![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
NEUROPHARMACOLOGY
Serono Research Institute, Rockland, Massachusetts (S.D.M., C.K., M.Y., X.J., R.K.C.); Serono Pharmaceutical Research Institute, Plan-les-Ouates, Geneva, Switzerland (G.F., P.-A.V.); and Istituto di Ricerche Biomediche "A Marxer", Ivrea, Italy (V.A., R.C.)
Received January 11, 2007; accepted May 9, 2007.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
and 
chains of an inactive version of the heterodimeric protein hormone, human chorionic gonadotropin. The resulting molecule, termed TNF-soluble high-affinity receptor complex (SHARC), self-assembles into a heterodimeric protein containing two functional TBP-1 moieties. The TNF-SHARC is a potent inhibitor of TNF- bioactivity in vitro and has a prolonged pharmacokinetic profile compared with monomeric TBP-1 in vivo. Consistent with the long half-life, the duration of action in an lipopolysaccharide-mediated proinflammatory mouse model is prolonged similarly. In a collagen-induced arthritis mouse model, this molecule demonstrates improved efficacy over monomeric TBP-1. Based on these results, we demonstrated that inactivated heterodimeric protein hormones are flexible and efficient scaffolds for the creation of soluble high-affinity receptor complexes.
(TNF-) and TNF- are naturally produced proinflammatory cytokines involved in mediating a variety of immune and inflammatory processes. TNF- is expressed by a wide variety of cells, whereas TNF- is expressed mainly by leukocytes (Paul and Ruddle, 1988
; Vilcek and Lee, 1991). In certain disease conditions, including arthritis, psoriasis, and endometriosis, TNF has been demonstrated to play a pathological role. TNF levels are elevated in the synovium of arthritic joints (Saxne et al., 1988) and in the peritoneum of endometriotic women (Overton et al., 1996). Antibodies capable of neutralizing TNF production and/or bioactivity are known to be efficacious in treating certain inflammatory diseases (Beutler, 1999; Feldmann, 2002). Although structurally distinct, both forms of TNF mediate their actions through either of two types of receptor proteins known as TNFR1 (p55) and TNFR2 (p75). These receptors occur naturally as monomeric subunits on cell membranes. In addition, both p55 and p75 also exist as soluble TNF-binding proteins (TBP-1 and TBP-2, respectively) that are generated by proteolytic cleavage of the membrane versions (Nophar et al., 1990).
The natural role of soluble TBPs has generally been postulated as negative modulators of TNF bioactivity, although some data also support a role for TBPs as stabilizers of TNF bioactivity (Aderka et al., 1992). Under normal conditions, these shed receptors are found at serum concentrations that are up to 1000-fold higher than TNF concentrations, whereas in certain disease conditions, such as sepsis, lupus, and rheumatoid arthritis, the concentrations of TBPs can elevate to even higher levels in serum and synovial fluid (Cope et al., 1992; Heilig et al., 1992). It is noteworthy that although both TBP-1 and TBP-2 preferentially inhibit TNF- versus TNF-, the relative TNF- selectivity of TBP-1 (177-fold) is much greater than that of TBP-2 (only 9-fold) (Terlizzese et al., 1996). Although TNF- has been postulated to be the more important form to target in rheumatoid arthritis, as evidenced by the efficacy of the purely TNF--targeting antibody Remicade, it is unclear whether some increased benefit in efficacy may be afforded by targeting TNF- as well. Therapeutically, soluble forms of both receptors, TBP-1 and TBP-2, have been explored as inhibitors of TNF activity for the treatment of inflammatory diseases. Etanercept, an immunoglobulin Fc fusion molecule containing two TBP-2 domains, inhibits bioactivity of both TNF- and TNF- and has been demonstrated to be efficacious in patients suffering from rheumatoid arthritis and other inflammatory diseases (Mease et al., 2000; Moreland et al., 2006). Likewise, the extracellular domain of TNFR1 (or TBP-1) has been evaluated as a clinical candidate. This molecule has been demonstrated to be active in various models in both an unfused monomeric version (D'Antonio et al., 2000) as well as in the form of an immunoglobulin Fc fusion molecule (Mori et al., 1996).
