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bioRxiv · 10.1101/2025.09.23.678000

High-throughput cell-free profiling of SARS-CoV-2 RBD variants enables rapid and quantitative in vitro affinity landscape mapping

Grasemann, L.·Han, J.·Tischler, J.·Arefi, F.·Castillo, M. A. G.·Irvine, E. B.·Chen, N.·Reddy, S. T.·Maerkl, S. J.
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Abstract

SARS-CoV-2 variants continue to threaten public health, necessitating the study of cumulative and epistatic effects of receptor-binding domain (RBD) mutations on antibody evasion. We present a high-throughput platform combining cell-free protein synthesis and microfluidics to quantify the affinity of a large number of RBD triplet mutants covering the evolutionary space between wild-type and Omicron against two therapeutic antibodies and one engineered binder. Using rapid in vitro gene assembly and cell-free synthesis, we expressed 518 RBD variants and obtained 31,740 quantitative affinity measurements to generate three comprehensive binding energy landscapes. This approach enables rapid and large-scale in vitro affinity profiling and machine learning-based predictions, providing a valuable tool for studying emerging variants.

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Grasemann, L., Han, J., Tischler, J., Arefi, F., Castillo, M. A. G., Irvine, E. B., Chen, N., Reddy, S. T., Maerkl, S. J.. 2025-09-23. High-throughput cell-free profiling of SARS-CoV-2 RBD variants enables rapid and quantitative in vitro affinity landscape mapping. https://doi.org/10.1101/2025.09.23.678000

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