DNA origami–based artificial antigen-presenting cells for adoptive T cell therapy

Nanosized artificial antigen-presenting cells (aAPCs) with efficient signal presentation hold great promise for in vivo adoptive cell therapy. Here, we used DNA origami nanostructures as two-dimensional scaffolds to regulate the spatial presentation of activating ligands at nanoscale to construct high-effective aAPCs. The DNA origami–based aAPC comprises costimulatory ligands anti-CD28 antibody anchored at three vertices and T cell receptor (TCR) ligands peptide–major histocompatibility complex (pMHC) anchored at three edges with varying density. The DNA origami scaffold enables quantitative analysis of ligand-receptor interactions in T cell activation at the single-particle, single-molecule resolution. The pMHC-TCR–binding dwell time is increased from 9.9 to 12.1 s with increasing pMHC density, driving functional T cell responses. In addition, both in vitro and in vivo assays demonstrate that the optimized DNA origami–based aAPCs show effective tumor growth inhibiting capability in adoptive immunotherapy. These results provide important insights into the rational design of molecular vaccines for cancer immunotherapy.


Figures and Tables
Table S1.The sequences of oligonucleotides for the triangular DNA origami used in this work

Name
The sequences (from 5 to 3)

Fig. S1 .
Fig. S1.Synthesis and characterization of biotinylated DNA origamis.Scheme and AFM images of biotinylated DNA origamis and their corresponding cross-section height analysis results.Biotin (SA binding site) was represented as the green dot.Scale bar: 50 nm.

Fig. S2 .
Fig. S2.Synthesis and characterization of SA-modified DNA origamis.Scheme and AFM images of SA-modified DNA origamis and their corresponding cross-section height analysis results.SA was represented as the gray dot.Scale bar: 50 nm.

Fig. S4 .
Fig. S4.Synthesis and characterization of DNA origami-based aAPCs.Scheme and AFM images of four types of DNA origami-based aAPCs, including aAPC-T3, aAPC-T6, aAPC-T9, aAPC-T12, and their corresponding cross-section height analysis results.pMHC was represented as the red dot.aCD28 was represented as the blue dot.Scale bar: 50 nm.

Fig. S7 .
Fig. S7.Stability analysis of DNA origami-based aAPCs in PBS.The long-term stability analysis of DNA origami-based aAPCs in PBS for different times.(M: M13mp18 scaffold)

Fig. S10 .
Fig. S10.Ca 2+ signaling analysis after stimulation.Ca 2+ signaling measurements by fluorescence spectrometry and fluorescence microscopy after stimulation of T cells with aAPC-T12 and PBS for 1 h.

Fig. S13 .
Fig. S13.Synthesis and performance of cross-based aAPC.(a) Scheme and AFM image of DNA origami-based aAPC assembled by a cross DNA origami platform (denoted as cross-based aAPC, aAPC-C12).The ligands of pMHC and aCD28 were on the same side of the cross origami, and the copy numbers of pMHC and aCD28 of crossbased aAPC were the same as those of aAPC-T12 and the inter-pMHC spacing at each edge of the cross origami was 20 nm.Scale bar: 50 nm.(b) Flow cytometry analysis of CD69 expression of T cells after stimulation by aAPC-C12 and aAPC-T12.Data are shown as means ± s.d.(n = 3)

Fig. S17 .
Fig. S17.Size analysis of DNA origami-based aAPCs-TCR complexes clustering.Representative TIRF images for DNA origami-based aAPCs-TCR complexes clustering and size distributions of aAPCs-TCR complexes clusters at the T-cell-SLB interface.Images were recorded 30 min after T cell seeding.Data of size distributions of aAPCs-TCR complexes clusters are from 100 to 150 regions from 30 to 50 cells per condition.Data are from three independent experiments.Scale bar: 2 m.i: aAPC-T3,ii: aAPC-T6, iii: aAPC-T9, iv: aAPC-T12.

Table S2 .
The sequences of oligonucleotides for the cross DNA origami used in this work