title: Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies.
abstract: Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/β TCR. Replacing CH1 with Cα and CL with Cβ, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cβ with a canonical β-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the CH1/CL and Cα/Cβ domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases.
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: Kai
authors.initials: K
authors.lastname: Zhang
authors.forename: Micheal
authors.initials: M
authors.lastname: Batt
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Fitchett
authors.forename: Dongmei
authors.initials: D
authors.lastname: He
authors.forename: Heather L
authors.initials: HL
authors.lastname: Rick
authors.forename: Elaine M
authors.initials: EM
authors.lastname: Conner
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Demarest
mesh_terms: D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D018033:Antibodies, Bispecific; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins
keywords: DSC, differential scanning calorimetry; FG loop; HC, heavy chain; Ha, heavy chain containing Ca in place of CH1; Hb, heavy chain containing Cb in place of CH1; LC, light chain; La, heavy chain containing Ca in place of CL; Lb, heavy chain containing Cb in place of CL; RU, resonance units; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; SPR, surface plasmon resonance; T cell receptor; TCR, T cell receptor; bispecific antibody; protein chimera; protein design