Figure 4
From: A stable, engineered TL1A ligand co-stimulates T cells via specific binding to DR3
Fc-scTL1A was Optimized for Monodispersity. (A) Structural depiction of the crystal structure of the TL1A trimer, adapted from PDB ID 2RE9, showing the three subunits of TL1A. One subunit is shown in blue ribbons while the other two subunits are shown as gray surfaces. The position of the C162-C202 disulfide bond, which is critical for maintenance of DR3 binding is highlighted pink. (B) Analytical SEC analysis of Fc-scTL1A after protein A capture (gray line) showing target dimer species (blue fill), undesired tetrameric species (green fill), and undesired heterogeneous oligomeric species (red fill). Elution profile of Fc-scTL1A under reducing conditions is shown as the black trace. (C) ELISA analysis of the abilities of C162 / C202 mutants to bind DR3. TL1A variants were designed as His-TL1A (non-covalent trimers), and identities are indicated in the graph. (D) Analytical SEC analysis of Fc-scTL1A after protein A purification in non-reducing buffer (black trace), after redox in 1 × PBS (red trace) and after redox in low salt buffer (green trace). (E) ELISA comparison of the binding of Fc-TL1A purified by gel filtration (black) or after redox in low salt buffer to recover dimeric target species (green). (F) Solution X-ray scattering analysis of the Fc-scTL1A molecule showing the scTL1A moieties oriented away from the Fc. Manual docking of TL1A and Fc components of TL1A W14 into bead model for 11.85 mg/mL concentration. Fc dimer generated from PDB ID 1L6X. TL1a trimers are from PDB ID 2RE9. Structural images were generated using Pymol (Schrödinger): https://pymol.org/2/.
