All experiments were performed at 37 C

All experiments were performed at 37 C. and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the crucial mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability. scFv stability. Menbutone Empirical analyses of VL-CDR3 point mutants and high-resolution scFv-CXCL13 co-crystal structures were used to elucidate contributing factors. The four VL-CDR3 substitutions were examined in isolation, and subsequently in combination. Each mutant was expressed as scFv-Fc and characterized in comparative kinetic and thermal stability analyses. These empirical data exhibited that distinct individual residue changes impact binding affinity and/or VH-VL interface stability. In parallel, high-resolution crystal structures of both scFvs were solved in isolation, and in complex with human CXCL13. In addition to providing the first description of the human CXCL13 structure, these analyses show a novel mode of VH-CDR3 engagement that clearly demonstrates why this loop was not amenable to mutation. They also elegantly support the empirical observations from kinetic and stability analyses and confirm the key residues mediating both affinity and stability improvements. In summary, we have exhibited that as few as three amino acid substitutions, confined to the VL-CDR3, are sufficient to mediate the affinity optimization and stability improvements necessary to facilitate high concentration formulation. This study shows that: 1) Darwinian protein optimization can achieve significant scFv stability and affinity optimization with minimal mutational weight. 2) A single CDR amino acid side-chain clash affecting packing at the VH-VL interface can lead to dramatic differences in the biophysical behavior of human scFvs with therapeutic potential. 3) Importantly, these improvements were not directly mediated by new side-chain contacts between the antibody v-domains or with CXCL13, but were significantly impacted through the resolution of delicate repulsive causes in the antibody combining site. These findings are of broad importance in the antibody engineering field as they highlight that this CDRs of human scFvs are not just crucial mediators of affinity and specificity but may also be the primary drivers of biotherapeutic developability. Experimental Procedures ScFv-Fc Expression, Purification, and in Vitro Analyses ScFv-Fc fusion proteins Sh3pxd2a were expressed transiently in Expi293F cells and purified from filtered conditioned medium using ProPlus suggestions from Phynexus around the automated Phynexus MEA. The producing proteins were buffer-exchanged into PBS using 40-kDa cut-off Zeba columns (Thermo Scientific) and quantified using a Micro BCA kit (Thermo Scientific). Thermal stability ELISAs and DSC analyses were performed as previously explained (13). Binding Kinetics Analyses Biacore analysis was performed using a T-200 biosensor, series S CM5 chips, an amine-coupling kit, 10 mm sodium acetate Menbutone immobilization buffer, pH 5.0, 1 HEPES-buffered saline EDTA-phosphate running buffer containing an additional 250 mm NaCl (final NaCl concentration 400 mm), and 3 m MgCl2 (regeneration answer) (GE Healthcare). Approximately 8000 response models of an anti-human IgG Fc (GE Healthcare) were covalently immobilized to circulation cells 1 and 2 of the CM5 chip at pH 5.5. Then 50C100 response models of 3B4/3B4 variant scFv-Fc fusion (diluted in 1 running buffer) were captured on circulation cell 2. Human CXCL13 (100C25 nm) diluted in running buffer was flowed across both circulation cells Menbutone at 100 l/min with a contact phase of 120 s and a dissociation phase of Menbutone 600 s, followed by a 5-s regeneration pulse with 3 m MgCl2. All experiments were performed at 37 C. Data were corrected for instrument and bulk artifacts by double referencing (15) a surface-immobilized with capture antibody without scFv-Fc-scFv using Scrubber version 2.0c software (BioLogic Software). The transformed data were fit to a 1:1 binding model in Biacore T200 evaluation software v1.0 (GE Healthcare), which includes a parameter for mass transfer (16). Production and Purification of 3B4 and 3B4-CXCL13 Complex 2 liters of 0.22 m filtered 3B4-TEV-Fc Menbutone conditioned medium was purified on HiTrap rProtein A (GE Healthcare) using low pH elution under standard conditions. The eluted portion was concentrated and dialyzed into 50 mm Tris, pH 8.0, for His-TEV protease cleavage (Life Technologies). Cleaved Fc was removed with a second HiTrap rProtein A run, and His-TEV protease was removed using Ni2+-nitrilotriacetic acid capture (GE Healthcare). 3B4 was further purified on a HiLoad 26/600 Superdex.