Stability engineering of scFvs for the development of bispecific and multivalent antibodies

Single-chain Fvs (scFvs) are commonly used building blocks for creating engineered diagnostic and therapeutic antibody molecules. Bispecific antibodies (BsAbs) hold particular interest due to their ability to simultaneously bind and engage two distinct targets. We describe a technology for producing stable, scalable IgG-like bispecific and multivalent antibodies based on methods for rapidly engineering thermally stable scFvs. Focused libraries of mutant scFvs were designed using a combination of sequence-based statistical analyses and structure-, and knowledge-based methods. Libraries encoding these designs were expressed in E. coli and culture supernatants-containing soluble scFvs screened in a high-throughput assay incorporating a thermal challenge prior to an antigen-binding assay. Thermally stable scFvs were identified that retain full antigen-binding affinity. Single mutations were found that increased the measured Tm of either the VH or VL domain by as much as 14°C relative to the wild-type scFv. Combinations of mutations further increased the Tm by as much as an additional 12°C. Introduction of a stability-engineered scFv as part of an IgG-like BsAb enabled scalable production and purification of BsAb with favorable biophysical properties.