Transfer of engineered biophysical properties between different antibody formats and expression systems

Abstract

Recombinant antibodies and their derivatives are receiving ever increasing attention for many applications. Nevertheless, they differ widely in biophysical properties, from stable monomers to metastable aggregation-prone mixtures of oligomers. Previous work from our laboratory presented the combination of structure-based analysis with family consensus alignments as being able to improve the properties of immunoglobulin variable domains. We had identified a series of mutations in the variable domains that greatly influenced both the stability and the expression level of single-chain Fv (scFv) fragments produced in the periplasm of Escherichia coli. We now investigated whether these effects are transferable to Fab fragments and immunoglobulin G (IgG) produced in bacteria, Pichia pastoris, and mammalian cells. Taken together, our data indicate that engineered mutations can increase functional expression levels only for periplasmic expression in prokaryotes. In contrast, stability against thermal and denaturant-induced unfolding is improved by the same mutations in all formats tested, including scFv, Fab and IgG, independent of the expression system. The mutations in V(H) also influenced the structural homogeneity of full-length IgG, and the reducibility of the distant C(H)1-C(L) inter-chain disulfide bond. These results confirm the potential of structure-based protein engineering in the context of full-length IgGs and the transferability of stability improvements discovered with smaller antibody fragments.

Analysis of IgG kinetic stability by differential scanning calorimetry, probe fluorescence and light scattering

“…For the analysis of the kinetic stability of IgG with our previously derived mathematical model we chose the well‐characterized antibody IgG6B3, a human IgG1, that contains a heavy chain of subclass V H 6 and a light chain of the V λ 3 family …”

“…The well‐characterized IgG6B3 binds myoglobin and had been selected from the Human Combinatorial Antibody Library (HuCAL) . Protein was produced from a stable HEK293 clone as described previously as well as from transiently transfected FreeStyle 293‐F cells (Thermo Fisher Scientific) according to the protocol for HEK cell lines summarized by Hacker et al Subsequently, IgGs were affinity‐purified from cleared supernatants on Protein A columns (GE Healthcare Life Sciences). Eluted fractions were pooled and dialyzed against PBS, pH 7.4, (Thermo Fisher Scientific) before proteins were used for subsequent analyses.…”

Glypican-3–Targeted 89Zr PET Imaging of Hepatocellular Carcinoma

“…Additionally, heat denaturing of the 89 Zr-αGPC3 limited binding in size-matched HepG2 tumors to levels less than in the negative control RH7777 tumors, further confirming the necessity of αGPC3 mAb for tumor targeting. The substantial increase in liver and splenic activity in the heat-denatured animal is due to the well-known phenomenon of antibody aggregation when subjected to increased tem-which leads to an increase in size (31) and uptake by large-molecule clearance organs. αGPC3 was created using human GPC3 protein and demonstrated high-affinity binding during development.…”

Novel multispecific heterodimeric antibody format allowing modular assembly of variable domain fragments

“…Since it is well described that biophysical properties of variable domains can be transferred between different antibody formats, 45 we were motivated to combine these highly stable functional Fv modules in a single molecule to produce a novel class of heterodimeric, tetraspecific antibody derivatives whose assembly is exclusively driven by interactions between cognate variable domains on each protein subunit. Such a design would obviate the need for functionally irrelevant 2 or undesirable dimerization domains, which would normally confer distribution-impairing bulk, the potential for off-target activity, and, possibly, immunogenicity.…”