Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

Figures & data

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).

Table 1. Biochemical characterization of initial IgG_TCRs

Construct Name^a	Expression Level (μg/mL)	Apparent LC association by SDS-PAGE	Monomer/ Aggregate by SEC/LS^b	Activity by kinetic Biacore
Anti-17 IgG4	70	+	64/36	100
Hα1Lβ1_G4	81	+	63/37	35
Hα2Lβ2_G4	56	+	67/33	54
Hα2Lβ1_G4	29	+	64/36	35
Hα1Lβ2_G4	74	+	65/35	48
Hβ1Lα1_G4	34	−	∼25/∼75	-^c
Hβ2Lα2_G4	60	−	25/75	17
Hβ2Lα1_G4	44	−	∼25/∼75	-^c
Hβ1Lα2_G4	27	−	∼25/∼75	-^c

^aNomenclature can be dissected as follows. ‘H’ and ‘L’ stand for heavy chain or light chain, respectively. ‘α’ and ‘β’ indicates, whether Cα or Cβ, respectively are embedded in the heavy chain or light chain, respectively. ‘1’ indicates full-length Cα or Cβ sequence, while ‘2’ indicates an N-terminal truncation.

^bBased on 2 mL expressed/purified material. The pH 3.0 elution from protein G magnetic beads induced some aggregation including with the IgG4 control.

^cNot measured.

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.

Table 2. Occupancy of predicted N-linked glycosylation sites

Potential Site	Tryptic Peptide	%Occupancy N-linked Glycan
LC_Cβ_N186 (Kabat #)	EQPALN^186DSR	24.6
HC_Cα_N151 (Kabat #)	SVC^aLFTDFDSQTN^151VSQSK	100.0
HC_Cα_N185 (Kabat #)	SNSAVAWSN^185K	97.3
HC_Cα_N196 (Kabat #)	SDFAC^aANAFN^196NSIIPEDTFFPSPEPK	100.0
HC_CH2_N297 (EU#)	EEQYN^297STYR	99.9

^aCysteines were reduced and alkylated with iodoacetamide.

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.