction in body weight of about 10% (from 42 to 38 g) for over 3 months. These results also show that after an initial weight gain of about 14% (from 44 to 51 g) for mice on an ineffective dosing schedule of 0.1 mg/kg three times weekly, switching to a new regimen of s.c. injections of 1 mg/kg once every 4 days resulted in a mean reduction in body weight of about 33% (from 51 to 34 g) in 4 weeks, after which the mean reduction in body weight leveled off at about 37% (32 g).
Treatment of lean mice and db/db mice with muFcmuLeptin
For comparison with ob/ob mice, normal C57BL/6J, C57BL/KS and Balb/C mice, and also diabetic C57BL/KS db/db mice, all received daily (Monday through Friday) i.p. or s.c. injections of 0.25 or 1 mg/kg of leptin in the form of muFcmuLeptin. As shown in Table I, muFcmuLeptin at both dosage levels had no effect on db/db mice, as would be expected, since these mice lack the receptor for leptin. In normal C57BL/6J, C57BL/KS and Balb/C mice, the low dose had only a very modest effect. The high dose, however, resulted in a significant reduction in body weight over 19 days (Table I), independent of the ages and initial body weights of the mice.
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The data presented in Figure 5a clearly demonstrate the greatly enhanced efficacy in vivo of the human Fcleptin constructs in comparison with recombinant human leptin. It is interesting that even at the highest dosage of recombinant leptin (5.0 mg/kg), only a very modest weight loss (about 7%) could be obtained over the 12-day treatment period. By contrast, a 50-fold lower dose of huFc2hhuLeptin (0.1 mg/kg) was sufficient to obtain a very significant weight loss (about 33%) over the same treatment period. Even at a 200-fold lower dosage (0.025 mg/kg), huFc
2hhuLeptin was somewhat more effective than recombinant human leptin (5 mg/kg). Based on the results shown in Figure 5a, therefore, huFc
2hhuLeptin administered to ob/ob mice is at least two orders of magnitude more potent in vivo than recombinant human leptin.
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Figure 5b and c show the effect of huFc2hhuLeptin in AKR/J mice under chow (Figure 5b) and high-fat (Figure 5c) diets. Since these mice are not leptin deficient, higher doses were required to see significant weight loss over a relatively short treatment period. In spite of this, however, huFc
2hhuLeptin administered to AKR/J mice led to significant weight losses over the first few days of the treatment period, displaying a far greater potency in vivo than recombinant human leptin. After 56 days, however, the efficacy of these huFchuLeptin constructs was limited, presumably by the production of antibodies against these non-murine proteins. On both diets, even the highest doses of human leptin (as high as 10.0 mg/kg) failed to achieve any significant weight loss in the AKR/J mice over the 12-day treatment period. By contrast, huFc
2hhuLeptin at a much lower dose (1.0 mg/kg) induced a weight loss of about 12% for mice on the chow diet and as much as 3% for mice on the high-fat diet, over the same treatment period. These results are particularly encouraging insofar as they indicate that the clinical administration of huFc
2hhuLeptin may potentially benefit even the non-leptin-deficient majority of obese individuals.
Pharmacokinetics
We compared the circulating half-lives of the recombinant mouse leptin and the Fcleptin fusion protein after intravenous injection. The circulating half-lives of muFcmuLeptin and murine leptin in mice were determined to be 8.8 h and 18 min. respectively. The huFchuLeptin constructs were found to have circulating half-lives of over 10 h in mice. As shown in Figure 6, western blot analysis revealed that even after 24 h in the mouse blood circulation, the huFc2hhuLeptin construct remained essentially intact, with no detectable cleavage of the leptin domain from the Fc fragment.
