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

Abstract

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 T(m) of either the V(H) or V(L) domain by as much as 14 degrees C relative to the wild-type scFv. Combinations of mutations further increased the T(m) by as much as an additional 12 degrees 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.

Deep Mutational Scans as a Guide to Engineering High Affinity T Cell Receptor Interactions with Peptide-bound Major Histocompatibility Complex

“…Accordingly, the present study identified mutations that increased surface levels up to about 6-fold. Similar observations have been made about the role of residues in the thermal stability of antibody scFv fragments (32,44,45).…”

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity

“…41 We previously reported a new and robust BsAb platform that utilizes stability-engineered scFvs appended to full length IgGs to target two tumor necrosis factor receptor family members or two distinct epitopes of IGF-1R. [42][43][44] Both BsAbs displayed IgGlike pharmaceutical properties, dual specificity and improved anti-tumor activity. Here, we describe the development of a novel BsAb targeting EGFR and IGF-1R using this platform technology.…”

Biomedicals from a soil bug

“…From an ethical point of view the phage display approach can help to reduce the amount of animal experiments in medical research. Furthermore, because the DNA sequence encoding the antibody is available, the small fragments offer numerous ways for the engineering and modification of biophysical properties to suit their operation purpose 3,4 and are eligible for production at low costs and fast production cycles in microbial systems. 5 These advantages however, are impaired by the often poor levels of soluble antibody fragments in prokaryotic production systems and the necessity of time consuming improvement approaches like codon-optimization or excessive screening of culture and expression conditions.…”