Design rationale for the BsAbs
The BsAb-1 and BsAb-2 molecules were constructed with a scFv domain
fused to the C-terminal end of the HC of a mAb via a peptide linker. A
schematic of the constructs is shown in Figure 1A. The BsAbs were
designed to bind to the same two soluble ligands both of which have
minimal peripheral concentrations in normal cynomolgus monkeys. The BsAb
molecules consisted of a target binding orientation switch, in which the
Fab region (of mAb-1) of BsAb-1 binds to the same ligand as the scFv
(scFv-1) component of BsAb-2. Relatedly, the Fab region (of mAb-2) of
BsAb-2 binds to the same ligand as scFv-2 in BsAb-1. The orientation
switch did not influence the binding to either ligand thus, both BsAbs
bind to each soluble ligand with no statistically significant changes in
affinity (data not shown). Each BsAb is composed of a human
IgG4 with the same CH1,
CH2 and CH3 regions; thus, the variable
region is the only difference between the mAbs in BsAb-1 and BsAb-2.
Each of these molecules also included mutations in the Fc region,
S228P/L234A/L235A (European Union (EU) numbering system), to eliminatein vivo arm exchange with endogenous IgGs and Fcγ receptor
interactions.17, 18 Fusion of the scFv elements to the
C-terminus of the intended IgG4 HC was facilitated by a
flexible glycine-serine linker to generate each BsAb.