The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chirinos-Rojas, C. L.
Right arrow Articles by Partidos, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chirinos-Rojas, C. L.
Right arrow Articles by Partidos, C. D.
The Journal of Immunology, 1998, 161: 5621-5626.
Copyright © 1998 by The American Association of Immunologists

A Peptidomimetic Antagonist of TNF-{alpha}-Mediated Cytotoxicity Identified from a Phage-Displayed Random Peptide Library1

Carlos L. Chirinos-Rojas*, Michael W. Steward* and Charalambos D. Partidos2,{dagger}

* Department of Infections and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; and {dagger} Department of Pathology and Infectious Diseases, Royal Veterinary College, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phage-displayed peptide libraries represent a vast collection of peptide sequences that can be used to identify novel therapeutic molecules. In this report, a 15-mer phage-displayed peptide library was used to identify potential TNF-{alpha} antagonists. After direct interaction of recombinant human TNF-{alpha} with the library, four randomly selected phage clones were shown to inhibit in a dose-dependent fashion both mouse and human TNF-{alpha}-induced cytotoxicity in vitro. DNA sequencing of the positive clones revealed a common amino acid sequence that does not bear any structural similarity to the known primary structures of the extracellular domains of either 55-kDa or 75-kDa TNF receptors. This sequence was synthesized, and the peptidomimotope was shown i) to bind to the recombinant human TNF-{alpha} using surface plasmon resonance (biosensor) technology and ii) to inhibit both recombinant mouse and human TNF-{alpha}-induced cytotoxicity in vitro in a dose-dependent fashion.

These findings highlight the potential of phage-displayed random peptide libraries for the identification of novel low molecular antagonistic molecules that can block the biologic activities of TNF-{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor-{alpha} is a potent cytokine that acts as mediator in inflammatory and immunologic reactions and plays an important role in host defenses against infection (1, 2). The active form of TNF-{alpha} is a trimeric molecule that exerts its activities after binding to each of two types of receptors, the 55-kDa and the 75-kDa (3). Both receptors have marked sequence similarity in their extracellular domains, while their cytoplasmic region sequences are entirely unrelated, suggesting different modes of signaling and function (4).

The recently elucidated crystal structure of the complex of soluble human 55-kDa receptor with the human TNF-ß (which has similar overall structure with TNF-{alpha} despite their low sequence identity) (5) reveals that the binding of the cytokine to its receptor occurs over large surface areas of both the ligand and the receptor. However, only three short clusters of amino acids from the receptor were shown to contribute to the binding to each corresponding TNF-ß subunit (5). This suggests that, if the interaction between ligand and receptor requires only a small cluster of residues, there is the potential to design small molecules that can act as inhibitors. The development of molecules mimicking the binding site of the TNF-{alpha} receptor(s) could be useful in dampening the potentially lethal or debilitating effects of an overproduction of TNF-{alpha}, as has been shown in septic shock, rheumatoid arthritis, or other inflammatory processes (6). Several studies in experimental animals have highlighted the potential of recombinant proteins expressing TNF-{alpha} receptor for therapy in the above described conditions (7, 8, 9, 10, 11). However, the use of such large recombinant molecules may limit their therapeutic application since they are produced in small quantities, can be unstable in vivo, can be immunogenic, and could exert an agonistic effect by acting as TNF-{alpha} carriers (12). Instead, small molecules that can be prepared synthetically and can be administered at high concentrations might be a more appropriate alternative for therapy.

Recent progress in peptide technology has allowed the development of combinatorial peptide libraries expressed either on a solid phase support (13) or displayed on bacteriophages (14). The high molecular diversity displayed by these libraries provides the potential to map the specificity of mAbs (15) and to study ligand:receptor interactions (16). In this study, we describe the use of a phage-displayed peptide library to identify antagonists of TNF-{alpha} that can inhibit TNF-{alpha}-induced cytotoxicity in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Recombinant mouse TNF-{alpha} (rmTNF-{alpha})3 (1.0–2.0 x 108 IU/mg) and recombinant human TNF-{alpha} (rhTNF-{alpha}) (1.73 x 106 IU/50 µg) containing BSA were purchased from R&D Systems Europe (Abingdon, U.K.) and Genentech (San Francisco, CA), respectively. Actinomycin D-manitol was purchased from Sigma (St. Louis, MO).