Whereas immunoglobulin fusion molecules are increasingly used as a method to improve the half-life and avidity of binding protein moieties, it is well known that the constant region of immunoglobulins can mediate effector functions, such as antibody-dependent cell cytotoxicity and complement-dependent cytoxicity. In cases in which the ligand may be found in association with cell membranes, as TNF can be, this may result in undesired cell death. In this case, alternative and flexible scaffolds may be desired. In an effort to generate a version of soluble TBP-1 that is long-acting, potent, and lacking an immunoglobulin constant domain, we have fused TBP-1 moieties to both the and chains of a biologically inert version of the heterodimeric protein hormone, chorionic gonadotropin (hCG). Results are presented describing the biological properties of this molecule.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
(amino acids Ala1-Cys87) or hCG- (amino acids Ser1-Gln145) via a flexible amino acid linker (AGAAPG -chain linker; AGAG -chain linker) (see Fig. 1), using the calcium phosphate method. The D- vector used for stable CHO transfection has been described in more detail (Kelton et al., 1992). Bioreactors were seeded with selected CHO cell pools on Asahi Hi-charge microcarriers (Cytopore 2; GE Healthcare, Piscataway, NJ) and were fed via perfusion as necessary to maintain approximately 1 x 106 cells/ml of media volume/day with minimum essential medium (–) containing 10% dialyzed fetal bovine serum (Invitrogen, Carlsbad, CA), 2 mM l-glutamine, and 1.0 µm of methotrexate. When cultures neared growth plateau (days 8–11), growth medium was switched to ProCHO-5 medium (Lonza Walkersville, Inc., Walkersville, MD) with 4 mM L-glutamine and hypoxanthine and thymidine supplements. Expression levels of the heterodimeric TNF-SHARC protein were determined by hCG ELISA (DSL, Inc., Webster, TX).
|
Biophysical Characterization. Purified protein was evaluated by SDS-PAGE using the Invitrogen NuPAGE Bis-Tris and Tris acetate gel systems at 30 mA/gel constant current. Reduction of protein samples was performed by incubation at 70°C for 10 min in the presence of 50 mM dithiothreitol. Boiled samples were prepared by incubation for 10 min at 100°C. For purity determination by Coomassie Blue staining, the gels were loaded at 5 to 10 µg of protein. Bio-Rad Precision-Plus All Blue calibration standard was used to determine apparent molecular weight (Bio-Rad, Hercules, CA). For Western blot analysis, proteins were transferred to polyvinylidene difluoride in an Invitrogen Transfer apparatus. The Vector ABC-AP protocol was used along with antibodies BHS107 (biotinylated sheep-polyclonal anti-hCG and hCG- subunit; Chromaprobe, Maryland Heights, MO), INN-hFSH-158 (mouse anti-human CG5; Oxford Biotech, Oxfordshire, UK), and T2065 (goat-polyclonal anti-human-TNF sRI; Sigma Chemical, St. Louis, MO). Gels and blots were scanned with a calibrated densitometer (Bio-Rad Model GS-800) and analyzed with Quantity One software (Bio-Rad).