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Discussion |
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The use of the Fc fragment as the N-terminal fusion partner results in efficient expression and secretion of leptin from mammalian cells, resulting in expression cell lines that produce about 380 mg of Fcleptin per ml of tissue culture supernatant. This level of expression exceeds the highest yields of recombinant leptin that can be obtained from Escherichia coli (Fawzi et al., 1996). In contrast to the complicated process involved in renaturing leptin from bacterial inclusion bodies, the expression protocol described here yields soluble Fcleptin in its native, biologically active form, that can be readily purified by protein A affinity chromatography.
To date, large-scale production of recombinant leptin has been done in E.coli (Fawzi et al., 1996) and the denaturing and renaturing protocols that are used lead to products that vary widely in yield and potency. In addition, leptin contains an intramolecular disulfide bond, so the refolding process must be carefully controlled in order to minimize the formation of intermolecular disulfide bonds and insoluble aggregates. It is interesting that one of the cysteine residues forming the disulfide bond is located at the C-terminus. The proximity of this cysteine to the Fc moiety may explain why both leptinFc and leptinlinkerFc fusion proteins are not efficiently secreted.
In a recent clinical trial (Friedman and Halaas, 1998), the use of unfused recombinant leptin required high doses of the protein to be injected three times daily for 6 months to achieve the desired weight reduction. It is presumed that these frequent, high doses were necessitated by the combination of the low potency and short serum half-life of leptin. This observation is also reflected in the current ob/ob mouse models in which a daily intraperitoneal injection of 520 mg/kg of leptin was needed to demonstrate a significant reduction in body weight (Halaas et al., 1995
; Pelleymounter et al., 1995
; Chehab et al., 1996
; Mounzih et al., 1997
). To overcome the sub-optimal pharmacokinetics of leptin, a chronic subcutaneous infusion of leptin at 400 ng/h was needed to achieve a physiological plasma level of leptin in mice (Halaas et al., 1997
).
The use of Fcleptin obviates the need for frequent and high doses. As demonstrated in the ob/ob mouse model, a daily intraperitoneal or subcutaneous injection of 0.1 mg/kg of leptin in the form of muFcmuLeptin was enough to achieve reductions in body weight comparable to those obtained using the more frequent, high-dose regimen with unfused leptin. The frequency of injection could be lowered if a higher dosage was used. For example, 0.25 mg/kg three times weekly or 1 mg/kg once every 4 days was also sufficient to maintain optimal body weight for the ob/ob mice in our study. Furthermore, ob/ob mice injected daily with muFcmuLeptin for over 1 year were healthy and fertile, with decreased appetite, normalized blood glucose levels (as shown in Figure 7) and increased thermogenesis and locomotor activities. Although ob/ob mice are immunodeficient, it has been shown that the administration of exogenous leptin can restore immune function (Lord et al., 1998). In spite of this fact, the ob/ob mice receiving 0.5 mg/kg of huFchuLeptin (Figure 5a) developed only low, non-neutralizing antibody titers against the human fusion proteins, even after more than 1 year of treatment (data not shown). This is also interesting in view of the finding that leptin has been shown to suppress Th2 cytokine production (Lord et al., 1998
), a response generally thought to be essential for promoting antibody production (Mosmann and Coffman, 1989
).
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The frequent doses of leptin required for treatment appear to be a product of the intrinsic properties of the protein itself. With a molecular weight of 16 kDa, leptin is small enough to be cleared by renal filtration, hence a high dose is necessary to compensate for its reduced serum half-life. Fcleptin, with a molecular weight of 96 kDa, exhibits a much longer serum half-life. For example, muFcmuLeptin has a circulating half-life of about 9 h in ob/ob mice, compared with 18 min for murine leptin. The circulating half-lives of the huFchuLeptin constructs in mice are about 10 h and this is likely to be even longer in humans.
The dosage of leptin that can be administered in clinical trials is limited by its solubility. Attempts to improve the solubility of leptin have included the mutation of certain residues to aspartates or glutamates, thereby lowering the isoelectric point of leptin from 5.84 to below 5.5 (DiMarchi et al., 1998). The use of Fc as a fusion partner avoids the creation of such a leptin mutein, since the Fc domain is glycosylated and highly charged at neutral pH and hence acts as a carrier to solubilize leptin. As a result, Fcleptin exhibits a much greater solubility than leptin.
