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.

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.

Figure 1.

Schematic diagram of an IgG-Fab BsAb (A). Diagrams to the right of the correctly assembled IgG-Fab are potential mispairings related to the lack of LC specificity for a particularly HC F_d region. (B) Schematic diagram of the domain architecture of a α/β TCR. The receptor is a heterodimer consisting of 2 chains that each comprise a V-class and a C-class Ig-fold, much like an immunoglobulin Fab. (C) Superposition of the structures of an IgG1 C_H1/Cκ heterodimer (pdb id: 3HC0) and the constant domains of an α/β TCR (pdb id: 3ARB). The structural homology between Cβ and Cκ as well as that between Cα and C_H1 is apparent. (D) Diagram demonstrating the exchange of the C_H1/Cκ domains with the TCR Cα/Cβ domains within an IgG1 antibody (denoted IgG_TCR).

Figure 2.

Characterization of initial IgG_TCR constructs. (A, B) SDS-PAGE analysis of IgG_TCR constructs under reducing, 10 mM DTT, (A) and non-reducing conditions (B). 15 μL of protein G magnetic bead purified protein from 2 mL 293F purifications was added to each lane. β1 and β2 differ in the N-terminal residues of the β-domain (β1 starts with E117, while β2 starts with K121). Similarly, α1 and α2 differ in the N-terminal residues of the α-domain (α1 starts with P116, while α2 starts with I118). (C) Non-reduced (left side of gel) and reduced (right side of gel) SDS-PAGE analysis of protein G pull-downs from supernatants expressed using mismatched and matched pairs of IgG and IgG_TCR heavy and light chains. (D) Cation exchange separation of IgG_TCR proteins secreted with 0 (1st peak), 1 (middle peak), or 2 (3rd peak) associated LCs. The inset shows the SDS-PAGE analysis of the 3 cation exchange fractions. (E) Binding activity of the protein fractions separated in (D), demonstrating the importance of LC association for binding to antigen. The association of the IgG_TCR protein can be observed between 300–600 s, while the antigen (IL-17 in this case) association can be observed between 800–1000 s. (F) Improvement in the uniform expression of fully paired (HC₂LC₂) IgG_TCR proteins after truncating the C-terminal tail of the β-constant domain of the LC. HC₂LC₂ elution time was at 13.5 minutes based on static light scattering analysis.

Figure 3.

Effect of deleting N-linked glycosylation on HEK293 expression of IgG_TCR proteins. Reduced SDS-PAGE analysis (A) and analytical SEC (B) of fully glycosylated (WT) IgG_TCR, single N-linked glycosylation deletion mutants, double mutants, and a triple mutant after protein G pull-down from 2 mL HEK293 expression supernatants. (C) DSC analysis of fully glycosylated (WT) and single N-linked glycosylation deletion mutants of IgG_TCR proteins after 100 mL HEK293 scale-up and protein A purification.

Figure 4.

Replacement of the FG loop from the β-constant domain with common β-turn motifs. (A) Stick diagram of the structure of the β-constant domain (from pdb 3QEU) where the FG loop is colored orange. Non-reduced (top) and reduced (bottom) SDS-PAGE analyses (B) and analytical SEC (C) of WT IgG_TCR, FG loop-deleted IgG_TCR, and IgG_TCRs with the FG loop replaced with a PS (proline_serine, Type I), NG (Type I’), and GN (Type II’) β-turn. The analyses in (B) and (C) were performed on IgG_TCR proteins expressed at the 2 mL scale in HEK293 and pulled down using protein G magnetic beads. (D) DSC analyses of WT IgG_TCR and FG loop replaced IgG_TCR after scale-up and protein A purification.

Figure 5.

Biophysical characterization of HER2×HER2 IgG-Fab BsAbs produced using IgG_TCR modalities to direct LC assembly. Panels A and B are non-reduced (left) and reduced (right) SDS-PAGE analysis and analytical SEC, respectively, of C-BsAb and N-BsAbs. Panels E and F are an evaluation of the HER-2 binding properties of trastuzumab IgG_TCR, pertuzumab IgG_TCR, C-BsAb, and N-BsAb by analyzing their ability to block 40 nM HER-2 from binding surfaces labeled with IgG1 trastuzumab (C) or pertuzumab (D). Panels E and F are intact mass spectrometry analyses of C-BsAb and N-BsAb, respectively under reducing conditions. The N-BsAb contained the V_L_Y36F mutation and V_L_Q38D/V_H_Q39K to reduce the affinity of the trastuzumab LC for the pertuzumab F_d containing Cα/Cβ. The spectra show the levels of LC within the IgG-Fab BsAbs. The HC was heavily N- and O-glycosylated; therefore, non-reduced spectra were complex.

Figure 6.

Biological activity of IgG_TCR BsAbs. The effect of IgG_TCR BsAbs on the FBS-driven proliferation of (A) BT-474 breast cancer cells (top) and N87 gastric cancer cells (bottom). (B) Western blot analyses of the phosphorylated state of EGFR, HER-2, HER-3, Akt, and Erk from N87 tumor cells grown for 48 hours in FBS in the presence of various anti-HER-2 monoclonal and bispecific antibodies. Additionally, the presence of cleaved PARP was evaluated on the blot. Actin was probed to demonstrate the normalized amount of protein loaded into each well.