TNF Inhibition Assay. In vitro TBP activity was measured using the A549 cell assay. Human A549 lung carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium (DMEM) highglucose containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin (Invitrogen). Cells were seeded into 96-well plates at a density of 5 x 104 cells/well in 100 µl of growth medium. Cells were allowed to grow 24 h at 37°C. All samples were diluted in growth medium plus 25 µg/ml cyclohexamide. For TBP-1, a 12-point 1:2 serial dilution starting from 1 x 10–7 M was prepared. The TNF-SHARC samples were prepared in dilution medium with appropriate concentrations. An equal volume of a predetermined cytotoxic concentration of TNF- medium [DMEM high-glucose containing 2 ng/ml human TNF- from R&D Systems, Inc. (Minneapolis, MN)] was added to each of the TBP-1 and TNF-SHARC samples. Growth medium was removed from the cells, and 100 µl of the test samples was added to the appropriate wells. Each sample was assayed in triplicate. A 1:1 mixture of the dilution medium and TNF- medium was added to the last three wells as blanks. After incubating overnight in a cell-culture incubator, samples were removed and the cells were washed twice with PBS. To each well was added 100 µlof 4% paraformaldehyde. After 1.5 h, wells were washed twice with PBS (without magnesium and calcium). A volume of 40 µl of 0.5% crystal violet in 30% methanol was added to each well and incubated for 1 h. The plate was washed twice by submerging in a beaker of water and dried. Crystal violet uptake was measured at the optical density at 540 nm (minus optical density of turbidity at 690 nm) using a SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA). IC50 was determined for TNF-SHARC based on standard four-parameter curve fitting using SoftMax Pro 2.1 software (Molecular Devices). As TBP-1 failed to achieve complete inhibition at the highest doses tested, the IC50 was determined by assigning the maximal absorbance plateau to that determined experimentally for TNF-SHARC, because by visual inspection of the cells, it was observed that the maximal absorption values for TNF-SHARC were associated with complete inhibition of TNF-induced cytotoxicity.
hCG Bioassay. In vitro hCG activity was assessed using CHO cells stably transfected with the human luteinizing hormone (LH)/chorionic gonadotropin receptor. Growth medium for the cells was minimum essential medium supplemented with 10% fetal bovine serum, and 600 µg/ml G418 (Geneticin; all from Invitrogen). Assay medium was DMEM/F12 (phenol-free) supplemented with 1 mg/ml bovine serum albumin (Sigma Chemical) and 0.1 M IBMX (3-isobutyl-1-methylxanthone phosphodiesterase inhibitor; Sigma Chemical). Cells were seeded into 96-well plates at a density of 2 x 104 cells/well in growth medium, leaving the first two rows empty for the cAMP standards. After 24 h at 37°C in a cell-culture incubator, growth medium was removed and 25-µl assay medium was added to the cells. After 15 min at 37°C, test samples diluted in assay medium were added in a volume of 20 µl. A 12-point serial 1:3 dilution of hCG starting at 2.25 x 10–8 M was included as a positive control for the assay. After 1 h at 37°C, assay medium was discarded, and 60 µlof assay lysis buffer from the cAMP-Screen Direct System kit (Applied Biosystems, Foster City, CA) was added. Subsequent steps for assay of cAMP production by the cells were done according to the protocol supplied in the assay kit.
Pharmacokinetics. All in vivo studies were approved by the Institutional Animal Care and Use Committees and the respective governmental regulatory authorities. Eight to 10-week-old male CD-1 mice (Charles River Laboratories, Wilmington, MA) were stratified by body weight into groups of three mice per group. Mice received a single subcutaneous injection of 30 µg of TBP-1 or TNF-SHARC in 100 µl of PBS and were anesthetized with isoflurane for blood sampling at either 0, 0.5, 1, 2, 4, 8, 12, 24, or 48 h. Blood samples were collected by heart puncture with heparinized needles. Plasma samples were stored at –20°C for assaying those test compounds with anti-hCG ELISA (DSL). Pharmacokinetic parameters were determined using PK Solutions software (Summit PK, Montrose, CA).
Proinflammatory Cytokine Inhibition Assay. Serum IL-6 concentrations were determined by ELISA (R&D Systems, Inc.) following collection of serum from 8-week-old C3H/Hen female mice (Elevage Janvier, Le Genest Saint Isle, France) 150 min after intraperitoneal administration of LPS (0111:B4) at 0.9 mg/kg in saline. Mice (n = 6 per group) were pretreated with either TBP-1 (5 mg/kg s.c.) or TNF-SHARC (5 mg/kg s.c.) 1, 6, or 24 h before the LPS challenge.