It has been shown that leptin is able to enter the brain via a saturable transport process (Banks et al., 1996) where it is recognized by the biologically active, long-form leptin receptor (Ob-Rb) in the arcuate nucleus of the hypothalamus (Chen,G. et al., 1996
) and from where it exerts its neuroendocrine effects on the regulation of appetite (Schwartz et al., 2000
) and the storage of triglycerides (Cohen et al., 2002
). Although it has not been directly shown that Fcleptins are able to traverse the bloodbrain barrier, it seems most likely that these molecules are able to gain access to the relevant areas of the hypothalamus in much the same manner as the normal, unfused leptin protein, based on the potent activity in vivo, of the Fcleptin constructs described in this paper. Indeed, it is well established that the diffusion of even relatively large proteins into the hypothalamus from the circulation is facilitated by the proximity of the hypothalamus to the median eminence, an area of the brain outside the bloodbrain barrier whose capillaries lack the tight junctions that are characteristic of the endothelial cells in the blood vessels of the brain (Gloor et al., 2001
).
It is plausible that Fcleptin may have a very favorable tissue distribution, especially in view of its long serum half-life and the high dose of soluble protein that can be administered. The successful treatment of non leptin-deficient mice with Fcleptin raises some hope for the successful treatment of the non-leptin-deficient majority of obese humans with Fcleptin, analogous to the insulin treatment of type II (insulin-resistant) diabetes. The data from subcutaneous injections in mice suggest that intramuscular injections in humans should be equally successful, while other routes of administration such as aerosols delivered nasally and gene therapy approaches (Chen,G. et al., 1996) could also be explored.
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References |
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---|
Baumann,H., et al. (1996) Proc. Natl Acad. Sci. USA, 93, 83748378.
Brann,D.W., Wade,M.F., Dhandapani,K.M., Mahesh,V.B. and Buchanan,C.D. (2002) Steroids, 67, 95104.[CrossRef][ISI][Medline]
Bush,K.A., Farmer,K.M., Walker,J.S. and Kirkham,B.W. (2002) Arthritis Rheum., 46, 802805.[CrossRef][ISI][Medline]
Chehab,F.F., Lim,M.E. and Lu,R. (1996) Nat. Genet., 12, 318320.[ISI][Medline]
Chen,G., et al. (1996) Proc. Natl Acad. Sci. USA, 93, 1479514799.
Chen,H., et al. (1996) Cell, 84, 491495.[CrossRef][ISI][Medline]
Christadoss,P. and Goluszko,E. (2002) J. Neuroimmunol., 122, 186190.[CrossRef][ISI][Medline]
Cohen,P., et al. (2002) Science, 297, 240243.
Cole,M.S., Anasetti,C. and Tso,J.Y. (1997) J. Immunol., 159, 36133621.[Abstract]
Considine,R.V., et al. (1996) N. Engl. J. Med., 334, 292295.
DiMarchi,R.D., Hermeling,R.N. and Hoffmann,J.A. (1998) Anti-obesity proteins. US patent 5,7,19,266. EI: Lilly & Company.
Ducy,P., et al. (2000) Cell, 100, 197207.[ISI][Medline]
Fawzi,A.B., Zhang,H. van Heek,M. and Graziano,M.P. (1996) Horm. Metab. Res., 28, 694697.[ISI][Medline]
Friedman,J.M. and Halaas,J.L. (1998) Nature, 395, 763770.[CrossRef][ISI][Medline]
Gainsford,T., et al. (1996) Proc. Natl Acad. Sci. USA, 93, 1456414568.
Ghilardi,N. and Skoda,R.C. (1997) Mol. Endocrinol., 11, 393399.
Ghilardi,N., Ziegler,S., Wiestner,A., Stoffel,R., Heim,M.H. and Skoda,R.C. (1996) Proc. Natl Acad. Sci. USA, 93, 62316235.