Cells

The mouse fibroblast cell line L929 was used to measure TNF-{alpha}-mediated cytotoxicity. Cells were maintained in DMEM (Life Technologies, Paisley, Scotland) supplemented with 10% heat inactivated FCS, 2 mM L-glutamine, 0.1 M HEPES, and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin).

Library screening and clone selection

A peptide library composed of random 15-mer peptides displayed on filamentous phages at the N terminus of the major coat protein pIII was used (14). Underivatized polystyrene beads (Pierce, Rockford, IL) were coated with 1 ml of rhTNF-{alpha} (0.5 mg/ml) in 0.1 M carbonate bicarbonate buffer (pH 9.6) overnight at 4°C. The beads were washed with PBS/0.05% Tween 20 and blocked with 1% gelatin for 1 h at 37°C, dried, and stored at -20°C. Two beads were incubated overnight at 4°C with the 15-mer library, which contained approximately 9 x 1010 clones (input phage). The beads were then washed repeatedly in PBS/0.05% Tween 20 to remove unbound phages, and the bound phage clones were subsequently eluted in 0.2 M HCL, adjusted to a pH of 2.2 with glycine. After neutralization with 1 M Tris-HCL (pH 9.1), the eluted phages were concentrated using an Amicon 30 micron filter (output phage) and amplified in Escherichia coli K91kan. The amplified phages were then harvested by NaCl/polyethylene glycol precipitation. Two further rounds of biopanning were performed using a fraction of the amplified phages from the previous rounds. However, the incubation period of the rhTNF-{alpha}-coated beads with the output phages was 4 h during the second biopanning and 1 h during the third biopanning. During the final round of biopanning, the phages were eluted from the polystyrene beads using a stepwise decrease in pH. Individual colonies containing phages were eluted at pH 2.0 and amplified as 20-ml cultures. The phages were precipitated using NaCl/polyethylene glycol. The input and output phages were titrated using luria broth (LB) media so that the percent enrichment of bound phages could be calculated. Percent enrichment was calculated as follows: output/input x 100.

DNA sequencing

DNA was purified from the phages by phenol chloroform extraction followed by ethanol precipitation. The amino acid sequence of the insert in the isolated clones was deduced using an ABI PRISM Dye Terminator Cycle Sequencing Reaction Kit sequencing on an ABI PRISM automated 377 DNA sequencer (Perkin-Elmer Corporation, Warrington, U.K.).

Peptide synthesis

Solid-phase peptide synthesis was performed by converting F-moc-protected amino acids to the hydroxybenzotriazole-activated esters by treatment with hydroxybenzotriazol (HOBT) and diisopropylcarbodiimide in dimethylformamide (DMF). The subsequent coupling reactions were performed in DMF, and the F-moc groups were removed with 20% piperidine in DMF followed by a series of washes in DMF. After synthesis, side chain protecting groups were removed and the peptide was cleaved from the support resin with trifluoroacetic acid in the presence of scavengers. After cleavage, the peptides were extracted into diethylether and purified by preparative HPLC. The purity of the peptides was assessed by analytical HPLC and fast atomic bombardment mass spectrometry.

Inhibition of TNF-{alpha}-mediated cytotoxicity

The inhibition of the cytotoxic effect of rmTNF-{alpha} and rhTNF-{alpha} by the phages or the mimotope was assayed using the murine L929 fibroblast cell line. Briefly, cells were plated into 96-well flat-bottom microtiter plates (NUNC, Roskilde, Denmark) at 3 x 105/ml of complete DMEM medium and cultured overnight at 37°C in the presence of 5% CO2. After removing the supernatant, TNF-{alpha} was incubated with various concentrations of each phage clone or mimotope in complete DMEM medium supplemented with 2 mg/ml actinomycin D-manitol for 2 h at 37°C in 5% CO2; thereafter, the TNF-{alpha}/mimotope or phage mixtures were added to the cell culture for overnight incubation at 37°C in 5% CO2. After removing the supernatant, cells were stained for 5 min with a 0.5% solution of crystal violet, washed, and allowed to dry at room temperature. Absorbance was measured at 550 nm in a Dynatech MR 5000 (Chantilly, VA) microplate reader. Each concentration of phage, or mimotope/TNF-{alpha} mixture was tested in triplicate. SD in triplicate cultures was consistently less than 10%. Controls of TNF-{alpha} with the cells, cells alone, and peptide or phages alone with the cells were also included in each assay. Inhibition of cytotoxicity was calculated as follows: percent inhibition = (OD test - OD TNF-{alpha})/(OD control - OD TNF-{alpha}) x 100.

