Self-adjuvanting nanoemulsion targeting dendritic cell receptor Clec9A enables antigen-specific immunotherapy
Bijun Zeng, … , Riccardo Dolcetti, Ranjeny Thomas
Bijun Zeng, … , Riccardo Dolcetti, Ranjeny Thomas
Published February 27, 2018
Citation Information: J Clin Invest. 2018;128(5):1971-1984. https://doi.org/10.1172/JCI96791.
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Self-adjuvanting nanoemulsion targeting dendritic cell receptor Clec9A enables antigen-specific immunotherapy

Abstract

Non–antigen-specific stimulatory cancer immunotherapies are commonly complicated by off-target effects. Antigen-specific immunotherapy, combining viral tumor antigen or personalized neoepitopes with immune targeting, offers a solution. However, the lack of flexible systems targeting tumor antigens to cross-presenting dendritic cells (DCs) limits clinical development. Although antigen–anti-Clec9A mAb conjugates target cross-presenting DCs, adjuvant must be codelivered for cytotoxic T lymphocyte (CTL) induction. We functionalized tailored nanoemulsions encapsulating tumor antigens to target Clec9A (Clec9A-TNE). Clec9A-TNE encapsulating OVA antigen targeted and activated cross-presenting DCs without additional adjuvant, promoting antigen-specific CD4+ and CD8+ T cell proliferation and CTL and antibody responses. OVA-Clec9A-TNE–induced DC activation required CD4 and CD8 epitopes, CD40, and IFN-α. Clec9A-TNE encapsulating HPV E6/E7 significantly suppressed HPV-associated tumor growth, while E6/E7–CpG did not. Clec9A-TNE loaded with pooled B16-F10 melanoma neoepitopes induced epitope-specific CD4+ and CD8+ T cell responses, permitting selection of immunogenic neoepitopes. Clec9A-TNE encapsulating 6 neoepitopes significantly suppressed B16-F10 melanoma growth in a CD4+ T cell–dependent manner. Thus, cross-presenting DCs targeted with antigen–Clec9A-TNE stimulate therapeutically effective tumor-specific immunity, dependent on T cell help.

Authors

Bijun Zeng, Anton P.J. Middelberg, Adrian Gemiarto, Kelli MacDonald, Alan G. Baxter, Meghna Talekar, Davide Moi, Kirsteen M. Tullett, Irina Caminschi, Mireille H. Lahoud, Roberta Mazzieri, Riccardo Dolcetti, Ranjeny Thomas

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Figure 3

OVA-Clec9A-TNE–mediated DC activation and IFN-α production require CD4+ and CD8+ T cell epitope presentation and CD40L-CD40 crosstalk.

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OVA-Clec9A-TNE–mediated DC activation and IFN-α production require CD4+ ...
(A) C57BL/6 or RAG1–/– mice were injected i.v. with OVA-Clec9A-TNE, SIINFEKL-Clec9A-TNE, OVA323–339-Clec9A-TNE, or SIINFEKL-OVA323–339-Clec9A-TNE. Six hours later, surface expression of CD86 by CD8+ DCs was analyzed by flow cytometry (n = 3–8 from 2 separate experiments). (B) C57BL/6 mice were injected i.v. with OVA-Clec9A-TNE, SIINFEKL-Clec9A-TNE, OVA323–339-Clec9A-TNE, SIINFEKL-OVA323–339-Clec9A-TNE, or OVA-isotype-TNE. Serum was collected 2, 4, and 24 hours after injection, and IFN-α levels were quantified by ELISA (n = 4). (C) C57BL/6 or CD40–/– mice were injected i.v. with OVA-Clec9A-TNE. Six hours later, surface expression of CD40, CD80, and CD86 by CD8+ DCs was analyzed by flow cytometry (n = 4). (D) C57BL/6 or CD40–/– mice were adoptively transferred with equal numbers of unpulsed CFSElo and SIINFEKL-pulsed CFSEhi target cells 6 days after i.v. injection with OVA-Clec9A-TNE. The percentage of SIINFEKL peptide–specific lysis in spleen is depicted (n = 4). (E) Splenic CD11c+ DCs were purified from C57BL/6 mice, then cultured for 5 hours with OT-I OVA-specific CD8+ and OT-II OVA-specific CD4+ T cells, in the presence or absence of OVA-Clec9A-TNE or OVA-isotype-TNE. Histograms depict intracellular IFN-α levels in gated DC populations. Splenic CD11c+ DCs were purified from C57BL/6 (WT), CD40–/–, or TLR9–/– mice, then cultured for 24 hours with OT-I OVA-specific CD8+ and OT-II OVA-specific CD4+ T cells, in the presence or absence of OVA-Clec9A-TNE or OVA-isotype-TNE. (F) Increase in CD40L expression by OT-II cells in the presence of OVA-Clec9A-TNE relative to no TNE control. (G) Increase in IFN-α secretion into cell culture supernatant in the presence of OVA-Clec9A-TNE relative to no TNE control. Representative data from 6 mice are shown. *P < 0.05; **P < 0.01; ****P < 0.0001 by Tukey’s multiple-comparisons test.