Designing Cell-Targeted Therapeutic Proteins Reveals the Interplay between Domain Connectivity and Cell Binding

Abstract

The therapeutic efficacy of cytokines is often hampered by severe side effects due to their undesired binding to healthy cells. One strategy for overcoming this obstacle is to tether cytokines to antibodies or antibody fragments for targeted cell delivery. However, how to modulate the geometric configuration and relative binding affinity of the two domains for optimal activity remains an outstanding question. As a result, many antibody-cytokine complexes do not achieve the desired level of cell-targeted binding and activity. Here, we address these design issues by developing a computational model to simulate the dynamics and binding kinetics of natural and engineered fusion proteins such as antibody-cytokine complexes. To verify the model, we developed a modular system in which an antibody fragment and a cytokine are conjugated via a DNA linker that allows for programmable linker geometry and protein spatial configuration. By assembling and testing several anti-CD20 antibody fragment-interferon ? complexes, we showed that varying the linker length and cytokine binding affinity controlled the magnitude of cell-targeted signaling activation in a manner that agreed with the model predictions, which were expressed as dose-signaling response curves. The simulation results also revealed that there is a range of cytokine binding affinities that would achieve optimal therapeutic efficacy. This rapid prototyping platform will facilitate the rational design of antibody-cytokine complexes for improved therapeutic outcomes.

Interplay of Affinity and Surface Tethering in Protein Recognition

“…Tethered ligand–receptor complexes are common in protein recognition , and cellular adhesion . Surface-bound ligand–protein complexes are also the basis for biotechnological applications, such as biosensors − and cell-targeted therapeutic proteins, , as well as for single-molecule techniques that probe the dynamics and thermodynamics of protein binding. − Yet, how the presence of spatial constraints imposed by the surface and/or the tether affects the thermodynamics and, especially, kinetics of binding is largely an open experimental question. Most of the current insight into this topic comes from theoretical − and computational ,− studies.…”

“…Surface-bound ligand–protein complexes are also the basis for biotechnological applications, such as biosensors − and cell-targeted therapeutic proteins, , as well as for single-molecule techniques that probe the dynamics and thermodynamics of protein binding. − Yet, how the presence of spatial constraints imposed by the surface and/or the tether affects the thermodynamics and, especially, kinetics of binding is largely an open experimental question. Most of the current insight into this topic comes from theoretical − and computational ,− studies. However, experimental examinations of tethered ligand–protein interactions are mostly limited to measuring macroscopic intermolecular forces, − equilibrium dissociation constants, and effective protein concentrations. , …”

Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses

“…This question is motivated by experimental evidence showing that the kinetics , and dynamics − of protein recognition depend on the flexibility and length of the tether, which immobilizes one binding partner to a surface. However, most of the literature pertaining to this topic comes from computational − and theoretical − studies. Furthermore, we highlight that in many cases evaluating these sensors’ performance solely relies on the resistive-pulse technique (Figure a), limiting our knowledge of their quantitative and functional traits.…”