Journal: mAbs

Article Title: Molecular basis for antagonistic activity of anifrolumab, an anti-interferon–α receptor 1 antibody

doi: 10.1080/19420862.2015.1007810

Figure Lengend Snippet: Limited proteolytic digestion of IFNAR1. ( A ) Coomassie-stained SDS-PAGE and Western blot of fragmented human IFNAR1. Recombinant soluble IFNAR1 was treated with endoproteinase Lys-C for 15 min or 1h. The resulting fragments were separated as 8 bands (with 15 min treatment) labeled by arrows on the SDS-PAGE gel. Anifrolumab retained binding to 3 protein bands (1, 4, and 5). Western blot band number 4 after 15 min digestion appears as a so-called “ghost band” likely due to sample or detection antibody overloading as previously described. A ∼38 kDa band observed in the untreated IFNAR1 lane of the western blot was not detectable in the SDS-PAGE gel. This band is likely a minor degradation product that pre-existed in the initial protein preparation and was digested into smaller fragments upon endoproteinase treatment. ( B ) Schematic representation of the positions of digested IFNAR1 fragments as determined by N-terminal Edman sequencing. Apparent molecular weight (as estimated by SDS-PAGE) of all protein fragments are in parentheses. The positive fragments which were recognized by anifrolumab are shown in solid lines, and the negative bands are shown in dotted lines. The smallest ∼12 kDa fragment recognized by anifrolumab was approximately mapped to SD3-4 after the cleavage of K 240 .

Article Snippet: The authors would like to thank Jihong Whang of MedImmune, LLC (Gaithersburg, MD, USA) for acquiring N-terminal Edman sequencing data.

Techniques: Staining, SDS Page, Western Blot, Recombinant, Labeling, Binding Assay, Sequencing, Molecular Weight