Collagen-Induced Arthritis Model. Induction of collagen-induced arthritis in mice was performed as described previously (Plater-Zyberk and Bonnefoy, 1995; Malfait et al., 1998). In brief, 8 to 12-week-old male DBA/1 mice (Charles River) were immunized intradermally at the tail base with a 0.2-ml emulsion of 100 µgof bovine type II collagen (Morwell Diagnostics, Zurich, Switzerland) in Complete Freund's adjuvant (Difco, Detroit, MI) containing 0.4 mg of Mycobacterium tuberculosis. At approximately 3 weeks after immunization, signs of inflammation were observed affecting one or more limbs. For clinical score, each animal was scored 0 to 2 for the number of inflamed digits (0 = no digits involved; 0.5 = 1–5 digits; 1 = 5–10 digits; 1.5 = 11–15 digits, and 2 = 15–18 digits). For all groups (n = 8 mice per group), treatment was initiated when individual animals reached a clinical score of 
1.5 and were treated for 7 consecutive days. Treatments included subcutaneous administration of TBP-1 (10 mg/kg in PBS) administered twice per day or TNF-SHARC (1, 3, or 10 mg/kg in PBS) administered once per day. For a positive control, one group received indomethacin p.o. at 2 mg/kg in water. Clinical score was evaluated daily, and results were analyzed by Kruskal-Wallis test followed by the pairwise Wilcox test.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
(amino acids 1–87) and (amino acids 1–145) chains via a short peptide-linker sequence (Fig. 1). The truncation of the c-terminal amino acids 88 to 92 of the full-length chain was included to eliminate native hCG bioactivity (Chen et al., 1992). The resulting TNF-SHARC protein ran as a single band with an apparent molecular mass of 87 kDa under nonreducing SDS-PAGE conditions and dissociated into subunits of 57 and 47 kDa upon heating and reduction (Fig. 2A). Western blot analysis confirmed that the heterodimeric nature of the TNF-SHARC as the single nonreduced band immunoreacted with antibodies to TBP-1, hCG- chain, and hCG- chain (Fig. 2B). Under boiled but not reduced conditions, the 57-kDa band and, to a lesser extent, the 47-kDa band were recognized by the anti-hCG--chain antibody, whereas the anti-hCG--chain antibody recognized the 47-but not 57-kDa band. As we have previously observed that, under these same conditions, the anti- antibody is highly selective in recognizing the chain of wildtype hCG, whereas the anti- antibody recognizes wild-type hCG- chain strongly with some cross-reactivity of the chain (data not shown), these data support the formation of a heterodimeric molecule created by the interaction of the TBP-1-hCG- chain with the TBP-1-hCG- chain. Similar to hCG itself, a significant proportion of the TNF-SHARC apparently did not form interchain disulfide bonds because boiling of the protein under nonreducing conditions was sufficient to dissociate the two subunits. Because the dissociation was not complete, it is unclear whether this was due to incomplete heat denaturation or to the possibility that some TNF-SHARC may exist as a disulfide-linked heterodimer.
|
In Vitro Bioactivity. Neutralization of TNF- bioactivity was determined as the inhibition of TNF-mediated cytotoxicity of A549 cells. As demonstrated in Fig. 3A, on a molar basis, TNF-SHARC was significantly more potent than monomeric TBP-1 (IC50: 0.4 nM versus 3.1 nM, respectively) in inhibiting TNF activity. Moreover, the relatively steep inhibition curve of TNF-SHARC was consistent with an increased avidity for TNF, probably because of the bivalent nature of the molecule. To confirm that the deletion of the terminal amino acids of the hCG- chain reduced hCG bioactivity, TNF-SHARC was added to human LH receptor expressing CHO cells. Whereas hCG induced cAMP with an EC50 of 16.8 pM (n = 4), TNF-SHARC failed to achieve EC50 at over 20 µM, indicating a reduction in bioactivity potency of over one million-fold (Fig. 3B).
