A nanoadjuvant that dynamically coordinates innate immune stimuli activation enhances cancer immunotherapy and reduces immune cell exhaustion

Although conventional innate immune stimuli contribute to immune activation, they induce exhausted immune cells, resulting in suboptimal cancer immunotherapy. Here we suggest a kinetically

activating nanoadjuvant (K-nanoadjuvant) that can dynamically integrate two waves of innate immune stimuli, resulting in effective antitumour immunity without immune cell exhaustion. The

combinatorial code of K-nanoadjuvant is optimized in terms of the order, duration and time window between spatiotemporally activating Toll-like receptor 7/8 agonist and other Toll-like

receptor agonists. K-nanoadjuvant induces effector/non-exhausted dendritic cells that programme the magnitude and persistence of interleukin-12 secretion, generate effector/non-exhausted

CD8+ T cells, and activate natural killer cells. Treatment with K-nanoadjuvant as a monotherapy or in combination therapy with anti-PD-L1 or liposomes (doxorubicin) results in strong

antitumour immunity in murine models, with minimal systemic toxicity, providing a strategy for synchronous and dynamic tailoring of innate immunity for enhanced cancer immunotherapy.

All data that support the findings of this study are provided within the paper and its Supplementary Information. The raw datasets generated during the study are provided within source data.

Source data are provided with this paper.

This work was supported by National Research Foundation (NRF) grants funded by the Korean government (grant numbers NRF-2020R1A2C3006888 and SRC-2017R1A5A1014560).

These authors contributed equally: Seung Mo Jin, Yeon Jeong Yoo.

SKKU Advanced Institute of Nanotechnology (SAINT), Department of Nano Science and Technology, Department of Nano Engineering, School of Chemical Engineering, and Biomedical Institute for

Convergence at SKKU, Sungkyunkwan University, Suwon, Republic of Korea

Seung Mo Jin, Yeon Jeong Yoo, Hong Sik Shin, Sohyun Kim, Sang Nam Lee, Chang Hoon Lee, Hyunji Kim, Jung-Eun Kim & Yong Taik Lim

Department of Biological Sciences, Science Research Center (SRC) for Immune Research on Non-lymphoid Organ (CIRNO), Department of Biological Science, Sungkyunkwan University, Suwon, Republic

of Korea

New Drug Development Center, Osong Medical Innovation Foundation, Cheongju, Republic of Korea

Y.T.L., S.M.J. and Y.J.Y. conceived and designed the experiments. S.M.J., Y.J.Y., H.S.S., S.K., S.N.L., C.H.L., H.K., J.-E.K., J.H. and Y.-W.N. performed the experiments. Y.T.L., S.M.J.,

Y.J.Y. and Y.-S.B. interpreted the data and developed the discussion. Y.T.L., S.M.J. and Y.J.Y. composed the manuscript. Y.T.L. supervised the entire project.

Nature Nanotechnology thanks Enrico Lugli and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

a-b. Schematic of (a) DC maturation and (b) sequential signal integration for IL-12(p70) production. c-e. IL-12(p70) production in BMDCs (c) after the treatment of indicated samples (IFN-γ,

50 ng ml−1; R848, 3.18 μM; t-TLR7/8a, 3.18 μM) for 24 h (n = 6), (d) at indicated times after stimulation with the indicated samples for 12 h and washing (n = 3), and (e) re-cultured for 36 

h with or without CD40L (10 μg ml−1) after stimulation with the indicated samples for 12 h and washing (n = 4). f. In vitro migratory capacity of BMDCs in the presence of CCL21 (100 ng ml−1)

and CXCL12 (100 ng ml−1) (n = 4). The concentration of IL-12(p70) and IL-10 after 36 h of incubation with indicated samples in BMDCs (n = 3). g. Comparison of migratory and IL-12(p70)

production capacities. h. Representative flow cytometry dot plots and the population of effector memory CD8+ T cells (CD44+CD62L−in CD3+CD8+) and IFN-γ-producing CD8+ T cells (IFN-γ+ in

CD3+CD8+) in splenocytes in vivo after 2 times vaccination of DCs pre-stimulated with indicated samples and OVA (100 μg ml−1) with 7 days interval (n = 4 for G3, n = 5 for G2, the rest n = 

6). i. Schedules and the average tumour growth curves for the prophylactic (n = 4 for G4, n = 5 for G2, the rest n = 6) and therapeutic (n = 5 for G1, the rest n = 4) DC vaccination. All

data are presented as the mean±s.d. Statistical significances in (d-e) were evaluated by two-way ANOVA with Sidak’s multiple-comparisons test. Statistical significances in (c, f and h) were

evaluated by one-way ANOVA with Tukey’s multiple-comparison test and in (i) were evaluated by unpaired two-tailed t-test (P values: n.s. not significant, * P