A phage clone identified by mapping a hepatitis B surface Ag mAb (kindly provided by Prof C. R. Howard, Royal Veterinary College, London) was used as a control.

Determination of the binding of the mimotope to the rhTNF-{alpha}

Using the BIAcore 2000 System (Biacore, Uppsala, Sweden), which allows quantitative analysis of molecular interactions in real time, the binding of the synthesized mimotope with the rhTNF-{alpha} was measured. The BIAcore 2000 system uses surface plasmon resonance (SPR) (17), a quantum mechanical phenomenon, that detects changes in optical properties at the surface of a thin gold film on a glass support (18).

For the immobilization of the mimotope, the sensor chip SA with four sensor flow cells was previously coated with streptavidin in HBS buffer (Biacore; 10 mM HEPES with 0.15 M NaCL, 3.4 mM EDTA, and 0.05% surfactant P20 at pH 7.4). Following streptavidin coating, the biotinylated mimotope or a control peptide from the 45-kDa Ag of Mycobacterium leprae (FEPAPQLVSRYCMDRRD) was injected until an optimum immobilization of around 85 resonance units (RU) was achieved. Each peptide was immobilized in a different flow cell of the same sensor chip. Binding was detected after injecting rhTNF-{alpha} in HBS buffer at a concentration of 1 µg/ml. Each run was performed at 25°C, at a flow rate of 5–10 µl/min and lasted 5 min. After each run, the surfaces were regenerated with 0.1% SDS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Screening the phage-displayed random peptide library

Phage clones binding to rhTNF-{alpha} were identified by reacting the 15-mer phage-displayed peptide library (9 x 1010 phage clones) with the rhTNF-{alpha} bound on to polystyrene beads. The progress of biopanning was monitored by using aliquots of eluted phage from each step of biopanning to infect E. coli strain K91kan and by plating the cells on LB agar plates containing tetracyclin. The percentage enrichment after the first, second, and third rounds of biopanning was 0.66 x 10-2, 68.1 x 10-7, and 14 x 10-2, respectively (Table IGo).


View this table:
[in this window]
[in a new window]
  		Table I. Successive enrichment obtained by screening the phage-displayed peptide library with rhTNF-{alpha}.

 
Selection of phage clones

Randomly selected phage clones after the third biopanning were tested for their potential to act as TNF-{alpha} antagonists by inhibiting TNF-{alpha}-induced cytotoxicity in vitro. From the 20 selected phage clones, only four inhibited rmTNF-{alpha}-induced cytotoxicity in L929 cells in vitro (Table IIGo). This inhibition was dose dependent at concentrations of rmTNF-{alpha} giving 38.68% (Fig. 1GoA) or 68.58% cytotoxicity (Fig. 1GoB). In addition, the positive clone 14 was also shown to inhibit rhTNF-{alpha}-induced cytotoxicity of L929 cells in vitro in a dose-dependent fashion (Fig. 2Go). No inhibition was observed with a control phage.


View this table:
[in this window]
[in a new window]
  		Table II. Inhibitory effect of randomly selected phage clones on TNF{alpha}-mediated cytotoxicity in L929 cells

 


View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 1. Inhibition of rmTNf-{alpha}-induced cytotoxicity in L929 cells by phage clone 8 ({blacktriangleup}, 14 ({blacktriangledown}), 19 ({blacksquare}), 20 (•), and control phage clone ({circ}). rmTNF-{alpha} was used at a concentration giving 38.68% (A) and 68.58% cytotoxicity (B). Each concentration of phage/TNF-{alpha} mixture was tested in triplicate.

