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MAbs. 2015;7(3):470-82. doi: 10.1080/19420862.2015.1022694.

Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

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a Eli Lilly Biotechnology Center ; San Diego , CA , USA.


A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.


BiTE, bispecific T cell engager; BsAb, bispecific antibody; CD, circular dichroism; DSC, differential scanning calorimetry; Fab interface design; Fab, antigen binding antibody fragment; Fv, variable domains antibody fragment; HC, antibody heavy chain; IgG-Fab; LC, antibody light chain; LCMS, liquid chromatography with in-line mass spectrometry; SEC-LC, size exclusion chromatography with in-line static light scattering; T cell; Tm, temperature at the midpoint of thermal unfolding; bispecific antibody; mAb, monoclonal antibody; scFv, single chain Fv; tandem Fab

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