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). In addition, both growth hormone and interferon have been fused to albumin in an attempt to prolong the activity of these two normally short half-lived molecules (Osborn et al., 2002a,b). The choice of hCG as a fusion partner in the current studies was based on several factors. In particular, the self-assembling heterodimeric nature and exposed N and C termini of the scaffold provide a flexible platform to which functional peptides can be fused. Hence, it may be possible to fuse distinct peptide moieties to the and chains, creating a multifunctional molecule. In addition, the N termini are approximately 10 Å apart so that the fusion of active peptide moieties to both chains may be sufficiently close to permit cooperativity between the binding sites, possibly leading to increased ligand avidity. This may be particularly important for soluble TNF because this ligand typically is present as a homotrimer (Smith and Baglioni, 1987; Wingfield et al., 1987).
hCG belongs to a family of gonadotropin hormones, including follicle stimulating hormone, LH, and thyroid stimulating hormone, which share a common chain and a hormonespecific chain. A unique feature of this family is that the heterodimer is stabilized by a 20 amino acid segment of the chain that wraps around the loop 2 domain of the chain (Xing et al., 2001). This "seatbelt" arrangement maintains the heterodimeric structure in the absence of intermolecular disulfide bonds. Studies described in the present report focused on the bioactivities of a version containing two TBP-1 moieties fused to the N termini of both hCG chains. As active molecules could be also generated when TBP-1 was fused to the C termini of both hCG chains (data not shown), it may be possible that a single molecule could be constructed that has up to four active and possibly different peptide moieties. One major obstacle of using a peptide scaffold comprising a heterodimeric protein hormone is the potential for bioactivity mediated by the scaffold itself. In this regard, the final TNF-SHARC molecule contains an hCG- chain in which the terminal five amino acids had been deleted, since it has been reported that these deletions in hCG eliminate interaction with the LH receptor without disruption of the holoprotein structure (Chen et al., 1992). Although other strategies have been published for reducing hCG binding to LH receptors, including specific point mutations (Chen et al., 1991; Chen and Puett, 1992), we selected the deletion strategy because it has less potential for immunogenicity than methods resulting in the alteration of the primary amino acid sequence. In our hands, the TNF-SHARC molecule containing the terminal -chain five amino acid deletion had negligible hCG bioactivity in an LH receptor-expressing cell bioassay even when added at one million times the active concentration of hCG itself.
TNF is recognized as a mediator of pathology in certain inflammatory diseases, and selective inhibitors of TNF are efficacious in patients suffering from these conditions. Although it is clear that TNF- plays a major role in this pathology, the role of TNF- is less clear. Infliximab, an antibody product specific for TNF-, has been demonstrated to be effective in treating patients with rheumatoid arthritis (Maini et al., 1999). Likewise, etanercept, the TBP-2-based immunoglobulin fusion molecule that inhibits both TNF- and TNF-, is also effective in this indication. Notably, of these therapeutics, only infliximab induces remission in Crohn's disease. Both of these treatments have been associated with increased incidence of opportunistic infections in patients (Keane, 2005). Aside from differences in TNF-/TNF- selectivity, another difference between these two therapeutics is the ability to bind membrane-associated TNF-; whereas infliximab readily binds both soluble and membrane TNF, etanercept is reported to be selective for the soluble form (Scallon et al., 2002). In a biological sense, this may be relevant since it has been demonstrated that antibodies binding to membrane-associated TNF can initiate signal transduction (Mitoma et al., 2005). Ultimately, it remains to be determined which TNF binding profile is optimal for greatest efficacy and least associated with adverse events. Similar to TBP-2, TBP-1 is a structurally complex protein, containing 24 cysteine residues as well as three n-glycosylation sites (Nophar et al., 1990; Jones et al., 1997). The fact that TBP-1 is reported to be up to 10-fold more potent in inhibiting TNF cytotoxicity (Hale et al., 1995) and has greater TNF- selectivity than does TBP-2 (Terlizzese et al., 1996) suggests that TBP-1-based therapeutics may have unique clinical utility. Several TBP-1-based TNF inhibitors have been explored as therapeutic candidates, including a mammalian cell-derived monomeric version (Trinchard-Lugan et al., 2001), an immunoglobulin constant region-based fusion (Furst et al., 2003), and a PEGylated Escherichia coli derived monomeric version (Moreland et al., 2000). Each of these molecules has been demonstrated to be efficacious in animal models and/or patients. Like the immunoglobulin fusion approach, the TBP-1-based TNF-SHARC has two potential binding sites, but unlike the IgG fusion, the TNF-SHARC would not be expected to induce antibody or complement dependent cytotoxicity upon interaction with membrane-bound TNF.
Fusion of TBP-1 to the hCG- and - chains imparts a 10-fold enhancement in pharmacokinetic exposure (area-under-the-curve) in mice, even though the half-life of hCG itself is only approximately 2-fold greater than that of TBP-1 (data not shown). This synergy of half-life is probably due to the fact that the molecular mass of the TNF-SHARC holoprotein (87 kDa) may be greater than the kidney-filtration molecular mass cut-off (roughly 70 kDa) observed for other proteins (Knauf et al., 1988), whereas those for TBP-1 and hCG are below this size. Based on this pharmacokinetic improvement, together with the increased specific activity, it is not surprising that the TNF-SHARC would demonstrate superior efficacy in the collagen-induced arthritis model compared with that of monomeric TBP-1. Perhaps more importantly, the heterodimeric nature of this scaffold suggests that inactivated hCG maybe an extremely flexible approach to designing novel biotherapeutics beyond the TBP-1 fusion.
A novel approach in the construction of a TNF antagonist has been described based on the fusion of the soluble TNF receptor TBP-1 with the N terminus of both chains of the heterodimeric hormone hCG. The resulting molecule is a potent inhibitor of TNF that has a prolonged duration of action and resulting enhanced efficacy in preclinical inflammatory models.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: TNF, tumor necrosis factor; TBP, TNF-binding protein; SHARC, soluble high-affinity receptor complex; TNFR, TNF receptor; LPS, lipopolysaccharide; Fc, constant fragment; PAGE, polyacrylamide gel electrophoresis; hCG, human chorionic gonadotropin; CHO, Chinese hamster ovary; LH, luteinizing hormone; ELISA, enzyme-linked immunosorbent assay; Cu-IMAC, copper-immobilized metal-affinity chromatography; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium.
Address correspondence to: Sean D. McKenna, Serono Research Institute, One Technology Place, Rockland, MA 02370. E-mail: sean.mckenna{at}serono.com
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aderka D, Engelmann H, Maor Y, Brakebusch C, and Wallach D (1992) Stabilization of bioactivity of tumor necrosis factor by its soluble receptors. J Exp Med 175: 323–329.
Beutler B (1999) The role of tumor necrosis in health and disease. J Rheumatol Suppl 57: 16–21.[Medline]
Chamow S and Ashkenazi A (1996) Immunoadhesins: principles and applications. Trends Biotechnol 14: 52–60.[CrossRef][Medline]
Chen F, Wang Y, and Puett D (1991) Role of the invariant aspartic acid 99 of human choriogonadotropin beta in receptor binding and biological activity. J Biol Chem 266: 19357–19361.
Chen F and Puett D (1992) A single amino acid residue replacement in the beta subunit of human chorionic gonadotropin results in the loss of biological activity. J Mol Endocrinol 8: 87–89.[Abstract]
Chen F, Wang Y, and Puett D (1992) The carboxy-terminal region of the glycoprotein hormone alpha subunit: contributions to receptor binding and signaling in human chorionic gonadotropin. Mol Endocrinol 6: 914–919.[Abstract]
Cope AP, Aderka D, Doherty M, Engelmann H, Gibbons D, Jones AC, Brennan FM, Maini RN, Wallach D, and Feldmann M (1992) Increased levels of soluble tumor necrosis factor receptors in the sera and synovial fluid of patients with rheumatic diseases. Arthritis Rheum 35: 1160–1169.[Medline]
D'Antonio M, Martelli F, Peano S, Papoian R, and Borrelli F (2000) Ability of recombinant human TNF binding protein-1 (r-hTBP-1) to inhibit the development of experimentally-induced endometriosis in rats. J Reprod Immunol 48: 81–98.[CrossRef][Medline]
Feldmann M (2002) Development of anti-TNF therapy for rheumatoid arthritis. Nat Rev Immunol 2: 364–371.[CrossRef][Medline]
Furst DE, Weisman M, Paulus HE, Bulpitt K, Weinblatt M, Polisson R, Zaug M, Kneer J, Van der Auwera P, and Stevens RM (2003) Intravenous human recombinant tumor necrosis factor receptor p55-Fc IgG1 fusion protein, Ro 45-2081 (lenercept): results of a dose-finding study in rheumatoid arthritis. J Rheumatol 30: 2123–2126.[Medline]
Hale KK, Smith CG, Baker SL, Vanderslice RW, Squires CH, Gleason TM, Tucker KK, Kohno T, and Russell DA (1995) Multifunctional regulation of the biological effects of TNF-a by soluble type I and type II TNF receptors. Cytokine 7: 26–38.[CrossRef][Medline]
Heilig B, Wermann M, Gallati H, Brockhaus M, Berke B, Egen O, Pezzutto A, and Hunstein W (1992) Elevated TNF receptor plasma concentrations in patients with rheumatoid arthritis. Clin Investig 70: 22–27.[CrossRef][Medline]
Jones MD, Hunt J, Liu JL, Patterson SD, Kohno T, and Lu HS (1997) Determination of tumor necrosis factor binding protein disulfide structure: deviation of the fourth domain structure from the TNFR/NGFR family cystein-rich region signature. Biochemistry 36: 14914–14923.[CrossRef][Medline]
Keane J (2005) TNF-blocking agents and tuberculosis: new drugs illuminating an old topic. Rheumatology 44: 714–720.
Kelton CA, Cheng SV, Nugent NP, Schweickhardt RL, Rosenthal JL, Overton SA, Wands GD, Kuzeja JB, Luchette CA, and Chappel SC (1992) The cloning of the human follicle stimulating hormone receptor and its expression in COS-7, CHO, and Y-1 cells. Mol Cell Endocrinol 89: 141–151.[CrossRef][Medline]
Knauf MJ, Bell DP, Hirter P, Luo Z-P, Young JD, and Katre NV (1988) Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin-2 chemically modified with water-soluble polymers. J Biol Chem 263: 15064–15070.
Maini R, St Clair EW, Breedveld F, Furst D, Kalden J, Weisman M, Smolen J, Emery P, Harriman G, Feldmann M, et al. (1999) Infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomized phase III trial. AT-TRACT Study Group. Lancet 354: 1932–1939.[CrossRef][Medline]
Malfait AM, Butler DM, Presky DH, Maini RN, Brennan FM, and Feldmann M (1998) Blockade of IL-12 during induction of collagen-induced arthritis (CIA) markedly attenuates the severity of arthritis. Clin Exp Immunol 111: 377–383.[CrossRef][Medline]
Mease PJ, Goffe BS, Metz J, VanderStoep A, Finck B, and Burge DJ (2000) Etanercept in the treatment of psoriatic arthritis and psoriasis: a randomized trial. Lancet 356: 385–390.[CrossRef][Medline]
Mitoma H, Horiuchi T, Hatta N, Tsukamoto H, Harashima S, Kikuchi Y, Otsuka J. Okamura S, Fujita S, and Harada M (2005) Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-alpha. Gastroenterology 128: 376–392.[CrossRef][Medline]
Moreland LW, McCabe DP, Caldwell JR, Sack M, Weisman M, Henry G, Seely JE, Martin SW, Yee CL, Bendele AM, et al. (2000) Phase I/II trial of recombinant methionyl human tumor necrosis factor binding protein PEGylated dimer in patients with active refractory rheumatoid arthritis. J Rheumatol 27: 601–609.[Medline]
Moreland LW, Weinblatt ME, Keystone EC, Kremer JM, Martin RW, Schiff MH, Whitmore JB, and White BW (2006) Etanercept treatment in adults with established rheumatoid arthritis: 7 years of clinical experience. J Rheumatol 33: 854–861.[Medline]
Mori L, Iselin S, De Libero G, and Lesslauer W (1996) Attentuation of collagen-induced arthritis in 55-kDa TNF receptor type 1 (TNFR1)-IgG1-treated and TNFR1-deficient mice. J Immunol 157: 3178–3182.[Abstract]
Nophar Y, Kemper O, Brakebusch C, Engelmann H, Zwang R, Aderka D, and Holtmann H (1990) Soluble forms of tumor necrosis factor receptors (TNF-Rs)-the cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both cell surface and soluble forms of the receptor. EMBO J 9: 3269–3278.[Medline]
Osborn BL, Olsen HS, Nardelli B, Murray JH, Zhou JX, Garcia A, Moody G, Zaritskaya LS, and Sung C (2002a) Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys. J Pharmacol Exp Ther 303: 540–548.
Osborn BL, Sekut L, Corcoran M, Poortman C, Sturm B, Chen G, Mather D, Lin HL, and Parry TJ (2002b) Albutropin: a growth hormone-albumin fusion with improved pharmacokinetics and pharmacodynamics in rats and monkeys. Eur J Pharmacol 456: 149–158.[CrossRef][Medline]
Overton C, Fernandez-Shaw S, Hicks B, Barlow D, and Starkey P (1996) Peritoneal fluid cytokines and there relationship with endometriosis and pain. Hum Reprod 11: 380–386.[Medline]
Paul NL and Ruddle NH (1988) Lymphotoxin. Annu Rev Immunol 6: 407–438.[CrossRef][Medline]
Plater-Zyberk C, and Bonnefoy JY (1995) Marked amelioration of established collagen-induced arthritis by treatment with antibodies to CD23 in vivo. Nat Med 1: 781–785.[CrossRef][Medline]
Saxne T, Palladino MA, Heinegard D, Talal N, and Wollheim FA (1988) Detection of tumor necrosis factor alpha but not tumor necrosis factor beta in rheumatoid arthritis synovial fluid and serum. Arthritis Rheum 31: 1041–1045.[Medline]
Scallon B, Cai A, Solowski N, Rosenberg A, Song XY, Shealy D, and Wagner C (2002) Binding and functional comparisons of two types of tumor necrosis factor antagonists. J Pharmacol Exp Ther 301: 418–426.
Smith RA and Baglioni C (1987) The active form of tumor necrosis factor is a trimer. J Biol Chem 262: 6951–6954.
Terlizzese M, Simoni P, and Antonetti F (1996) In vitro comparison of inhibiting ability of soluble TNF receptor p75 (TBP II) vs soluble TNF receptor p55 (TBP I) against TNF-a and TNF-b. J Interferon Cytokine Res 16: 1047–1053.[Medline]
Trinchard-Lugan I, Ho-Nguyen Q, Bilham WM, Buraglio M, Ythier A, and Munafo A (2001) Safety, pharmacokinetics and pharmacodynamics of recombinant human tumor necrosis factor-binding protein-1 (onercept) injected by intravenous, intramuscular and subcutaneous routes into healthy volunteers. Eur Cytokine Netw 12: 391–398.[Medline]
Vilcek J and Lee TH (1991) Tumor necrosis factor. New insights into the molecular mechanisms of its multiple actions. J Biol Chem 266: 7313–7316.
Wingfield P, Pain RH, and Craig S (1987) Tumor necrosis factor is a compact trimer. FEBS Lett 211: 179–184.[CrossRef][Medline]
Xing Y, Williams C, Campbell RK, Cook S, Knoppers M, Addona T, Altarocca V, and Moyle WR (2001) Threading of a glycosylated protein loop through a protein hole: implications for combination of human chorionic gonadotropin subunits. Protein Sci 10: 226–235.
This article has been cited by other articles:
|
|
 |
|
  E. M. Grund, D. Kagan, C. A. Tran, A. Zeitvogel, A. Starzinski-Powitz, S. Nataraja, and S. S. Palmer Tumor Necrosis Factor-{alpha} Regulates Inflammatory and Mesenchymal Responses via Mitogen-Activated Protein Kinase Kinase, p38, and Nuclear Factor {kappa}B in Human Endometriotic Epithelial Cells Mol. Pharmacol., May 1, 2008; 73(5): 1394 - 1404. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
