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

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.

Selection against super-4/ IL-4Rα/biotinylated γ c ternary complex on beads Selection against anti-cmyc antibody to enrich for full-length clones Clear against biotinylated γ c tetramers Clear against super-4/biotinylated IL-4Rα binary complex tetramers Selection against super-4/ IL-4Rα/biotinylated γ c ternary complex tetramers Isolate individual clones to characterize sequences and functional properties

Figure S2
IL-4 ternary complex stapler selection strategy. Flow diagram detailing the selection scheme used to to isolate stapler scFvs from a naïve human nonimmune yeast-displayed scFv library. Note the use of super-4 in place of the IL-4 cytokine to stabilize the target complex.

Figure S3
IL-4 stapler variable domain sequences. Sequences of the variable heavy (V H ) and variable light (V L ) chain third complementarity determining regions (CDR3s) for the three isolated IL-4 stapler scFvs.

Figure S4
Evolved stapler scFvs selectively bind the active IL-4 cytokine-receptor ternary complex. Binding of yeast-displayed scFv A8 (A) and scFv A11 (B) staplers to soluble cytokine-receptor complexes or components thereof. Both stapler scFvs specifically recognize the fully assembled super-4/IL-4Ra/g c TC. Surface plasmon resonance kinetic binding profiles detailing the interactions between scFv A8 or scFv A11 and the super-4/IL-4Ra/g c TC are shown at right. K D values values were calculated by fitting equilibrium titration curves to a logistic model via non-linear regression. The top curve represents a concentration of 20 µM and subsequent curves represent threefold serial dilutions.

Figure S6
Stapler heavy and light chains exhibit partitioned engagement of the IL-4Ra and g c receptor subunits. Crystal structure (3.1 Å resolution) of the full IL-4 Stapler Fab (HC shown in magenta and LC shown in olive) bound to the super-4 (brown)/IL-4Ra (blue)/g c (yellow) ternary complex. Details of the IL-4 Stapler light chain/g c heavy chain/IL-4Ra (left) and the IL-4 Stapler light chain/g c (right) interfaces are shown overlaid with a composite, simulated-annealing omit 2mFo -DFc map contoured at 1 sigma (navy).

Figure S7
Stapler binds IL-4Ra/g c heterodimer through contacts in all three complementarity determining regions (CDRs) of both heavy and light chains. A surface representation of the super-4 (brown)/IL-4Ra (blue)/g c (yellow) ternary complex with IL-4 Stapler Fab epitopes colored in gray is shown at top. Enlarged views of the interfaces between IL-4 Stapler V H (magenta, left) and V L (olive, right) chains and the IL-4 receptor subunits are presented with interacting residues on the IL-4 Stapler Fab show as sticks and labeled. Below, two-dimensional interaction maps between amino acids on IL-4 Stapler V H (magenta), IL-4 Stapler V L (olive), IL-4Ra (blue), and g c (yellow) are provided for each of the four IL-4 Stapler variable domain/IL-4 receptor subunit interfaces. Interactions between side chains are represented as lines whereas interactions between side chains and backbone are shown as arrows with the arrowheads pointing to the backbone. Black lines represent Van der Waals and hydrophobic contacts and red lines denote hydrogen bonds or electrostatic interactions.

IL-2Ra
Quaternary Complex: K D ≈10 pM  g c IL-2 Ternary Complex: K D ≈1 nM Figure S8 IL-2 cytokine-receptor quaternary and ternary complex structures. Crystal structures of the quaternary (left) and ternary (right) IL-2 cytokine-receptor complexes. The quaternary complex consists of the IL-2 cytokine and the IL-2Ra (cyan), IL-2Rb (navy), and g c (yellow) subunits, whereas the ternary complex lacks the IL-2Ra subunit. Note that the quaternary complex has 100-fold stronger affinity than the ternary complex.

IL-2 binary complex stapler selection strategy.
Flow diagram depicting the selection strategy implemented on a naïve human non-immune yeastdisplayed scFv library to evolve antibody fragments that stabilize IL-2/IL-2Rb binary complex formation. Note the use of Super-2 in place of the IL-2 cytokine to stabilize the target complex.