 


View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 2. Inhibition of rhTNF-{alpha}-induced cytotoxicity in L929 cells by phage clone 14 ({blacksquare}) and control phage clone ({blacktriangledown}). Each concentration of phage/TNF-{alpha} mixture was tested in triplicate.

 
Sequence analysis of the selected phage clones

The reverse complement of the insert DNA was obtained and then translated to give the amino acid product of the DNA insert. As shown in Table IIIGo, all four inhibiting clones had the same amino acid sequence. Comparison of the amino acid sequence of the identified mimotope with the sequence of the human and mouse TNF receptors 55-kDa and 75-kDa using the PROASD computer program (kindly provided by C.Hackett and D. Horowitz, Wistar Institute, Philadelphia) revealed no significant structural similarity (data not shown). This program compares a peptide segment with a large amino acid sequence, taking into account the structural dissimilarity between the two sequences by using the average structural dissimilarity formula (19) and a dissimilarity table (20). The mimotope sequence was also analyzed by the Basic Local Alignment Search Tool for Proteins (BLASTP available in the internet: http://www.genome.ad.IP/htbin/nph-blast) program for peptide homologies using the Swissprot database release 34.0, including 59,021 sequences. The program was tailored for sequence similarity searching biologically interesting database matches and correlating this interest with the statistical significance estimates. The database sequences are sorted by increasing p value (probability in the range of 0–1). The analysis has shown that only three proteins from the database showed some similarity: 1) hypothetical protein in MUTB 3' region (ORF-C) (p = 0.990); 2) myelin basic protein (MBP) (p = 0.998); and 3) PAB-dependent poly(A)-specific ribonuclease (p = 0.999). None of these proteins relate with the TNF receptors or their family.


View this table:
[in this window]
[in a new window]
  		Table III. Nucleotide and amino acid sequences of positively identified phage clones1

 
Inhibition of TNF-{alpha}-induced cytotoxicity by the mimotope

The peptide representing the amino acid sequence from the identified positive clones was synthesized and used to test its ability to inhibit TNF-{alpha}-induced cytotoxicity in vitro. Recombinant mTNF-{alpha} and rhTNF-{alpha} were used in three different concentrations able to induce 15%, 30%, and 75% and 20%, 30%, and 95% cytotoxicity, respectively. As shown in Fig. 3Go, A and B, the DEF mimotope showed a clear inhibitory effect on rmTNF-{alpha}- and rhTNF-{alpha}-induced cytotoxicity, and levels of inhibition were dependent on the concentration of TNF-{alpha} used. Moreover, the peptide was shown to inhibit both rhTNF-{alpha}- and rmTNF-{alpha}-mediated cytotoxicity in vitro in a dose-dependent fashion (Fig. 4Go). No inhibition was observed with equimolar concentrations of a control peptide representing sequence 398–412 from measles virus fusion protein.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 3. Inhibition of rmTNF-{alpha}- (A) and rhTNF-{alpha}- (B) mediated cytotoxicity in L929 cells by the DEF mimotope () or a control peptide ({square}). Three concentrations of rmTNF-{alpha} and rhTNF-{alpha} giving 15%, 30%, and 75% and 20%, 30%, or 95% cytotoxicity, respectively, were incubated with 110 nm of DEF or control peptide for 2 h, and then the peptide/TNF-{alpha} mixtures were added to the L929 cells. Each concentration was tested in triplicate. Results are expressed as mean ± SD percentage of inhibition of cytotoxicity. No direct cytotoxic effect was observed when peptide was added alone to the cells.

 


View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 4. Dose-response inhibition of rhTNF-{alpha}- ({blacksquare}) and rmTNF-{alpha}- ({square}) induced cytotoxicity in L929 cells by the mimotope peptide and control peptide ({circ}). Each concentration of peptide/TNF-{alpha} mixture was tested in triplicate.

