Abstract
The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab′)2-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.
Highlights
It has been shown that cysteines introduced into various places on monoclonal antibodies allow for the production of site-specific, homogeneous drug-antibody conjugates with many useful properties [1,2,3]
One important contribution provided by these bis-Fab molecules is a specific understanding that changes in the orientation of the Fv-regions relative to one another in an antibody molecule can result in profoundly different biological activities
Using our technology to systematically alter the arrangement of one Fab arm relative to the other arm, we have been able to show that a robust structure-activity relationship exists between different antibody-isomers
Summary
It has been shown that cysteines introduced into various places on monoclonal antibodies allow for the production of site-specific, homogeneous drug-antibody conjugates with many useful properties [1,2,3]. From a combinatorial chemistry point of view, the availability of specific, highly reactive sites at various places on the Fab domain of antibodies should allow for facile generation of Fab92-like molecules containing the Fabs from two different antibodies (e.g. bispecific molecules) or containing two different cysteine mutations from the same parent antibody (e.g. monospecific molecules). In both cases one has exquisite control of the orientation or geometry between the two Fab arms as defined by the location of the cysteine mutations on each Fab. In addition, control over the flexibility between the Fab arms of the molecule and the distance between the antigen binding sites is possible. In this report we describe our bis-Fab technology and its application to the generation and modulation of activity of bispecific and monospecific Fab92-like molecules directed against receptors of the epidermal growth factor family
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