Higher Order Structures Of Adalimumab, Infliximab And Their Complexes With Tnf Alpha Revealed By Electron Microscopy

Adalimumab and Infliximab are recombinant IgG1 monoclonal antibodies (mAbs) that bind and neutralize human tumor necrosis factor alpha (TNF alpha). TNF alpha forms a stable homotrimer with unique surface-exposed sites for Adalimumab, Infliximab, and TNF alpha receptor binding. Here, we report the structures of Adalimumab-TNF alpha and Infliximab-TNF alpha complexes modeled from negative stain EM and cryo-EM images. EM images reveal complex structures consisting of 1:1, 1:2, 2:2, and 3:2 complexes of Adalimumab-TNF alpha and Infliximab-TNF alpha. The 2:2 complex structures of Adalimumab-TNF alpha and Infliximab-TNF alpha show diamond-shaped profiles and the 2D class averages reveal distinct orientations of the Fab domains, indicating different binding modes by Adalimumab and Infliximab to TNF alpha. After separation by size exclusion chromatography and analysis by negative stain EM, the 3:2 complexes of Adalimumab-TNF alpha or Infliximab-TNF alpha complexes are more complicated but retain features recognized in the 2:2 complexes. Preliminary cryo-EM analysis of 3:2 Adalimumab-TNF alpha complex generated a low-resolution density consistent with a TNF alpha trimer bound with three Fab domains from three individual antibody molecules, while each antibody molecule binds to two molecules of TNF alpha trimer. The Fc domains are not visible in the reconstruction. These results show the two mAbs form structurally distinct complexes with TNF alpha.