Increasing robustness, reliability and storage stability of critical reagents by freeze-drying.

Aim: Stabilization of critical reagents by freeze-drying would facilitate storage and transportation at ambient temperatures, and simultaneously enable constant reagent performance for long-term bioanalytical support throughout drug development. Freeze-drying as a generic process for stable performance and storage of critical reagents was investigated by establishing an universal formulation buffer and lyophilization process. Results: Using a storage-labile model protein, formulation buffers were evaluated to preserve reagent integrity during the freeze-drying process, and to retain functional performance after temperature stress. Application to critical reagents used in pharmacokinetics and anti-drug antibodies assays demonstrated stable functional performance of the reagents after 11 month at +40°C. Conclusion: Stabilization and storage of critical assay reagents by freeze-drying is an attractive alternative to traditional deep freezing.

Brain Shuttle Antibody for Alzheimer's Disease with Attenuated Peripheral Effector Function due to an Inverted Binding Mode.

Receptors show promise for the transport of monoclonal antibodies (mAbs) across the blood-brain barrier. However, safety liabilities associated with peripheral receptor binding and Fc effector function have been reported. We present the Brain Shuttle-mAb (BS-mAb) technology, and we investigate the role of Fc effector function in vitro and in an Fcγ receptor (FcγR)-humanized mouse model. Strong first infusion reactions (FIRs) were observed for a conventional mAb against transferrin receptor (TfR) with a wild-type immunoglobulin G1 (IgG1) Fc. Fc effector-dead constructs completely eliminated all FIRs. Remarkably, no FIR was observed for the BS-mAb construct with a native IgG1 Fc function. Using various BS-mAb constructs, we show that TfR binding through the C-terminal BS module attenuates Fc-FcγR interactions, primarily because of steric hindrance. Nevertheless, BS-mAbs maintain effector function activity when binding their brain target. Thus, mAbs with full effector function can be transported in a stealth mode in the periphery while fully active when engaged with their brain target.

Crystal structure of an anti-Ang2 CrossFab demonstrates complete structural and functional integrity of the variable domain.

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.