 
Binding of the mimotope to rhTNF-{alpha}

To determine the binding of the mimotope to the rhTNF-{alpha}, the peptide was used as a ligand in SPR studies with the BIAcore system. For this purpose, the mimotope and the control peptide were biotinylated by manual coupling of activated biotin at the amino-terminus of the deprotected peptide bound on the resin. The successful coupling of biotin was confirmed by ELISA. Using biotinylated mimotope or control peptide as a solid phase Ag and rabbit anti-biotin IgG Abs (at a dilution of 1/500) for detection, the OD values at 492 nm were 0.796 and 0.55, respectively, whereas OD values of anti-biotin Abs against nonbiotinylated mimotope or control peptide were 0.08 and 0.089, respectively. Following injection of rhTNF-{alpha} in HBS buffer at a concentration of 1 µg/ml, binding with the immobilized mimotope was demonstrated (Fig. 5Go). The binding of rhTNF-{alpha} resulted in an increase of the refractive index close to the sensor chip, which was translated in an increase of the response in RU. The signal due to the binding of the rhTNF-{alpha} to the mimotope at the beginning of the dissociation was 18 RU, far above to the 3 RU observed with the control peptide. After injecting 5 µg/ml of rhTNF-{alpha} in HBS buffer, peptide DEF was shown to bind to rhTNF-{alpha} with a Kd ~1.62 mM as compared with the described Kd ~0.59 nM for the interaction between human TNF-{alpha} and 55-kDa receptor (21).



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 5. Interaction between rhTNF-{alpha} and immobilized mimotope or control peptide using SPR analysis. After optimal immobilization of biotinylated peptides on different cells (baseline), rhTNF-{alpha} was injected and a different sensogram for each peptide was recorded. Sensograms were overlaid for comparison showing the baseline (left) and the binding achieved (right) in RU.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A high affinity interaction between TNF-{alpha} with its cell-bound receptors is critical for this molecule to exert its biologic activities. In addition to cell-bound receptors, soluble extracellular domains of TNF receptors occur naturally in body fluids (22), and it has been suggested that their role is to regulate TNF activities in vivo (23). Several attempts have been made to identify regions from TNF-{alpha} receptors that are involved in ligand:receptor interaction and could be used as potential antagonists. Synthetic peptides representing sequences from the extracellular domain of the two TNF-{alpha} receptors have been used to identify regions that could bind to TNF-{alpha} and inhibit TNF-{alpha}-induced cytotoxicity in vitro (24, 25). However, the use of these linear sequences as TNF-{alpha} antagonists is of limited value since they are unlikely to mimic the conformation of the receptor binding site.

The use of phage-displayed random peptide libraries has become a powerful tool in identifying novel molecules mimicking conformational epitopes from pathogens (15), receptor binding sites from hormones (26), or cytokines (16). In this study the potential of a 15-mer phage-displayed peptide library was tested to identify antagonists of TNF-{alpha}-receptor interaction. Following screening of the phage library by direct interaction with rhTNF-{alpha}, four clones were identified that were capable of inhibiting in a dose-dependent fashion both high and low concentrations of recombinant mouse and human TNF-{alpha}-induced cytotoxicity in vitro. The ability of the phage to inhibit TNF-{alpha} so effectively might be as a result of the presence of five copies of the 15-mer peptide sequence appropriately presented at the tip of the phage filaments. These peptide sequences may all bind TNF-{alpha} molecules in close proximity to each other on the fluid phase and so give an enhanced functional binding affinity for the phage. The work described here extends our previous observations on the potential of solid phase displayed peptide libraries for the identification of antagonists of TNF-{alpha} activities (27).

