Fig. 2: DutaFab libraries.
From: DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously
a DSC data of different human VH3-Vk1 DutaFab dummy scaffolds. Left side: Up to a Fab thermostability of 99 °C, co-operative melting of all 4 domains in the Fab is observed (blue trace). Above 99 °C, a left shoulder starts to appear, representing the unfolding of CH1-Ck constant domains in the presence of folded Fv (red, green and brown traces of Fab unfolding data). Right side: In case of high Fv stabilities, constant domain unfolding can be analyzed independently; the red trace represents the data for a Fab fragment with an Fv TM of 105.7 °C, the green trace the non-2-state fit for the earlier melting constant domain unfolding event and the blue trace the non-2-state fit for the later melting Fv unfolding event (fitted with Origin software). b Top-down view on a DutaFab scaffold showing conserved structural residues that maintain stability or conformation. DutaFab heavy and light chains are colored dark and light gray, respectively. Residues conferring high stability or restricting HCDR3 conformation are colored according to panel c. The structure is based on PDB 6T9D, which is discussed in further detail below. c Typical DutaFab library design, showing H-side diversified residues (dark red X), optionally diversified H-side enhancing residues (light red X), L-side diversified residues (dark blue X), optionally diversified L-side enhancing residues (light blue X), stability residues (pink #) and HCDR3 conformational residues (green $). Underlined: all positions designated as CDRs in either the Kabat, Chothia, and Contact definitions43,44,45.
