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The trimer to monomer transition of Tumor Necrosis Factor-Alpha is a dynamic process that is significantly altered by therapeutic antibodies.

ABSTRACT: The cytokine tumor necrosis factor-alpha (TNF-?) readily forms homotrimers at sub-nM concentrations to promote inflammation. For the treatment of inflammatory diseases with upregulated levels of TNF-?, a number of therapeutic antibodies are currently used as scavengers to reduce the active TNF-? concentration in patients. Despite their clinical success, the mode-of-action of different antibody formats with regard to a stabilization of the trimeric state is not entirely understood. Here, we use a biosensor with dynamic nanolevers to analyze the monomeric and trimeric states of TNF-? together with the binding kinetics of therapeutic biologics. The intrinsic trimer-to-monomer decay rate k?=?1.7 × 10^-3?s^-1 could be measured directly using a microfluidic system, and antibody binding affinities were analyzed in the pM range. Trimer stabilization effects are quantified for Adalimumab, Infliximab, Etanercept, Certolizumab, Golimumab for bivalent and monovalent binding formats. Clear differences in trimer stabilization are observed, which may provide a deeper insight into the mode-of-action of TNF-? scavengers.

SUBMITTER: Daub H

PROVIDER: S-EPMC7283243 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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The trimer to monomer transition of Tumor Necrosis Factor-Alpha is a dynamic process that is significantly altered by therapeutic antibodies.

Daub Herwin H Traxler Lukas L Ismajli Fjolla F Groitl Bastian B Itzen Aymelt A Rant Ulrich U

Scientific reports 20200609 1

The cytokine tumor necrosis factor-alpha (TNF-α) readily forms homotrimers at sub-nM concentrations to promote inflammation. For the treatment of inflammatory diseases with upregulated levels of TNF-α, a number of therapeutic antibodies are currently used as scavengers to reduce the active TNF-α concentration in patients. Despite their clinical success, the mode-of-action of different antibody formats with regard to a stabilization of the trimeric state is not entirely understood. Here, we use a ...[more]

PMID: 32518229

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Project description:The expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-α are needed to deepen our understanding of inflammatory dynamics in dairy cows. The objective of this study was to generate a monoclonal antibody (mAb) pair that could be used to quantify bovine TNF-α in cell culture supernatants and plasma and to detect cytoplasmatic TNF-α in bovine leukocyte populations. One mouse was immunized with a recombinant fusion protein of bovine TNF-α and equine IL-4 generated in Chinese hamster ovary cells. Murine monoclonal antibodies specific to bovine TNF-α were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-α protein. Clones 197-1 and 65-2, both murine IgG1 isotypes, detected the bovine TNF-α fusion protein as well as the native protein produced by peripheral blood mononuclear cells (PBMC) stimulated with a combination of phorbol myristate acetate and ionomycin. Both mAbs were tested for and lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines (IFN-γ, IL-10, and CCL5) and were used to develop a fluorescent bead-based assay. The range of bovine TNF-α detection in the assay was 0.2 to 620 ng/mL, and the test was used to quantify native bovine TNF-α in cell culture supernatants of stimulated PBMC and in plasma from ex vivo whole-blood stimulations. Sample matrices were spiked with TNF-α, with subsequent recovery rates (mean ± SD) of 89% ± 9 (n = 3) in culture medium and 94% ± 12 (n = 3) in heat-inactivated fetal bovine serum. Serial dilutions of plasma and cell culture supernatants from stimulated whole blood or PBMC indicated excellent accuracy for quantification of native TNF-α in bovine samples. Both bovine TNF-α mAbs also detected intracellular TNF-α in bovine CD14+ monocytes and CD4+/CD8+ lymphocytes. In conclusion, we demonstrated that the mAbs generated provide valuable new tools to quantify native bovine TNF-α in a wide concentration range and to characterize intracellular TNF-α expression in bovine leukocytes.

Dataset Information

The trimer to monomer transition of Tumor Necrosis Factor-Alpha is a dynamic process that is significantly altered by therapeutic antibodies.

Publications

The trimer to monomer transition of Tumor Necrosis Factor-Alpha is a dynamic process that is significantly altered by therapeutic antibodies.

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