Protein engineering offers unique solutions for protein scaffolding for a variety of uses. Tandem repeat proteins (TRPs) are small proteins with repeated sequences. TRPs are useful for protein engineering as they are compact, have high thermal stability, good solubility characteristics, and are relatively easy to design and express. Additionally, TRPs can be conjugated to various forms of functional cargo at defined positions. Circular TRPs (cTRPs) can be made from repeating motifs, allowing protein cargo to be attached at regular intervals around the structure. The Bradley, Stoddard, Riddell and Kaiser labs teamed up to further develop cTRPs with functional domains. The results of their engineering efforts were recently published in Nature Structural & Molecular Biology.
The authors computationally designed and expressed a cTRP with 24 tandem loop repeats, named cTRP24. Shaped like a donut, cTRP24 has an outer diameter of approximately 100 angstroms and inner diameter of approximately 60 angstroms. The authors modified their construct to express repeats of 3, 4, 6, or 12 loops to see if these smaller units could self-assemble into the full cTRP of 24 repeats. Only the 12-loop construct could assemble into a dimer of the full 24 looped structure, however dimerization was not complete. The authors sought to stabilize the interaction by modifying the protein termini to contain cysteines allowing disulfide bonds between multimers. Both the 12-loop and 6-loop constructs were stabilized by this modification and could self-assemble into dimers or tetramers, respectively. Additionally, the authors used a mammalian expression system to broaden their ability to test modifications to cTRP24 that might not be well expressed in bacterial systems.
Next, the authors wanted to test if cargo could be bound to cTRP24 and retain function. Numerous protein cargo types were tested: peptide-binding domains, protein ligation domains, fluorescent proteins, as well as single-chain MHC molecules and single-chain Fv or receptor domains targeting important immune modulators. Constructs containing peptide-binding or protein ligation domains were able to successfully bind their respective partners, allowing flexibility in loading many cargo types onto cTRP24. MHC tetramers are useful immunologic reagents used to identify and stain T cells specific for a certain peptide-MHC complex. The authors used their 6-loop construct to produce cTRP tetramers conjugated to four peptide-MHC complexes presenting a peptide derived from cytomegalovirus (CMV). This tetrameric cTRP-MHC complex stained CMV-specific T cells to a similar degree to traditional tetramer reagents. T cell stimulation and growth in vitro requires co-stimulation from its TCR and co-receptors like CD28. The authors engineered a 6-loop cTRP construct with a single-chain Fv domain capable of binding CD28. This construct self-assembled and when added to T cell culture could stimulate T cell growth to the same degree as free CD28 antibody, indicating its function.
This scaffolding system based on self-assembling cTRPs allows great flexibility for engineering small proteins with many different functional domains. Dr. Stoddard explained: “This work described a novel protein nanoparticle that can be used to organize and deliver a wide variety of functional molecular ‘cargo’ for applications ranging from cell manufacture to vaccine development to potential reagents for therapeutic applications. Because an increasing number of biological molecules for such purposes are composed of proteins (instead of small molecules) there is a pressing need for easily adaptable protein-based scaffolds for display and delivery of such molecules.” Their initial findings using T cell stimulatory cTRPs for T cell growth have already drawn great interest from industry and could improve efficiency and cost of manufacturing for T cell-based immunotherapies.
This study was supported by the National Institutes of Health and Fred Hutch.
UW/Fred Hutch Cancer Consortium members Stanley Riddell, Barry Stoddard, and Philip Bradley contributed to this work.
Colin E Correnti, Jazmine P Hallinan, Lindsey A Doyle, Raymond O Ruff, Carla A Jaeger-Ruckstuhl, Yuexin Xu, Betty W Shen, Amanda Qu, Caley Polkinghorn, Della J Friend, Ashok D Bandaranayake, Stanley R Riddell, Brett K Kaiser, Barry L Stoddard, Philip Bradley. 2020. Engineering and Functionalization of Large Circular Tandem Repeat Protein Nanoparticles. Nat Struct Mol Biol. Apr;27(4):342-350. doi: 10.1038/s41594-020-0397-5.