Structures of a pan-specific antagonist antibody complexed to different isoforms of TGFβ reveal structural plasticity of antibody-antigen interactions

Abstract

Various important biological pathways are modulated by TGFβ isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan-TGFβ neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen-binding fragment of fresolimumab (GC1008 Fab) in complex with TGFβ3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFβ1 and TGFβ2 was insufficiently understood. We report the crystal structure of the single-chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFβ1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFβ2 to 2.83 Å. The structures provide further insight into the details of TGFβ recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan-specificity and provide potential starting points for the development of isoform-specific antibodies using a fresolimumab scaffold.

Keywords: X-ray crystallography; antibody; fibrosis; ligand; pan-specific inhibitor; protein complex; receptor; transforming growth factor beta.

Figure 1

The crystal structures of the complexes of: A: GC1008 scFv (chocolate and salmon) with TGFβ1 (blue and slate); B: GC1008 Fab with TGFβ2 (colors as in A); C: GC1008 Fab with TGFβ3 (colors as in A—from PDB ID 3EO1); D: TGFβ1 (colors as in A—from PDB ID 3KFD) in ternary complex with its Type I (green) and Type II (orange) receptor.

Figure 2

A: The epitope surface of TGFβ2. The “tip” region is circled in blue; the “hydrophobic patch” region is circled in red; the “helix three” region is circled in green. Note the tip and hydrophobic patch comprise mainly the epitope supplied by the TGFβ2 monomer rendered in magenta, while the helix three regions come entirely from the second monomer, rendered in orange. B: A sequence alignment of all three TGFβ isoforms, showing only stretches of sequence containing residues involved in GC1008 Fab interaction. Those epitope residues (defined as within 4 Å of any GC1008 atoms) are shown in bold. They are colored as per the tip/hydrophobic patch/helix three scheme in panel A.

Figure 3

A: An overlay of a structural alignment of all three GC1008 scFv/GC1008 Fab:TGFβ complexes focusing on residue 60 of the helix three region. Shown only are the heavy chain for GC1008 scFv/GC1008 Fab and helix three of the three TGFβ isoforms. The replacement of threonine in TGFβ3 with either lysine (TGFβ1) or arginine (TGFβ2) causes rotation of the GC1008 Fab away from the helix three region. The fulcrum of rotation is the hydrophobic patch (not shown). K60 and R60 both make close contacts with GC1008 scFv/Fab heavy chain residue E74. B: An overlay of a structural alignment of all three GC1008 scFV/GC1008 Fab:TGFβ complexes focusing on residues 67 and 68 of the helix three region. Here, TGFβ1 has Q67 and H68 whereas TGFβ2 and TGFβ3 have T67 and I/L68. The heavy chain residue Y27 reorients in the TGFβ1-bound structure to make a hydrogen-bonding interactions with Q67 and H68, causing a rearrangement of the CDR loop with respect to the TGFβ2 and TGFβ3 structures. Oxygen atoms are colored red; nitrogens are in blue, carbons are colored as follows: TGFβ1 in slate, TGFβ2 in yellow, TGFβ3 in salmon. GC1008scFv heavy chain in green, GC1008 Fab heavy chain complexed to TGFβ2 in cyan, GC1008 Fab heavy chain complexed to TGFβ3 in brown.