All four phage clones had the same amino acid sequence, which suggests that the same phage was consistently propagated. However, the sequence of the identified mimotope had no significant homology or structural similarity with the amino acid sequences of TNF-{alpha} receptors or other protein sequences. This suggests that the identified sequence is likely to mimic a conformational region of one binding site from the extracellular domain of TNF-{alpha} receptor(s). The synthesized mimotope also inhibited TNF-{alpha}-induced cytotoxicity in vitro, although less effectively than the phage-displayed mimotope. Taking into account that TNF-{alpha} is a trimer (28) and that there is a requirement of cross-linking of cell surface TNF-{alpha} receptors for signal transduction, the multimeric presentation of the mimotope on the surface of the filamentous phage is likely to result in much higher functional affinity of binding to TNF-{alpha} molecule than the monomer mimotope. Therefore, it may function as a more potent inhibitor of the biologic activities of TNF-{alpha}. This is in agreement with observations that dimeric constructs of TNF-{alpha} receptors are more effective inhibitors of TNF-{alpha} in vitro, as compared with the monomer receptors (29). However, the possibility exists that the free monomer peptide is able to adopt a larger number of conformations than its counterpart on the phage filament. As a consequence, there is a much lower number of favorable conformations available for interaction with the TNF-{alpha} molecule. This is further supported by the finding that prediction methods did not reveal any particular secondary structure preference for the mimotope and by the effectiveness of the peptide to inhibit low concentrations as compared with high concentrations of TNF-{alpha}-induced cytotoxicity.

The inhibitory effect on rhTNF-{alpha}-mediated cytotoxicity demonstrated on the mouse L929 cell line suggests that the mimotope is likely to mimic the binding site of the 55-kDa TNF-{alpha} receptor since hTNF-{alpha} cannot bind to the murine 75-kDa receptor (30). However, the possibility exists that the mimotope might also mimic the binding site of the human 75-kDa receptor since the two receptors have clear sequence similarity in their extracellular domains (31).

In conclusion, this study highlights the potential of random combinatorial peptide libraries for the identification of novel low m.w. molecules to block the deleterious biologic effects of TNF-{alpha}. In addition, our findings are consistent with recent findings by Takasaki et al. (32), who recently demonstrated that exocyclic peptidomimetics designed on the basis of the crystal structure of the TNF-ß/55-kDa TNF-{alpha} receptor complex (5) can inhibit TNF-{alpha} binding to its receptor and TNF-{alpha}-mediated cytotoxicity in vitro.


   Acknowledgments
 
We thank Dr. Mark Domin (Department of Pharmaceutical Chemistry, School of Pharmacy, London University), Dr Tanya Parrish, and Miss Maggie Long (Department of Clinical Sciences, London School of Hygiene and Tropical Medicine) for performing the fast atomic bombardment mass spectrometry, DNA sequencing, and peptide synthesis, respectively. We also thank Professor Colin R. Howard and Miss Felicity D’Mello (Department of Pathology and Infectious Diseases, Royal Veterinary College) for kindly providing the phage-displayed peptide library and for constructive discussions and advice on the use of phage libraries, respectively. We are also grateful to Dr. Wallis Jones and Dr. Sylvie Cot (Biocore) for their help and suggestions in determining the binding of the mimotope to TNF-{alpha}.


   Footnotes
 
1 C.L.C.-R. is supported by a scholarship from the Venezuelan Council for Development of Science and Technology (CONICIT) and the InterAmerican Bank for Development. Back

2 Address correspondence and reprint requests to Dr. Charalambos D. Partidos, Department of Pathology and Infectious Diseases, Royal Veterinary College, Royal College Street, London NW1 OTU, U.K. E-mail address: Back

3 Abbreviations used in this paper: rmTNF-{alpha}, recombinant mouse TNF-{alpha}; rhTNF-{alpha}, recombinant human TNF-{alpha}; DMF, dimethylformamide; SPR, surface plasmon resonance; RU, resonance unit. Back

