De novo design of protein minibinder agonists of TLR3

Abstract

Toll-like Receptor 3 (TLR3) is a pattern recognition receptor that initiates antiviral immune responses upon binding double-stranded RNA (dsRNA). Several nucleic acid-based TLR3 agonists have been explored clinically as vaccine adjuvants in cancer and infectious disease, but present substantial manufacturing and formulation challenges. Here, we use computational protein design to create novel miniproteins that bind to human TLR3 with nanomolar affinities. Cryo-EM structures of two minibinders in complex with TLR3 reveal that they bind the target as designed, although one partially unfolds due to steric competition with a nearby N-linked glycan. Multivalent forms of both minibinders induce NF-κB signaling in TLR3-expressing cell lines, demonstrating that they may have therapeutically relevant biological activity. Our work provides a foundation for the development of specific, stable, and easy-to-formulate protein-based agonists of TLRs and other pattern recognition receptors.

Fig. 1. Computational design of TLR3 minibinders.

a Left, TLR3 is natively dimerized and activated by dsRNA (PDB ID: 7WV5). Right, two hydrophobic patches on the TLR3 monomer were targeted for minibinder design (PDB ID: 1ZIW). b Polyvaline scaffolds were used in the RifDock pipeline to design de novo miniprotein binders. Several structural metrics were used as filters to select 23,789 designs for experimental screening. c Binders were identified using yeast surface display. During Sort 5, binding was clearly observed at 150 nM receptor but approached levels of background signal at 100 nM.

Fig. 2. Biochemical characterization and affinity maturation of lead TLR3 minibinders.

a Left, Design models of minibinders 7 (yellow) and 8 (purple) in complex with TLR3 (gray). Right, the details of the predicted interface are shown. b Left, Size exclusion chromatograms of each minibinder on a Superdex 75 Increase 10/300 GL. Right, Affinity determination for each minibinder by BLI. The concentrations of hTLR3 used are listed. The black lines represent experimental data, and the colored lines represent fits. KD values are given. c Site saturation mutagenesis heat maps of interface residues. The originally designed amino acid at each position is provided at the bottom and in the white square. Red indicates affinity improvement and blue indicates affinity reduction. SC-50 is the midpoint concentration in the binding transition, a proxy for the KD, and is described in detail in ref. . d Bio-layer interferometry of affinity-matured minibinders 7.1 and 8.6. KD values are given. e CD of affinity-matured constructs at various temperatures. Solid line, 25 °C; dashed line, 95 °C; dotted line, 95 °C followed by 25 °C.

Fig. 3. Structural characterization of TLR3 minibinders.

a Two different views of the cryo-EM structures of human TLR3 in complex with minibinder 7.7 (left) or minibinder 8.6 (right). TLR3, glycans, minibinder 7.7, and minibinder 8.6 are in gray, brown, yellow, and purple, respectively. b Comparison of experimental and AlphaFold2-predicted structures of the minibinders in complex with TLR3. c–h Close-up views of key molecular interactions in TLR3/minibinder 7.7 (c–e) or TLR3/minibinder 8.6 (f–h). Each box is a close-up view of the same colored box in (a). Residues involved in the TLR3/minibinder interaction are displayed as sticks and labeled.

Fig. 4. Multimerization of minibinders leads to NF-κB activation.

a Multivalent minibinders were generated by fusing two or more tandem repeats of the minibinder together using 12, 16, or 40-residue (GlySer) linkers. Antiparallel 8.6 dimers were generated by fusing minibinder 8.6 to the C terminus of an antiparallel coiled-coil derived from myosin 10. b TLR3 is expressed on the cell surface and in the endosome of TLR3hi cells, which express an NF-κB-linked GFP reporter. GFP levels are measured with flow cytometry. The schematic was rendered using BioRender.com. c Left, Histograms showing GFP signal in stimulated TLR3hi cells. The identities and concentrations of the stimuli are provided on the right and left sides of each panel, respectively. Right, mean fluorescence intensity (MFI) values from assay duplicates.