Higher Order Structures of Adalimumab, Infliximab and Their Complexes with TNFα Revealed by Electron Microscopy (original) (raw)
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Journal of Biological Chemistry, 2012
Background: Human TNF-␣ is a cytokine involved in many disease-related cellular processes. Results: High affinity binding of therapeutic antibody (inhibitor) to native and molten globule-like TNF-␣ conformation is driven by specific noncovalent interactions. Conclusion: Binding-coupled conformational changes are crucial for antibody-TNF-␣ recognition. Significance: This work helps learn which forces drive unfolding of TNF-␣ and its recognition by monoclonal antibodies and how they affect TNF-␣ activity regulation.
Journal of Biological Chemistry, 1998
A human tumor necrosis factor-␣ (TNF-␣) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-Å resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the Nterminal seven amino acids that is disordered in the structure of wild-type TNF-␣. Compared with wild-type TNF-␣, it exhibits 11-and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-␣. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra-and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe 56 projects into a hydrophobic cavity, while Ile 52 is exposed to the bulk solvent. Ile 52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-␣. It is also expected that the 1.8-Å resolution structure will serve as an accurate model for explaining the structure-function relationship of wildtype TNF-␣ and many TNF-␣ mutants reported previously and for the design of new TNF-␣ mutants.
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.
Humanization and Characterization of an Anti-Human TNF-α Murine Monoclonal Antibody
PLoS ONE, 2011
A murine monoclonal antibody, m357, showing the highly neutralizing activities for human tumor necrosis factor (TNF-a) was chosen to be humanized by a variable domain resurfacing approach. The non-conserved surface residues in the framework regions of both the heavy and light chain variable regions were identified via a molecular modeling of m357 built by computer-assisted homology modeling. By replacing these critical surface residues with the human counterparts, a humanized version, h357, was generated. The humanized h357 IgG 1 was then stably expressed in a mammalian cell line and the purified antibody maintained the high antigen binding affinity as compared with the parental m357 based on a soluble TNF-a neutralization bioassay. Furthermore, h357 IgG 1 possesses the ability to mediate antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity upon binding to cells bearing the transmembrane form of TNF-a. In a mouse model of collagen antibody-induced arthritis, h357 IgG significantly inhibited disease progression by intra-peritoneal injection of 50 mg/mouse once-daily for 9 consecutive days. These results provided a basis for the development of h357 IgG as therapeutic use.
Future Journal of Pharmaceutical Sciences, 2021
Tumour necrosis factor ligand superfamily member 8 (TNFL8) is a cytokine that plays vital roles in immune activations and inflammatory responses through its interaction with the tumour necrosis factor superfamily member. Despite multiple studies on the involvement of its receptor in the inflammatory response, there is limited information on the molecular characterization and structural elucidation of the cytokine. Considering the significance of the cytokine, the three-dimensional structure of TNFL8 model was generated by homology modelling through the Iterative Threading ASSEmbly Refinement (I-TASSER) server and validated through PROCHECK and Qualitative Model Energy Analysis (QMEAN) servers. The predicted structure has 90.00% of residues in the most favoured region of the Ramachandran plot while the QMEAN value gives − 3.06. The sequence and structural alignment between the generated model of the cytokine and template (1XU2) reveal that similar active site residues such as ILE-142...
J Biol Chem, 2009
Monoclonal antibodies have recently started to deliver on their promise as highly specific and active drugs; however, a more effective, knowledge-based approach to the selection, design, and optimization of potential therapeutic antibodies is currently limited by the surprising lack of detailed structural information for complexes formed with target proteins. Here we show that complexes formed with minimal antigen binding single chain variable fragments (scFv) reliably reflect all the features of the binding interface present in larger Fab fragments, which are commonly used as therapeutics, and report the development of a robust, reliable, and relatively rapid approach to the determination of high resolution models for scFv-target protein complexes. This NMR spectroscopy-based approach combines experimental determination of the interaction surfaces and relative orientations of the scFv and target protein, with NMR restraint-driven, semiflexible docking of the proteins to produce a reliable and highly informative model of the complex. Experience with scFvs and Fabs targeted at a number of secreted regulatory proteins suggests that the approach will be applicable to many therapeutic antibodies targeted at proteins, and its application is illustrated for a potential therapeutic antibody targeted at the cytokine IL-1. The detailed structural information that can be obtained by this approach has the potential to have a major impact on the rational design and development of an increasingly important class of biological pharmaceuticals. * This work was supported by UCB-Celltech and Wellcome Trust grants (to M. D. C.). The atomic coordinates and structure factors (code 2KH2) have been deposited in the Protein Data Bank,
Nature Communications, 2021
We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer–small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF–TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein–protein interactions.