Abstract
BackgroundSelective inhibition of TNFR1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, while leaving TNFR2 untouched, thus allowing for cell survival and tissue homeostasis. ATROSAB is a humanized antagonistic anti-TNFR1 antibody developed for the treatment of inflammatory diseases.Methodology/Principal FindingsThe epitope of ATROSAB resides in the N-terminal region of TNFR1 covering parts of CRD1 and CRD2. By site-directed mutagenesis, we identified Arg68 and His69 of TNFR1 as important residues for ATROSAB binding. ATROSAB inhibited binding of 125I-labeled TNF to HT1080 in the subnanomolar range. Furthermore, ATROSAB inhibited release of IL-6 and IL-8 from HeLa and HT1080 cells, respectively, induced by TNF or lymphotoxin alpha (LTα). Different from an agonistic antibody (Htr-9), which binds to a region close to the ATROSAB epitope but elicits strong TNFR1 activation, ATROSAB showed a negligible induction of IL-6 and IL-8 production over a broad concentration range. We further verified that ATROSAB, comprising mutations within the Fc region known to abrogate complement fixation and antibody-mediated cellular effector functions, indeed lacks binding activity for C1q, FcγRI (CD64), FcγRIIB (CD32b), and FcγRIII (CD16) disabling ADCC and CDC.Conlusions/SignificanceThe data corroborate ATROSAB’s unique function as a TNFR1-selective antagonist efficiently blocking both TNF and LTα action. In agreement with recent studies of TNFR1 complex formation and activation, we suggest a model of the underlying mechanism of TNFR1 inhibition by ATROSAB.
Highlights
Tumor necrosis factor (TNF) plays an important role in the development of inflammatory diseases like rheumatoid arthritis, Crohn’s disease and the relapsing phases of multiple sclerosis
Antagonistic TNF Receptor One-Specific Antibody (ATROSAB) is a humanized IgG1 antibody with a mutated heavy chain described to prevent the induction of Complement-dependent Cytolysis (CDC) and Antibody-dependent Cellular Cytotoxicity (ADCC) [38]
We could show that ATROSAB lacks binding to C1q and FccRIA and does not induce ADCC or CDC in TNFR1-expressing cells
Summary
Tumor necrosis factor (TNF) plays an important role in the development of inflammatory diseases like rheumatoid arthritis, Crohn’s disease and the relapsing phases of multiple sclerosis. Selective inhibition of signaling through TNFR1 holds the potential to greatly reduce the pro-inflammatory activity of TNF, while leaving TNFR2 untouched, allowing for cell survival, tissue homeostasis and, for the CNS, myelin regeneration [21,22] This change of concept in the treatment of TNF-mediated inflammatory diseases, from global ligand inhibition to selective receptor blockade, has gained increasing attention [23] and has led to the development of a number of TNFR1-selective inhibitors. We identified critical amino acids within the ATROSAB epitope of TNFR1 and studied in detail kinetic binding constants by QCM as well as functional activities in comparison to an agonistic TNFR1-specific antibody that binds an epitope in close proximity to the ATROSAB epitope Based on these data and previous results on TNFR1-TNF signal complex formation, we suggest a model of the underlying mechanism of antibodymediated TNFR1 inhibition and activation, respectively
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