title: Fab-based bispecific antibody formats with robust biophysical properties and biological activity.
abstract: 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.
authors.affiliation: a Eli Lilly Biotechnology Center ; San Diego , CA , USA.
authors.forename: Xiufeng
authors.initials: X
authors.lastname: Wu
authors.forename: Arlene J
authors.initials: AJ
authors.lastname: Sereno
authors.forename: Flora
authors.initials: F
authors.lastname: Huang
authors.forename: Steven M
authors.initials: SM
authors.lastname: Lewis
authors.forename: Ricky L
authors.initials: RL
authors.lastname: Lieu
authors.forename: Caroline
authors.initials: C
authors.lastname: Weldon
authors.forename: Carina
authors.initials: C
authors.lastname: Torres
authors.forename: Cody
authors.initials: C
authors.lastname: Fine
authors.forename: Micheal A
authors.initials: MA
authors.lastname: Batt
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Fitchett
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Glasebrook
authors.forename: Brian
authors.initials: B
authors.lastname: Kuhlman
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Demarest
mesh_terms: D018033:Antibodies, Bispecific / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D006801:Humans; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies
keywords: 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