Comparing domain interactions within antibody Fabs with kappa and lambda light chains

Abstract

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

Keywords: Antibody; Fab; kappa light chain; lambda light chain; stability.

Figure 1.

Schematic diagrams of Fab constructs produced to evaluate cooperative inter-domain interactions within antibody Fabs. On top, are wild-type Fab and T cell receptor diagrams. All three proteins comprise a similar structure with 2 V-class domains responsible for antigen binding and 2 C-class domains linked by a C-terminal inter-chain disulfide bond. For the study described here, we utilized pertuzumab (pdb 1S78) as a representative κ LC Fab and PGT128 (pdb 3TV3) as a representative λ LC Fab. On the bottom, are schematics of the chimeric Fabs that were generated to evaluate the thermodynamic properties of variable and constant domains within Fabs.

Figure 2.

Representative SDS-PAGE and analytical size exclusion chromatography (SEC) of Fab constructs. (A) Non-reducing SDS-PAGE with the wild-type pertuzumab and PGT128 Fabs (lanes 2 and 3, respectively) and pertuzumab Fabs containing Cλ (lane 4) and Cα/Cβ (lane 6). Lane 5 contains a chimeric Fab with the pertuzumab HC and PGT128 LC. (B) Analytical SEC with in-line static light scattering confirmed the monodisperse nature of the constructs evaluated in this report. Shown are chromatograms of the wild-type pertuzumab Fab (bottom), pertuzumab Fab containing Cλ, and containing Cα/Cβ. The uppermost chromatogram is of the Cα/Cβ-containing pertuzumab Fab after enzymatic N-linked glycan removal.

Figure 3.

Differential scanning calorimetry (DSC) evaluation of the thermal unfolding of the κ LC-containing pertuzumab Fab. (A) DSC traces of the pertuzumab Fab (bottom), CH1/Cκ subunit (middle), and VH/Vκ pertuzumab scFv (top). The pertuzumab scFv was not stable when expressed on its own, but did express fairly well as an N-terminal fusion to IgG1 Fc. (B) DSC traces of the wild-type pertuzumab Fab (bottom), Cλ-containing pertuzumab Fab (middle), and Cα/Cβ-containing pertuzumab Fab both before (red) and after (orange) enzymatic deglycosylation (top). (C) DSC traces of Cλ-containing pertuzumab Fab before (bottom) and after introduction of destabilizing VH mutations G16E (middle) or V89F (top). These mutations were made to determine whether VH or VL was the less stable of the pertuzumab variable domains. Clearly VH is the less stable of the two. For all DSC traces, the identity of the domain(s) giving rise to each transition is labeled.

Figure 4.

Guanidinium HCl (GuHCl) chemical denaturations and 1-anilino-8-naphthalene sulfonate (ANS) binding of the pertuzumab Fabs. (A) GuHCl denaturation of the wild-type pertuzumab Fab (blue circles) and Cλ-containing pertuzumab Fab (red squares) at 1 mg/mL (open symbols) and 2 mg/mL (closed symbols). Only one transition was visible by Far UV circular dichroism (CD) at 234 nm; therefore, the curves were fit to a single unfolding event to obtain the m-value (slope of denaturation), which is correlated with the change in solvent exposed surface area for protein unfolding, and the GuHCl concentration where 50% of the protein is unfolded (C₅₀). (B) ANS fluorescence in the presence of the wild-type pertuzumab Fab (circles), Cα/Cβ-containing pertuzumab Fab (squares) in PBS buffer (blue closed symbols) and under acidic conditions (red open symbols). The protein α-lactalbumin was used as both a negative and positive control (diamonds). α-lactalbumin should not bind and induce fluorescence of ANS at neutral pH bound to Ca²⁺ (pH 8, 500 mM CaCl₂), but is known to partially unfold and bind ANS under acidic conditions (< pH 3).

Figure 5.

DSC evaluation of the thermal unfolding of the λ LC-containing PGT128 Fab. (A) DSC traces of the wild-type PGT128 Fab (bottom), CH1/Cλ subunit (middle), and VH/Vλ PGT128 scFv (top). (B) DSC traces of the wild-type PGT128 Fab (bottom), Cκ-containing PGT128 Fab (middle), and Cα/Cβ-containing PGT128 Fab (top). (C) DSC traces of the wild-type PGT128 Fab (blue), 10C12 Fab (green), and 3P6b_B01 Fab (red) all naturally containing fully λ LCs. For all DSC traces, the identity of the domain(s) giving rise to each transition are labeled.

Figure 6.

Comparison of 6 human scFvs and Fabs originating from antibodies with either κ or λ LCs. (A) Analytical SEC of matching scFvs and Fabs from 3 different human IgG1/κ mAbs. (B) Analytical SEC of matching scFvs and Fabs from 3 different human IgG1/λ mAbs. (C) DSC traces of matching scFvs and Fabs from 3 different human IgG1/κ mAbs. (D) DSC traces of matching scFvs and Fabs from 3 different human IgG1/λ mAbs.