Abstract
In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor α (sIL-6Rα) and IL-6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, ZIL-6_13 with an affinity (KD) for IL-6 of ∼500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, ZIL-6_13 was fused to either the N- or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira®). One AffiMab construct with ZIL-6_13 positioned at the N-terminus of the heavy chain, denoted ZIL-6_13-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the ZIL-6_13-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.
Keywords:
AffiMab; Antibody; IL-6; TNF; adalimumab; affibody; affinity; fusion; inflammation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adalimumab
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Animals
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Anti-Inflammatory Agents / chemistry
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Anti-Inflammatory Agents / immunology
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Anti-Inflammatory Agents / pharmacology
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Antibodies, Blocking / chemistry
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Antibodies, Blocking / immunology*
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Antibodies, Blocking / pharmacology
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Antibodies, Monoclonal, Humanized / chemistry
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Antibodies, Monoclonal, Humanized / immunology*
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Antibodies, Monoclonal, Humanized / pharmacology
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Antibody Affinity / immunology
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Cell Line, Tumor
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Cells, Cultured
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Cytokine Receptor gp130 / immunology
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Cytokine Receptor gp130 / metabolism
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Enzyme-Linked Immunosorbent Assay
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Humans
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Inflammation / blood
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Inflammation / immunology
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Inflammation / prevention & control
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Inflammation Mediators / antagonists & inhibitors
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Inflammation Mediators / immunology
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Inflammation Mediators / metabolism
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Interleukin-6 / antagonists & inhibitors
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Interleukin-6 / immunology
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Interleukin-6 / metabolism
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Interleukin-6 Receptor alpha Subunit / immunology
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Interleukin-6 Receptor alpha Subunit / metabolism
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Mice, Inbred BALB C
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Protein Binding / immunology
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / immunology*
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Recombinant Fusion Proteins / pharmacology
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Serum Amyloid A Protein / antagonists & inhibitors
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Serum Amyloid A Protein / immunology*
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Serum Amyloid A Protein / metabolism
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Tumor Necrosis Factor-alpha / antagonists & inhibitors
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Tumor Necrosis Factor-alpha / immunology
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Tumor Necrosis Factor-alpha / metabolism
Substances
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Anti-Inflammatory Agents
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Antibodies, Blocking
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Antibodies, Monoclonal, Humanized
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Inflammation Mediators
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Interleukin-6
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Interleukin-6 Receptor alpha Subunit
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Recombinant Fusion Proteins
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Serum Amyloid A Protein
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Tumor Necrosis Factor-alpha
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Cytokine Receptor gp130
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Adalimumab