Received for publication January 29, 1998. Accepted for publication June 30, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Balkwill, F. R., F. Burke. 1989. The cytokine network. Immunol. Today 10:299.[Medline]
  2. Larrick, J. W., S. L. Kunkel. 1988. The role of tumor necrosis factor and interleukin 1 in the immunoinflammatory response. Pharm. Research 3:129.
  3. Brockhaus, M., H. J. Schoenfeld, E. J. Schlaeger, W. Hunziker, W. Lesslauer, H. Loetscher. 1990. Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies. Proc. Natl. Acad. Sci. USA 87:3127.[Abstract/Free Full Text]
  4. Foxwell, B., K. Barret, M. Feldmann. 1990. Cytokine receptors: structure and signal transduction. Clin. Exp. Immunol. 90:161.
  5. Banner, D. W., A. D’Arcy, W. James, R. Gentz, H. J. Schoenfeld, C. Broger, H. Loetscher, W. Lesslauer. 1993. Crystal structure of the soluble human 55-kDa TNF receptor-human TNF-ß complex: implications for TNF receptor activation. Cell 73:431.[Medline]
  6. Tracey, K., A. Cerami. 1993. Tumor necrosis factor, other cytokines and disease. Annu. Rev. Cell Biol. 9:317.
  7. Evans, T. J., D. Moyes, A. Carpenter, R. Martin, H. Loetscher, W. Lesslauer, J. Cohen. 1994. Protective effect of 55- but not 75-kDa soluble tumor necrosis factor receptor-immunoglobulin G fusion proteins in an animal model of gram-negative sepsis. J. Exp. Med. 180:2173.[Abstract/Free Full Text]
  8. Korner, H., A. L. Goodsall, F. Lemckert, B. J. Scallon, J. Ghrayeb, A. L. Ford, D. Sedgwick. 1995. Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. USA 92:11066.[Abstract/Free Full Text]
  9. Dick, A. D., P. G. McMenamin, H. Korner, B. J. Scallon, J. Ghrayeb, J. V. Forrester, J. D. Sedgwick. 1996. Inhibition of tumor necrosis factor activity minimizes target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffic to the retina. Eur. J. Immunol. 26:1018.[Medline]
  10. Suffredini, A. F., D. Reda, S. M. Banks, M. Tropea, J. M. Agosti, R. Miller. 1995. Effect of recombinant dimeric TNF receptor on human inflammatory responses following intravenous endotoxin administration. J. Immunol. 155:5038.[Abstract]
  11. Lesslauer, W., H. Tabuchi, R. Gentz, M. Brockhaus, E. J. Schlaeger, G. Grau, P. F. Piguet, P. Pointaire, P. Vassalli, H. Loetscher. 1991. Recombinant soluble tumor necrosis factor receptor proteins protect mice from lipopolysaccharide-induced lethality. Eur. J. Immunol. 21:2883.[Medline]
  12. Mohler, K. M., D. S. Torrance, C. A. Smith, R. G. Goodwin, K. E. Stremler, V. P. Fung, H. Madani, M. B. Widmer. 1993. Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists. J. Immunol. 151:1548.[Abstract]
  13. Lam, K., K. Salmon, E. Hersh, V. Hruby, W. Kazmierski, R. Knapp. 1991. A new type of synthetic peptide library for identifying ligand binding activity. Nature 354:82.[Medline]
  14. Scott, J. K., G. P. Smith. 1990. Searching for peptide ligands with an epitope library. Science 249:386.[Abstract/Free Full Text]
  15. D’Mello, F., C. D. Partidos, M. W. Steward, C. R. Howard. 1997. Definition of the primary structure of hepatitis B virus (HBV) pre-S hepatocyte binding domain using random peptide libraries. Virology 237:319.[Medline]
  16. Yanofsky, S. D., D. N. Baldwin, J. H. Butler, F. R. Holden, J. W. Jacobs, P. Balasubramanian, J. P. Chinn, S. E. Cwirla, E. Peters-Bhatt, E. A. Whitehorn, E. H. Tate, A. Akeson, T. L. Bowlin, W. J. Dower, R. W. Barrett. 1996. High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries. Proc. Natl. Acad. Sci. USA 93:7381.[Abstract/Free Full Text]
  17. Lofas, S., B. Johnsson. 1990. A novel hydrogel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilization of ligands. J. Chem. Soc. Chem. Commun. 21:1526.
  18. Karlsson, R., A. Falt. 1997. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J. Immunol. Methods 200:121.[Medline]
