Engineering and functionalization of large circular tandem repeat protein nanoparticles

Abstract

Protein engineering has enabled the design of molecular scaffolds that display a wide variety of sizes, shapes, symmetries and subunit compositions. Symmetric protein-based nanoparticles that display multiple protein domains can exhibit enhanced functional properties due to increased avidity and improved solution behavior and stability. Here we describe the creation and characterization of a computationally designed circular tandem repeat protein (cTRP) composed of 24 identical repeated motifs, which can display a variety of functional protein domains (cargo) at defined positions around its periphery. We demonstrate that cTRP nanoparticles can self-assemble from smaller individual subunits, can be produced from prokaryotic and human expression platforms, can employ a variety of cargo attachment strategies and can be used for applications (such as T-cell culture and expansion) requiring high-avidity molecular interactions on the cell surface.

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Fig. 1: Properties of an engineered 24-repeat circular tandem repeat protein.
Fig. 2: Assembly of cTRP24x nanoparticles from smaller protein subunits, verified by SEC analyses.
Fig. 3: Computational models and characterization of functionalized 24-repeat cTRP constructs.
Fig. 4: Characterization of an scMHC tetramer expressed and purified from human 293 cells.
Fig. 5: Functional characterization of a cTRP246SS construct harboring an N-terminal single-chain Fv (scFv) specific for the T-cell costimulatory receptor CD28.
Fig. 6: Functional characterization of cTRP246SS constructs harboring single-chain trimers of tumor necrosis factor receptor ligands targeting T-cell costimulatory receptors 4-1BB, OX40 and CD27.

Data availability

The identities and sequences of all constructs used in this study are provided in Supplementary Table 1. All other data described in the article are provided in the main article and Extended Data figures. Source data for Figs. 3d and 5b are available with the paper online.

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Acknowledgements

A. Towlerton and E. H. Warren provided materials, advice and facilitation of scMHC tetramer-based flow cytometric experiments. R. Strong provided advice and facilitation of SPR (Biacore) binding experiments. J. Carter provided operations support for mammalian protein production and J. M. Olson provided laboratory support for protein expression. This work was funded by the NIH (grant no. R01 GM123378) and by the Fred Hutchinson Cancer Research Center.

Author information

Affiliations

Authors

Contributions

P.B. conducted the computational protein design work, including fold design and identification of point mutations leading to alteration of self-assembly properties, and generated the structural models used throughout the article. J.H., L.A.D., A.Q., C.P., B.K.K., B.L.S. and R.O.R. all conducted protein expression, purification and biochemical characterization experiments. B.W.S. conducted EM visualization studies. D.J.F. conducted SPR protein-binding studies. Y.X., C.A.J.-R., A.D.B. and S.R.R. designed and conducted T-cell staining studies. C.E.C. and B.L.S. designed functionalized cTRP constructs. C.E.C., B.K.K., B.L.S. and P.B. wrote the manuscript, which was edited extensively by all authors.

Corresponding authors

Correspondence to Brett K. Kaiser or Barry L. Stoddard or Philip Bradley.

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Competing interests

C.E.C., P.B., S.R.R. and B.L.S. are employees of the Fred Hutchinson Cancer Research Center; they are named inventors on intellectual property corresponding to the technology in this article. S.R.R. is a founder of Lyell Immunopharma Inc., which has recently licensed the technology for use in T-cell culture applications.

Additional information

Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Characterization of engineered cTRP24.

a, Circular dichroism (CD) spectra of cTRP24 at 22° and 95 °C shows preservation of secondary structure at high temperature. b, Small angle x-ray scattering (SAXS) spectra measured for cTRP24 (left), calculated SAXS spectrum derived from the atomic model of the designed protein construct (middle) and a superposition of the experimental and calculated spectra (right).

Extended Data Fig. 2 Design and electrophoretic gel visualization of self-assembling, disulfide-stapled cTRP24 nanoparticles.

a, The cTRP2412SS construct, assembled from dimerization of two identical protein subunits each harboring 12 repeats, with the N- and C-terminal repeat of each containing a cysteine residue (described in the main text and in Fig. 2) that enable disulfide stapling. SEC analyses (shown in Fig. 2c) and electrophoretic analyses (right panel) both indicate formation of a dimer that contains a mixture of one or two disulfide staples, both of which behave in solution similarly to a monomeric, single chain 24-repeat cTRP. b, A cTRP246SS construct, assembled from four identical protein subunits each harboring 6 repeats, with the N- and C-terminal repeat of each containing a cysteine residue (described in the main text and in Fig. 2) that enable disulfide stapling. SEC analyses indicate that expression and purification yield a cTRP24 that behaves in solution in a similar manner to a monomeric, single chain 24-repeat cTRP. Electrophoretic analyses of the same construct generated either via cytosolic expression in E. coli (and then oxidized in the presence of air during purification) or via secretion from human HEK cells (oxidized as part of eukaryotic disulfide bond formation mechanism during secretion) indicate that disulfides are formed in both cases. However, expression from bacteria generates a mixture of species (harboring 2, 3 or 4 disulfides), whereas expression and secretion from human cells generated a more homogeneous population of species primarily consisting of a full complement of disulfide bonds.

