A strategy for the selection of monovalent antibodies that span protein dimer interfaces

Abstract

Ligand-induced dimerization is the predominant mechanism through which secreted proteins activate cell surface receptors to transmit essential biological signals. Cytokines are a large class of soluble proteins that dimerize transmembrane receptors into precise signaling topologies, but there is a need for alternative, engineerable ligand scaffolds that specifically recognize and stabilize these protein interactions. Recombinant antibodies can potentially serve as robust and versatile platforms for cytokine complex stabilization, and their specificity allows for tunable modulation of dimerization equilibrium. Here, we devised an evolutionary strategy to isolate monovalent antibody fragments that bridge together two different receptor subunits in a cytokine-receptor complex, precisely as the receptors are disposed in their natural signaling orientations. To do this, we screened a naive antibody library against a stabilized ligand-receptor ternary complex that acted as a "molecular cast" of the natural receptor dimer conformation. Our selections elicited "stapler" single-chain variable fragments (scFvs) of antibodies that specifically engage the interleukin-4 receptor heterodimer. The 3.1 Å resolution crystal structure of one such stapler revealed that, as intended, this scFv recognizes a composite epitope between the two receptors as they are positioned in the complex. Extending our approach, we evolved a stapler scFv that specifically binds to and stabilizes the interface between the interleukin-2 cytokine and one of its receptor subunits, leading to a 15-fold enhancement in interaction affinity. This demonstration that scFvs can be selected to recognize epitopes that span protein interfaces presents new opportunities to engineer structurally defined antibodies for a broad range of research and therapeutic applications.

Keywords: antibody engineering; cell signaling; cytokine; dimerization; directed evolution; immunology; interleukin-2; interleukin-4; ligand-receptor interactions; structural biology.

Figure 1.

Novel evolutionary selection strategy isolates antibody-based “staplers” that dimerize cytokine receptor chains. A, schematic contrasting receptor heterodimerization and activation induced by cytokine, antibody, or “stapler” scFv binding, respectively. B, layout and selection scheme for yeast display-based evolution of cytokine–receptor complex staplers. A yeast-displayed scFv library is evolved through iterative rounds of selection, each of which comprises negative clearance steps against the individual complex components and positive selection for binders to the fully assembled ternary complex. C, flow cytometry histograms depicting the binding of yeast-displayed IL-4 stapler scFv to the indicated cytokine–receptor complexes and individual components thereof. TC, ternary complex. D, kinetic SPR binding profiles for the interactions between the immobilized IL-4 stapler scFv and soluble Super-4/IL-4Rα/γ_c TC components versus the fully assembled complex. All proteins were flowed at a concentration of 60 μm. E, surface plasmon resonance kinetic binding traces depicting the interaction between immobilized IL-4 stapler scFv and dilutions of the Super-4/Il-4Rα/γ_c ternary complex. The equilibrium dissociation constant (K_D) value was calculated by fitting the equilibrium titration curve to a logistic model via nonlinear regression. The top curve represents a concentration of 20 μm, and subsequent curves represent 3-fold serial dilutions.

Figure 2.

Stapler recognizes a composite epitope between two receptor subunits to bridge the dimer interface. A, orthosteric views of the crystallographic structure of the IL-4 stapler Fab fragment bound to the Super-4/IL-4Rα/γ_c ternary complex. B, enlarged views of the IL-4 stapler interfaces with the IL-4Rα and γ_c subunits. At the left, IL-4 stapler-binding sites on IL-4Rα and γ_c are depicted in gray, with interacting residues in the CDRs of the V_H (magenta) and V_L (olive) domains of the IL-4 stapler Fab shown as sticks. At the right, the CDRs on V_H (magenta) and V_L (olive) of the IL-4 stapler Fab are shaded.

Figure 3.

Evolved staplers specifically engage cytokine–receptor binary complex with engineerable affinity. A, schematic contrasting sequential assembly of a cytokine–receptor ternary complex in the absence (top) or presence (bottom) of a binary complex–stabilizing stapler scFv. B, yeast display platform diagram and selection strategy for evolution of cytokine–receptor binary complex stapler scFvs. C, binding of yeast-displayed IL-2B (left) or amIL-2B (right) stapler scFv to cytokine–receptor complexes and individual components thereof. Note the selective recognition of the IL-2/IL-2Rβ binary complex and compatibility with γ_c binding. TC, ternary complex. D, kinetic surface plasmon resonance binding profiles for the IL-2B stapler:IL-2 BC (left) and amIL-2B stapler:IL-2 BC (right) interactions. As shown in the traces, the affinity-matured stapler variant improves affinity 14-fold through deceleration of the dissociation rate. K_D values were calculated by fitting equilibrium titration curves to a logistic model via nonlinear regression.

Figure 4.

Binary complex staplers strengthen cytokine–receptor interactions to potentiate signaling of an affinity-impaired cytokine variant. A, equilibrium surface plasmon resonance titrations of the IL-2:IL-2Rβ binary complex interaction without scFv present (filled blue circles) and with IL-2BC stapler scFv (gold circles) or amIL-2B stapler scFv (cyan circles) compared with the Super-2:IL-2Rβ interaction (unfilled blue circles). K_D values were calculated by fitting equilibrium titration curves to a logistic model via nonlinear regression. IL-2B staplers increase the IL-2:IL-2Rβ interaction affinity ∼15-fold. B, YT-1 cell STAT5 phosphorylation responses to impaired affinity IL-2 (iIL-2) without scFv present (filled blue circles) and with saturating levels of IL-2B stapler (gold circles) or amIL-2B stapler (cyan circles) present compared with WT IL-2 (unfilled blue circles). EC₅₀ values are indicated. Error bars, S.D.