We have engineered a novel bsAb construct as an scFv fusion to the C-terminus of the light chain of an IgG. Fusing the scFv in this way should minimize the steric hindrance that could obstruct simultaneous binding of both target antigens that might result from an N-terminal fusion to the light and/or the heavy chain. To date, we have synthesized several versions of the construct with various IgG and scFv domains, and all molecules bind simultaneously to their respective targets and retain parental affinities within 2-fold. No linker-length optimization is required for expression and retention of scFv and IgG binding. The bispecific construct also exhibits IgG-like stability, blood clearance and in vivo tumor targeting. The bsAb construct appears to work generally to pair any stable and functionally expressing IgG and scFv into a bispecific format, while retaining IgG-like properties. However, it should be noted that all of the bsAb constructs tested in this study have IgG domains that bind to cell surface proteins and scFv domains that bind to haptens. While we believe that this bsAb construct will also work when the scFv specificity is a protein target due to flexibility in the Gly4Ser-based linker and in the hinge region of the IgG, this has yet to be tested.
Coloma and Morrison (1997
) also used an scFv for introducing additional specificity to an IgG, by attaching it to the C-terminus of the heavy chain of an IgG3. They report excellent results obtaining fully assembled monomeric functional protein from transfectoma supernatants. However, the IgG-scFv fusion results in notably faster clearance in an in vivo
mouse model compared with the parent IgG. This may be due to a decrease in FcRn binding possibly from steric hindrance of the attached scFv, or perhaps aggregation or instability driven by the scFv moiety.
We are interested in bsAbs for pretargeted radioimmunotherapy applications, in which the bispecific is administered first and allowed to localize to tumor cells before the addition of a second, radioactive molecule that only binds to the bispecific and otherwise clears rapidly from the body (Goodwin et al., 1988
; Le Doussal et al., 1989
; Chang et al., 2002
; Boerman et al., 2003
; Gruaz-Guyon et al., 2005
). The first bsAbs for pretargeting used streptavidin/antibody conjugates and have shown promise at the proof-of-concept stage (Axworthy et al., 2000
). However, recent pretargeted efforts have moved away from using streptavidin due to its immunogenicity, high kidney uptake (Wilbur et al., 2004
), and issues with endogenous biotin (Hamblett et al., 2002
). Several bispecific formats that do not utilize streptavidin have been used for pretargeted applications including chemically conjugated Fab and (Fab)2
formats (Kraeber-Bodere et al., 1999
; Schuhmacher et al., 2001
; Griffiths et al., 2004
), hybrid hybridoma bsAbs (van Schaijk et al., 2003
; van Schaijk et al., 2005
), recombinant diabodies and scFv fusions (DeNardo et al., 2001
; Rossi et al., 2005
), and a dock and lock tri-Fab (Sharkey et al., 2008
It is desirable to preserve the Fc region in a bsAb for pretargeting applications, which will result in prolonged plasma retention due to FcRn binding and hence increased tumor penetration (Olafsen et al., 2006
; Thurber et al., 2008
). Full IgG molecules have demonstrated significantly higher tumor accumulation compared with minibodies, diabodies and scFvs (Schneider et al., 2009
). The addition of the scFv to the C-terminus of the IgG light chain does not impact blood clearance, indicating that the scFv does not affect FcRn binding to the bispecific construct. The Fc-binding domain may also retain FcγR binding, for ADCC and additional therapeutic benefit.
The long-circulating half-life of the bsAb will result in increased tumor uptake as discussed above but may also result in significant residual antibody retention in the blood at the time of hapten dosing. Thus, a clearing agent to quickly clear antibody from the blood prior to hapten dosing may help to accelerate hapten clearance from the body. Rapid hapten clearance is necessary for high tumor to background ratios for imaging and low off-target radiation for therapy.
The Sm3e/C825 bsAb retains approximately 90% of its cmyc tag at 37°C in serum after 7 days, indicating little if any protease cleavage of the Gly4Ser linker. However, the binding activity of the C825 scFv decreases to about 60% after 7 days, with a rapid decrease during the first 24 h followed by a plateau. The decreased binding activity does not appear to be pH mediated as the pH of the serum solution remains neutral for the length of the experiment (data not shown). Nor does it appear to be simply due to thermal instability, because 90% of binding is retained after similar incubation at 37°C in PBSA. Thus, the loss of activity may be due to serum protein binding or protein- or enzyme-assisted denaturation.
One aspect of the bsAb format is an intermolecular disulfide bond between the VH and VL domains of the scFv. The open conformation of an scFv can be prone to aggregation (Reiter et al., 1994
; Worn and Pluckthun, 2001
). Disulfide stabilization of the scFv should prevent the scFv from assuming an open conformation and hence reduce the risk of aggregation. In addition, as one would expect, the stability of the scFv in the bispecific format is limited by the stability of the parent scFv. Thus, it is important to select highly stable scFvs.
It is interesting to note that attaching an scFv, or even an 18 amino acid flexible peptide, to the C-terminus of the light chain appears to disrupt formation of the disulfide bond between the light chain and the heavy chain of the human IgG1. This disulfide bond naturally exists at the C-terminal cysteine of the Cκ domain in IgGs. It does not appear to be necessary for function or stability of the bsAb, as all bsAbs tested retain parental affinities and exhibit excellent serum stability, even after protein A purification with acidic pH elution. We nevertheless added a disulfide bond between the VH and VL domains of the A33 IgG with peptide to determine if an interdomain disulfide bond can be introduced in this region to reform a covalent linkage between the LC and HC. Indeed, both disulfide stabilized versions of the A33 IgG with peptide exhibited significantly reduced dissociation after boiling, confirming that a covalent bond between the LC and HC can be reformed. While in this study, a covalent linkage between the heavy and light chains does not appear necessary for bsAb function or stability, it is possible that other IgGs may be less stable and require interdomain disulfide stabilization for stable bsAb construction.
While we have designed this bispecific format to be used for pretargeting approaches, this platform may be beneficial for other applications requiring bsAbs. We demonstrate that the light chain of an IgG can be extended with an scFv without affecting IgG function and stability. Other proteins or peptides, such as affibodies (Nygren, 2008
), single variable domains (Harmsen and De Haard, 2007
) and peptide toxins could be attached to IgGs in this site specific manner, to yield homogenous IgG fusion products for targeted delivery. This platform could be used in a straightforward fashion to modify current FDA-approved antibodies to add additional functionalities. Production and purification should scale directly with current antibody manufacturing methods. As a robust modular platform, this bsAb format obviates the need for extensive optimization of each new combination of binding domains and retains IgG-like properties both in vitro
and in vivo