Characterization of a Novel Bispecific Antibody With Improved Conformational and Chemical Stability

Abstract

Bispecific antibodies containing single-chain variable fragment (scFv) appended to immunoglobulins G offer unique development challenges. Here, we describe the stability of a novel bispecific format, BiS5, where the scFv is tethered to the C H 3 domain. BiS5 showed an improved conformational and chemical stability compared with that of BiS4 in which the scFv is appended in the hinge region between the F ab and F c. By switching the location of the scFv from hinge region to the C H 3, there was an improved stabilization of C H 2 and scFv domains. Interestingly, no noticeable impact was observed on the conformational stability of C H 3 and F ab domains. BiS4 and BiS5 showed different aggregation and fragmentation rates under accelerated temperature stress conditions. BiS4 showed higher fragmentation rates compared with BiS5 likely owing to fragmentation in the linker region on either side of the scFv while BiS5 is more resistant toward fragmentation owing to tethering of scFv to the C H 3 domain at its N and C terminus. In conclusion, the location of scFv affects both aggregation and fragmentation kinetics. These insights into the molecular structure and correlations with their physical and chemical stability will help formulation development of these novel bispecific antibodies.

Formulations That Suppress Aggregation During Long-Term Storage of a Bispecific Antibody are Characterized by High Refoldability and Colloidal Stability

“…The complexity of an IgG-scFv is needed to obtain certain desired biological features 3 . However, such artificially-created protein structures can exhibit reduced physical stability and increased aggregation propensity during storage in comparison to naturally occurring IgGs 4,5 .…”

“…The aggregation during storage of an IgG-scFv can be reduced by using suitable formulation conditions 3, 5 . A set of formulations must be tested to find the optimal formulation conditions, e.g.…”

Characterization of a novel bispecific antibody targeting tissue factor-positive tumors with T cell engagement

“…2 E). Based on IgG structural framework of TF-TCB and DSC thermogram of TF-011, the three peaks ( T m 1, T m 2 and T m 3) of TF-TCB may be associated with the unfolding of its scFv domain, CH2 domain and CH3/Fab domains, respectively 27 , 38 , 39 . And this could be further validated by comparing with the unfolding of CD3 scFv domain in future studies.…”

Rapid Multi-Residue Detection Methods for Pesticides and Veterinary Drugs

“…Bispecific antibodies contain two different antigen-binding sites, which can detect two distinct residues [ 45 ] ( Figure 2 C). Bispecific antibodies can be generated by chemical cross-linking, hybrid-hybridoma technology, or recombinant DNA techniques [ 46 ].…”