Computational design of non-porous, pH-responsive antibody nanoparticles

Abstract

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and important for targeted delivery of biologics. We describe the design of octahedral non-porous nanoparticles with the three symmetry axes (four-fold, three-fold, and two-fold) occupied by three distinct protein homooligomers: a de novo designed tetramer, an antibody of interest, and a designed trimer programmed to disassemble below a tunable pH transition point. The nanoparticles assemble cooperatively from independently purified components, and a cryo-EM density map reveals that the structure is very close to the computational design model. The designed nanoparticles can package a variety of molecular payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between to 5.9-6.7. To our knowledge, these are the first designed nanoparticles with more than two structural components and with finely tunable environmental sensitivity, and they provide new routes to antibody-directed targeted delivery.

Figure 1:. Design of symmetry-matched plugs to fill empty symmetry axes in protein nanoparticles.

A. 6 de novo tetramers (gray) and 12 dimeric Fc domains (purple) assemble into a porous octahedral O42 nanoparticle. The tetramers are aligned along the 4-fold symmetry axis and the Fc domains along the 2-fold symmetry axis. B. Combinations of helical repeat proteins were fused to each other and to the pH trimer subunit at regions of high backbone overlap between pairs of helices to generate fused trimer subunits large enough to fully occupy the void along the 3-fold axis in the original nanoparticle. C. These pH-dependent trimers were then docked into the nanoparticle by rotating and translating along its 3-fold axis. D. The resulting three-component nanoparticle has 8 new trimeric subunits (yellow) which occupy the three-fold symmetry axis of the octahedral architecture.

Figure 2:. Mixing independently purified components enables stable, efficient assembly with both Fc and IgG.

A. Design models with Fc or IgG (purple), designed nanoparticle-forming tetramers (gray), and pH-dependent plug-forming trimers (yellow). B. Overlay of representative SEC traces of full assembly formed by mixing designed tetramers, trimers, and Fc or IgG (black) with those of the single components in gray (tetramer), yellow (trimeric plug), or purple (Fc or IgG). C. Representative DLS of fractions collected from the O432 assembly peak shows average hydrodynamic diameters of 34 nm (polydispersity index or PDI: 0.05) and 40 nm (PDI: 0.05) for the O432 assemblies with Fc and full-length IgG, respectively. D. Negatively stained electron micrographs with reference-free 2D class averages along each axis of symmetry in inset; electron microscopy images were collected prior to SEC purification. Scale bars, 100 nm and 10 nm for the micrograph and 2D averages, respectively.

Figure 3:. Cryo-EM analysis of 72-subunit nanoparticles composed of three distinct structural components.

A. The O432 assembly with Fc before SEC purification was characterized by cryogenic electron microscopy. Computational design models viewed along each axis of symmetry of the octahedral architecture are shown. B. Representative 2D class averages along each axis of symmetry. Scale bar, 10 nm. C. 7 Å 3D EM density map reconstructed from the collected dataset. D. Overlay of the design model (gray) and the design model relaxed into the 3D reconstruction (pink) showing high agreement. Encouraged by these structural results, we next set out to redesign the nanoparticles to have either highly positively or highly negatively charged interiors to enable packaging of molecular cargoes via electrostatic interactions ^,,. We focused our redesign on selected interior surface residues of the trimeric plug, favorably weighting mutations to amino acids with the desired charge (or no charge), and unfavorably weighting mutations to amino acids with the undesired charge. We screened for packaging of positively charged GFP (pos36GFP) by mixing negatively charged trimeric plug variants exhibiting different magnitudes of interior surface charge with tetramer, Fc, and pos36GFP (Fig 4A). One variant interacted with supercharged GFP in these conditions, as shown by co-elution of the 488 nm signal and the 280 nm signal during SEC (Fig 4B), and will be referred to subsequently as O432-17(−). Including 1 M NaCl in the buffers used during packaging and SEC prevented packaging (Fig 4C), suggesting that GFP packaging was largely driven by electrostatic interactions between the cargo and nanoparticle interior.

Figure 4:. Plugged antibody nanoparticles electrostatically package cargoes and disassemble in response to acidification.

A. Positively or negatively charged variants of the designed trimers and designed tetramers were either assembled with pos36GFP and human Fc (top) or RNA and α-EGFR mAb (bottom). B. SEC chromatograms of in vitro packaging reactions with O432-17(−) were performed in either 200 mM NaCl or C. 1 M NaCl. Absorbance was monitored at 280 nm (black) and 488 nm (green). D. O432-17(+) assembled in vitro with RNA was treated with Benzonase, electrophoresed on non-denaturing 0.8% agarose gels, and stained with SYBR gold (nucleic acid) and E. Coomassie (protein). F. pH titration experimental design. O432-17 nanoparticles were assembled with AF647-conjugated trimeric plug variants, designed tetramer, sfGFP-Fc, and α-Myc antibody. Nanoparticles were incubated with Myc peptide-coated beads and split into titrated pH buffers. Beads were washed in TBS pH 7.5 and the remaining trimer and sfGFP-Fc fluorescence remaining on the beads was analyzed by flow cytometry. G. AF647 and H. sfGFP fluorescence were normalized to the minimum and maximum values across the titration and analyzed as a function of pH for each nanoparticle variant.

Figure 5:. Targeted receptor-mediated uptake of O432 nanoparticles.

A. Targeted and non-targeted variants of the O432-17(−) nanoparticle were assembled in vitro with trimeric plug conjugated with AF647, designed tetramer, and either premixed mRuby2-Fc and α-EGFR mAb (top) or mRuby2-Fc alone (bottom). B. Cellular uptake of O432-17-CTX and O432-17-Fc nanoparticles was measured by the AF647 and mRuby2 fluorescence within the cell area, identified by lysosome membrane immunostaining using LAMP2A antibodies, after 3 hours of incubation in A431 cells. Images correspond to single confocal planes, and grayscale panels correspond to each channel of the composite image showing lysosomal membranes (green), mRuby2-Fc (red), and Trimer-AF647 (gray). Scale bar, 10 μm.