Extended Data Fig. 3 Functional characterization of additional cTRP constructs.

a, A 24-repeat cTRP harboring four copies of the SpyCatcher protein domain (cTRP2412SS-Spy) is fully conjugated with four copies of SpyTagged Clover (a derivative of GFP). In this reducing SDS-PAGE gel two bands are observed as a result of addition of SpyCatcher, corresponding to capture of 1 or 2 copies of SpyTagged Clover by each 12-repeat protein subunit. b, Fluorescence of free Clover and cTRP2412SS-Spy-Clover nanoparticles, as a function of normalized Clover concentrations. Data shown as mean and s.d. for n=3 independent experiments. c, Four copies an engineered fluorescence activating protein (‘mFAP’) are inserted into four evenly distributed surface loops around the bottom face of the cTRP (‘cTRP246SS-mFAP’). d, Purification and fluorescence activity of cTRP246SS-mFAP. mFAP has previously been demonstrated to fluoresce in the presence of exogenous, bound DHFBI fluorophore. The two curves that demonstrate increasing fluorescence as a function of protein concentration correspond to the cTRP246SS-mFAP (blue) nanoparticle and to free mFAP (red); as in panel b the protein concentrations are normalized relative to the one-versus-four copies of mFAP per molecule. The three curves that do not increase in fluorescence as a function of protein concentration correspond to the ‘naked’ cTRP (cTRP246SS), cTRP246SS plus DHFBI, and DHFBI alone. For the latter two constructs, the DHFBI concentration is equivalent at each protein concentration to that which is present in the active constructs. Data shown as mean and s.d. for n=3 independent experiments.

Extended Data Fig. 4 Detection of CMV pp65-reactive CD8+ T-cells diluted into donor PBMCs using cTRP246SS-scMHC and streptavidin-allophycocyanin (APC) tetramers.

a, Forward versus side scatter plot showing an overlay of CMV pp65-reactive T-cells (red contour plot) and donor PBMCs (blue contour plot) and the lymphocyte gating strategy used for further analysis. This strategy was used to quantitate CMV pp65-reactive T-cells diluted into donor PBMCs as shown in panels c and d. b, Scatter plot showing an overlay of the DAPI-negative lymphocyte gates stained with an anti-His-APC secondary antibody (Biolegend #362605) confirms that the secondary antibody used to detect cTRP246SS-scMHC does not result in unwanted background staining of live cells. c, Representative flow cytometry scatter plot showing quantitation of CMV pp65-reactive T-cells diluted into donor PBMCs at a ratio of 1:4 respectively. d, Quantitation of CMV pp65-reactive T-cells at various dilutions using both the cTRP246SS-scMHC (detected using the anti-His-APC secondary) and streptavidin-APC scMHC tetramer (SA-Tetramer). Note that the ratios in the first column (CMV:PBMC) represent raw cell counts prior to staining.

Extended Data Fig. 5 Expression and preliminary characterization of a hexameric cTRP24 harboring N-terminal cargo and a tetrameric cTRP24 harboring both N- and C-terminal cargos.

a, Schematic showing the architecture of the cTRP244SS-scMHC and corresponding non-reducing and reducing SDS-PAGE following affinity purification. Under reducing conditions, the protein migrates as a monomer. b, Schematic showing the architecture of the cTRP246SS-scMHC-mFAP protein displaying four copies of a scMHC at each N-terminus and four copies of mFAP at each C-terminus, both decorating top of the cTRP scaffold. SEC and corresponding SDS-PAGE analysis confirms proper assembly of a functional tetramer.

Extended Data Fig. 6 Staining of 4-1BB receptor on activated CD8+ T-cells using cTRP246SS-scTrimer4-1BBL.

Flow cytometry histogram showing staining of activated CD8+ T-cells (4 days post stimulation with anti-CD3/anti-CD28 beads) with cTRP246SS-scTrimer4-1BBL using a secondary anti-His-iFluor647 antibody or unstained T cells (left) and a PE-labeled anti-4-1BB mAb (right).

Supplementary information

Source data

Source Data Fig. 3

Statistical source data (Excel format) for Fig. 3d.

Source Data Fig. 5

Statistical source data (Excel format) for Fig. 5b.

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Correnti, C.E., Hallinan, J.P., Doyle, L.A. et al. Engineering and functionalization of large circular tandem repeat protein nanoparticles. Nat Struct Mol Biol 27, 342–350 (2020). https://doi.org/10.1038/s41594-020-0397-5

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