A modular IgG-scFv bispecific antibody topology

Abstract

Here we present a bispecific antibody (bsAb) format in which a disulfide-stabilized scFv is fused to the C-terminus of the light chain of an IgG to create an IgG-scFv bifunctional antibody. When expressed in mammalian cells and purified by one-step protein A chromatography, the bsAb retains parental affinities of each binding domain, exhibits IgG-like stability and demonstrates in vivo IgG-like tumor targeting and blood clearance. The extension of the C-terminus of the light chain of an IgG with an scFv or even a smaller peptide does appear to disrupt disulfide bond formation between the light and heavy chains; however, this does not appear to affect binding, stability or in vivo properties of the IgG. Thus, we demonstrate here that the light chain of an IgG can be extended with an scFv without affecting IgG function and stability. This format serves as a standardized platform for the construction of functional bsAbs.

Fig. 1

Design of IgG light chain C-terminal scFv fusion. Pictorial representation of heavy chain, light chain and fully assembled bsAb with indicated N- and C- termini. The light chain is modified with an scFv fusion to the C-terminus, while a completely naïve heavy chain is preserved.

Fig. 2

Characterization of bsAb construct. (A) Gel electrophoresis of reduced (R) and non-reduced Sm3e/C825 bsAb and Sm3e IgG. (B) Size exclusion chromatography of Sm3e/C825 bsAb, Sm3e IgG and IgG purified from human plasma (Sigma).

Fig. 3

Simultaneous binding to cell surface antigen and hapten. Flow cytometry data of Sm3e/C825 bsAb (A), Sm3e/4m5.3 (B) and A33/4m5.3 (C) binding to cell surface antigen expressed on LS174T cells and soluble hapten. Legends show labeling scheme for sequential incubations.

Fig. 4

Disulfide stabilization of IgG. Western blot analysis detecting heavy and light chains of A33 IgG and A33 IgG extended by an 18 amino acid peptide (IgG + peptide) shows that extension of the light chain beyond the C-terminal Cκ cysteine disrupts disulfide bond formation. Two different pairs of cysteine mutations (ds1 and ds2) introduce disulfide stabilization of the IgG variable domains and demonstrate reformation of a covalent linkage between light and heavy chains.

Fig. 5

Thermal and serum stability. Thermal stability of various IgGs and bsAbs over 7 days at 37°C in PBSA detecting CEA binding (A) or A33 antigen binding (B). Serum stability of Sm3e/C825 bsAb over 7 days at 37°C in 50% mouse serum detecting cmyc retention (C) or DOTA-Y-647 binding (D).

Fig. 6

Blood clearance of Sm3e IgG and Sm3e/C825 bsAb in mice. Blood activity (±SD) time profile of Sm3e IgG (open diamonds) and Sm3e/C825 (closed diamonds) after a 500 µg intravenous dose in male nude mice, n = 3. The blood curves were fit by least squares regression to a biexponential function for Sm3e IgG (dotted line) and Sm3e/C825 (solid line).