title: An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo.
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.
authors.affiliation: a Division of Protein Technology; KTH Royal Institute of Technology ; AlbaNova University Center ; Stockholm , Sweden.
authors.forename: Feifan
authors.initials: F
authors.lastname: Yu
authors.forename: Lindvi
authors.initials: L
authors.lastname: Gudmundsdotter
authors.forename: Anastassja
authors.initials: A
authors.lastname: Akal
authors.forename: Elin
authors.initials: E
authors.lastname: Gunneriusson
authors.forename: Fredrik
authors.initials: F
authors.lastname: Frejd
authors.forename: Per-Åke
authors.initials: PÅ
authors.lastname: Nygren
mesh_terms: D000068879:Adalimumab; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D019138:Antibodies, Blocking / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D000915:Antibody Affinity / Q000276:immunology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D050822:Cytokine Receptor gp130 / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007249:Inflammation / Q000097:blood / Q000276:immunology / Q000517:prevention & control; D018836:Inflammation Mediators / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D015850:Interleukin-6 / Q000276:immunology / Q000378:metabolism; D053698:Interleukin-6 Receptor alpha Subunit / Q000276:immunology / Q000378:metabolism; D008807:Mice, Inbred BALB C; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D000685:Serum Amyloid A Protein / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D000093242:Interleukin-6 Inhibitors
chemical_list: D000068879:Adalimumab; D000893:Anti-Inflammatory Agents; D019138:Antibodies, Blocking; D061067:Antibodies, Monoclonal, Humanized; D050822:Cytokine Receptor gp130; D018836:Inflammation Mediators; D015850:Interleukin-6; D053698:Interleukin-6 Receptor alpha Subunit; D011993:Recombinant Fusion Proteins; D000685:Serum Amyloid A Protein; D014409:Tumor Necrosis Factor-alpha; D000093242:Interleukin-6 Inhibitors
keywords: AffiMab; Antibody; IL-6; TNF; adalimumab; affibody; affinity; fusion; inflammation