Application Of Ligand Complexes Based On DNA Origami In T Cell Detection And Activation

Antigen-specific T cells play a key role in the recognition and killing of tumor cells in adaptive immunity.Detection and activation of T cells allows monitoring of disease progression and the development of T cell-mediated adoptive immunotherapy.However,the ligand complexes currently used for T cell detection and activation cannot achieve the precise and controllable spatial arrangement of ligand molecules,limiting their interaction with T cells.Therefore,it remains challenging to analysis of low-affinity T cells and efficient in vivo adoptive immunotherapy.DNA origami has proven its utility in the precise organization of ligands on the nanoscale,owing to its high programmability and precise addressability.Based on this,we used the DNA origamis as scaffolds to develop highly precise monomer-ligand complexes（p MHC multimers）and dual-ligand complexes（artificial antigen-presenting cells）,enabling precise control over the stoichiometry,spacing,orientation,species of ligands.Due to the precise organization of ligands on DNA origami,the legends complexes can enhance their interaction with T cells and realize highly sensitive detection and efficient activation of T cells,which accelerates the development of T cell-mediated personalized immunotherapy.1.In Chapter 2,fifteen precise and controllable single-ligand complex p MHC multimers were synthesized using DNA origamis as scaffolds.The binding affinity and binding stability of those p MHC multimers to antigen-specific T cells were regulated by changing the p MHC valency or p MHC spacing,with apparent dissociation constants ranging from 1.8 to 86.9 n M and dissociation rates ranging from 2.55×10^-4 to 0.56×10^-4 s^-1.Among them,p MHC multimers with high p MHC valency（9-54 copies）and p MHC short spacing（20 nm）have higher binding affinity and slower dissociation kinetics than tetramer and dextramer.2.In Chapter 3,dorimers with high p MHC valency（9-54 copies）and p MHC short spacing（20 nm）were used to detect different types of antigen-specific T cells,including high-frequency antigen-specific T cells,human CMV-specific T cells,T cells with low TCR density,T cells with low-affinity TCR.By increasing the p MHC valency,dorimer can improve the sensitivity of detecting antigen-specific T cells,up to 99%for high frequency count antigenic T cells and up to 90%for low TCR density T cells.Compared with tetramer’s tetrahedral protein scaffold and dextramer’s flexible dextran chain scaffold,dorimer based on DNA origami has higher detection sensitivity,stronger signal intensity and lower background signal.Notably,Dorimer can detect low-affinity T cells that are difficult to detect with the commercial product tetramer,revealing antigen-specific populations that other technologies cannot.3.In Chapter 4,the DNA origami-based dual-ligand complex（artificial antigen-presenting cell）was developed to precisely control the spatial arrangement of two ligands at the nanoscale,fine tuning T cell activation.Using TIRF technology to analyze ligand-receptor interactions during T cell activation at the single-molecule level.The p MHC-TCR binding dwell time increased from 9.9 s to 12.1 s with increasing p MHC density.In addition,both in vitro and in vivo assays demonstrate that the optimized DNA origami-based a APCs show effective tumor growth inhibiting capability in adoptive immunotherapy.DNA origami-based artificial antigen-presenting cells provide an effective strategy for T cell-mediated cellular immunotherapy.In summary,this thesis focuses on the design and construction of precise ligand complexes and proposes the use of DNA origamis as scaffolds to achieve precise control of the number,valency,orientation and species of ligand molecules,which provides a reference and guidance for the rational design of ligand complexes for highly sensitive detection and efficient activation of T cells,as well as a flexible platform for disease course monitoring and the development of new immunotherapies.