Gillies,S.D., Lan,Y., Lo,K.M., Super,M. and Wesolowski,J. (1999) Cancer Res., 59, 21592166.
Gloor,S.M., Wachtel,M., Bolliger,M.F., Ishihara,H., Landmann,R. and Frei,K. (2001) Brain Res. Brain Res. Rev., 36, 258264.[ISI][Medline]
Green,E.D., et al. (1995) Genome Res., 5, 512.[Abstract]
Halaas,J.L., et al. (1995) Science, 269, 543546.[ISI][Medline]
Halaas,J.L., et al. (1997) Proc. Natl Acad. Sci. USA, 94, 88788883.
Heymsfield,S.B., et al. (1999) JAMA, 282, 15681575.
Ioffe,E., Moon,B., Connolly,E. and Friedman,J.M. (1998) Proc. Natl Acad. Sci. USA, 95, 1185211857.
Lee,G.H., et al. (1996) Nature, 379, 632635.[CrossRef][ISI][Medline]
Lo,K.M., et al. (1998) Protein Eng., 11, 495500.[CrossRef][ISI][Medline]
Lord,G.M., Matarese,G. Howard,J.K., Baker,R.J., Bloom,S.R. and Lechler,R.I. (1998) Nature, 394, 897901.[CrossRef][ISI][Medline]
Marti,A., Marcos,A. and Martinez,J.A. (2001) Obesity Rev., 2, 131140.[CrossRef]
Moschos,S., Chan,J.L. and Mantzoros,C.S. (2002) Fertil. Steril., 77, 433444.[CrossRef][ISI][Medline]
Mosmann,T.R. and Coffman,R.L. (1989) Annu. Rev. Immunol., 7, 145173.[CrossRef][ISI][Medline]
Mounzih,K., Lu,R. and Chehab,F.F. (1997) Endocrinology, 138, 11901193.
National Task Force on the Prevention and Treatment of Obesity (2000) Arch. Intern. Med. 160, 898904.
Pelleymounter,M.A., et al. (1995) Science, 269, 540543.[ISI][Medline]
Ravetch,J.V. (1997) Curr. Opin. Immunol. 9, 121125.[CrossRef][ISI][Medline]
Ring,B.D., et al. (2000) Endocrinology, 141, 446449.
Robinson,C.J., Gaines Das,R. and Woollacott,D. (2001) J. Mol. Endocrinol., 27, 6976.
Schwartz,M.W., Woods,S.C., Porte,D. Jr., Seeley,R.J. and Baskin,D.G., (2000) Nature, 404, 661671.[ISI][Medline]
Seufert,J., Kieffer,T.J. and Habener,J.F. (1999) Proc. Natl Acad. Sci. USA, 96, 674679.
Sierra-Honigmann,M.R., et al. (1998) Science, 281, 16831686.
Spiegelman,B.M. and Flier,J.S. (1996) Cell, 87, 377389.[CrossRef][ISI][Medline]
Tartaglia,L.A., et al. (1995) Cell, 83, 12631271.[CrossRef][ISI][Medline]
Thompson,D. and Wolf,A.M. (2001) Obesity Rev., 2, 189197.[CrossRef]
Unger,R.H, Zhou,Y.T. and Orci,L. (1999) Proc. Natl Acad. Sci. USA, 96, 23272332.
Vaisse,C., et al. (1996) Nat. Genet., 14, 9597.[ISI][Medline]
Weigle,D.S., et al. (1995) J. Clin. Invest., 96, 20652070.[ISI][Medline]
Zhang,Y., et al. (1994) Nature, 372, 425432.[CrossRef][ISI][Medline]
Zhang,F., et al. (1997) Nature, 387, 206209.[CrossRef][ISI][Medline]
Received April 7, 2004; revised November 12, 2004; accepted December 13, 2004.
Edited by Ian Tomlinson
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