A trimeric structural fusion of an antagonistic tumor necrosis factor-α mutant enhances molecular stability and enables facile modification (original) (raw)
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Structure-Activity Profiles of Ab-Derived TNF Fusion Proteins
The Journal of Immunology, 2006
TNF application in humans is limited by severe side effects, including life-threatening symptoms of shock. Therefore, TNF can be successfully applied as a tumor therapeutic reagent only under conditions that prevent its systemic action. To overcome this limitation, genetic fusion of TNF to tumor-selective Abs is a favored strategy to increase site-specific cytokine targeting. Because wild-type TNF displays its bioactivity as noncovalently linked homotrimer, the challenge is to define structural requirements for a TNF-based immunokine format with optimized structure-activity profile. We compared toxicity and efficacy of a dimerized CH2/CH3 truncated IgG1-TNF fusion protein and a single-chain variable fragment-coupled TNF monomer recognizing fibroblast-activating protein. The former construct preserves its dimeric structure stabilized by the natural disulfide bond IgG1 hinge region, while the latter trimerizes under native conditions. Analysis of complex formation of wild-type TNF and...
Tumor Necrosis Factor (TNF)-Soluble High-Affinity Receptor Complex as a TNF Antagonist
Journal of Pharmacology and Experimental Therapeutics, 2007
A novel high-affinity inhibitor of tumor necrosis factor (TNF) is described, which is created by the fusion of the extracellular domains of TNF-binding protein 1 (TBP-1) to both the ␣ 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 pharma-cokinetic 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. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
A TNFR2-Specific TNF Fusion Protein With Improved In Vivo Activity
Frontiers in Immunology
Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve the in vivo availability of TNFR2-specific TNF fusion proteins, we used here the neonatal Fc receptor (FcRn)-interacting IgG1 molecule as an oligomerizing building block and generated a new TNFR2 agonist with improved serum retention and superior in vivo activity.MethodsSingle-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obta...
Synthesis and characterization of a thermally-responsive tumor necrosis factor antagonist
Fusion protein Tumor necrosis factor alpha Soluble tumor necrosis factor receptor Drug depot Elastin-like polypeptide Numerous antagonists of tumor necrosis factor alpha (TNFα) have been developed to attenuate inflammation and accompanying pain in many disease processes. Soluble TNF receptor type II (sTNFRII) is one such antagonist that sequesters TNFα away from target receptors and attenuates its activity. Systemic delivery of soluble TNF receptors or other antagonists may have deleterious side effects associated with immune suppression, so that strategies for locally targeted drug delivery are of interest. Elastin-like polypeptides (ELPs) are biopolymers capable of in situ drug depot formation through thermally-driven supramolecular complexes at physiological temperatures. A recombinant fusion protein between ELP and sTNFRII was designed and evaluated for retention of bivalent functionality. Thermal sensitivity was observed by formation of supramolecular submicron-sized particles at 32°C, with gradual resolubilization from the depot observed at physiological temperatures. In vitro refolding of the sTNFRII domain was required and the purified product exhibited an equilibrium dissociation constant for interacting with TNFα that was seven-fold higher than free sTNFRII. Furthermore, anti-TNF activity was observed in inhibiting TNFα-mediated cytotoxicity in the murine L929 fibrosarcoma assay. Potential advantages of this ELP-sTNFRII fusion protein as an anti-TNFa drug depot include facility of injection, in situ depot formation, low endotoxin content, and functionality against TNFα.
Modifying TNF alpha for Therapeutic Use A Perspective on the TNF Receptor System
TNF alpha is an inflammatory mediator that is relevant to several autoimmune diseases. Macromolecular inhibitors of TNF alpha have proven therapeutically useful in some preliminary studies. We have developed small molecule TNF alpha antagonist based on the crystal structure of TNF receptor complex. The TNF alpha inhibitor is specific and mediates biological function similar to the inhibitory soluble TNF receptor. This review focuses on development of small molecule anti-TNF alpha mimetics by us and current status of other agents.
Biotechnology and Applied Biochemistry, 2009
Affibody molecules specific for human TNF-α (tumour necrosis factor-α) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-α is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-α-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteinefree affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilizedmetal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-α. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-α. For Z TNF-α:185 , subnanomolar affinity (K D = 0.1-0.5 nM) for human TNF-α was demonstrated, as well as significant binding to TNF-α from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-α receptor, since this interaction could be efficiently blocked by the Z TNF-α:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-α binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.
Structure-Expression Relationship of Tumor Necrosis Factor Receptor Mutants That Increase Expression
J. Biol. Chem., 2003
"The extracellular domain of the p55 TNF receptor (TNFrED) is an important therapeutic protein for targeting tumor necrosis factor- (TNF-). The expression level of the TNFrED is low for bioproduction, which is presumably associated with the complication of pairing 24 cysteine residues to form correct disulfide bonds. Here we report the application of the yeast display method to study expression of TNFrED, a multimeric receptor. Randomly mutated libraries of TNFrED were screened, and two mutants were identified that express severalfold higher protein levels compared with the wild type while still retaining normal binding affinity for TNF-. The substituted residues responsible for the higher protein expression in both mutants were identified as proline, and both proline residues are adjacent to cysteine residues involved in disulfide bonds. Analysis of the mutant residues revealed that the improved level of expression is due to conformational restriction of the substituted residues to that of the folded state seen in the crystal structures of TNFrED thereby forcing the neighboring cysteine residues into the correct orientation for proper disulfide bond formation."
Crystal structure of TNF-alpha mutant R31D with greater affinity for receptor R1 compared with R2
1997
Crystal structures have been determined of recombinant human tumor necrosis factor-α (TNF-α) and its R31D mutant that preferentially binds to TNF receptor R1 with more than seven times the relative affinity of binding to receptor R2. Crystals of the wild-type TNF were of space group P4 1 2 1 2 and had unit cell dimensions of a ⍧ b ⍧ 94.7 and c ⍧ 117.4 Å. Refinement of the structure gave an Rfactor of 22.3% at 2.5 Å resolution. The crystals of TNF R31D mutant diffracted to 2.3 Å resolution, and were of identical space group to the wild type with unit cell dimensions of a ⍧ b ⍧ 95.4 and c ⍧ 116.2 Å, and the structure was refined to an R-factor of 21.8%. The trimer structures of the wild-type and mutant TNF were similar with a root mean square (r.m.s.) deviation of 0.56 Å for Cα atoms; however, the subunits within each trimer were more variable with an average r.m.s. deviation of 1.00 Å on Cα atoms for pairwise comparison of subunits. Model complexes of TNF with receptors R1 and R2 have been used to predict TNF-receptor interactions. Arg31 in all three subunits of wild-type TNF is predicted to form an ionic interaction with the equivalent glutamic acid in both receptors R1 and R2. Asp31 of the TNF R31D mutant is predicted to interact differently with the two receptors. The side chain of Asp31 in two subunits of the TNF mutant is predicted to form hydrogen bond interactions with Ser59 or Cys70 of R1, while it has no predicted interactions with R2. The loss of three strong ionic interactions of Arg31 and the electrostatic repulsion of Asp31 with Glu in the receptors is consistent with the reduced binding of the R31D mutant to both receptors relative to wild-type TNF. The replacement of these ionic interactions by two weaker hydrogen bond interactions between Asp31 of the R31D mutant and R1, compared with no interactions with R2, is in agreement with the observed preferential binding of the R31D mutant to R1 over R2. Analysis of the structure and function of receptor-discriminating mutants of TNF will help understand the biological role of TNF and facilitate its use as an antitumor agent.