  19. Padlan, E. A.. 1977. Structural implications of sequence variability in immunoglobulins. Proc. Natl. Acad. Sci. USA 74:2551.[Abstract/Free Full Text]
  20. Sneath, P. H.. 1966. Relations between chemical structure and biological activity in peptides. J. Theor. Biol. 12:157.[Medline]
  21. Corti, A., C. Poiesi, S. Merli, G. Cassani. 1994. Tumor necrosis factor (TNF-{alpha}) quantification by ELISA and bioassay: effects of TNF-{alpha}-soluble TNF receptor (p55) complex dissociation during assay incubations. J. Immunol. Methods 177:191.[Medline]
  22. Engelman, H., D. Novick, D. Wallach. 1990. Two tumor necrosis factor binding proteins purified from human urine: evidence from immunological cross-reactivity with cell surface tumor necrosis factor receptors. J. Biol. Chem. 265:1531.[Abstract/Free Full Text]
  23. Lantz, M., S. Malik, M. L. Slevin, I. Olsson. 1990. Infusion of tumor necrosis factor (TNF) causes an increase in circulating TNF binding protein in humans. Cytokine 2:402.[Medline]
  24. Hwang, C., M. Gatanaga, E. K. Innis, R. S. Yamamoto, G. A. Granger, T. Gatanaga. 1991. A 20 amino acid synthetic peptide of a region from 55-kDa human tumor necrosis factor receptor inhibits cytolytic and binding activities of a recombinant human TNF in vitro. Proc. R. Soc. Lond. B. 245:115.[Medline]
  25. Lie, B.-L., D. Tunemoto, H. Hemmi, Y. Mizukami, H. Fukuda, H. Kikuchi, S. Kato, N. Numamo. 1992. Identification of the binding site of 55-kDa tumor necrosis factor receptor by synthetic peptides. Biochem. Biophys. Res. Commun. 188:503.[Medline]
  26. Wrighton, N. C., F. X. Farrell, R. Chang, A. K. Kashyap, F. P. Barbone, L. S. Mulcahy, D. L. Johnson, R. W. Barrett, L. K. Jolliffe, W. J. Dower. 1996. Small peptides as potent mimetics of the protein hormone erythropoietin. Science 273:458.[Abstract]
  27. Chirinos-Rojas, C. L., M. W. Steward, C. D. Partidos. 1997. Use of a solid-phase random peptide library to identify inhibitors of TNF-{alpha}-mediated cytotoxicity in vitro. Cytokine 9:226.[Medline]
  28. Eck, M. J., S. R. Sprang. 1989. The structure of tumor necrosis factor-{alpha} at 2.6 Å resolution. J. Biol. Chem. 264:17595.[Abstract/Free Full Text]
  29. Butler, D., B. Scallon, A. Meager, M. Kissonerghis, A. Corcoran, Y. Chernajovsky, M. Feldmann, J. Ghrayeb, F. Brennan. 1994. TNF receptor fusion proteins are effective inhibitors of TNF mediated cytotoxicity on human KYM-1D4 rhabdomyosarcoma cells. Cytokine 6:616.[Medline]
  30. Lewis, M., L. A. Tartaglia, A. Lee, G. L. Bennett, G. C. Rice, G. H. W. Wong, E. Y. Chen, D. V. Goeddel. 1991. Cloning and expression and cross-linking analysis of the murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc. Natl. Acad. Sci. USA 88:2830.[Abstract/Free Full Text]
  31. Dembic, Z., H. Loetscher, U. Gubler, Y.-C. E. Pan, H. W. Lahm, R. Gentz, M. Brockhaus, W. Lesslauer. 1990. Two human TNF receptors have similar extracellular but distinct intracellular domain sequences. Cytokine 2:231.[Medline]
  32. Takasaki, W., Y. Kajino, K. Kajino, R. Murali, M. I. Greene. 1997. Structure-based design and characterization of exocyclic peptidomimetics that inhibit TNF-{alpha} binding to its receptor. Nat. Biotechnol. 15:1266.[Medline]



This article has been cited by other articles:


Home page
 Protein Eng Des SelHome page
L. J. Holt, A. Basran, K. Jones, J. Chorlton, L. S. Jespers, N. D. Brewis, and I. M. Tomlinson
Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs
Protein Eng. Des. Sel., May 1, 2008; 21(5): 283 - 288.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chirinos-Rojas, C. L.
Right arrow Articles by Partidos, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chirinos-Rojas, C. L.
Right arrow Articles by Partidos, C. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS