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 May 1, 2018
Citation Information: J Clin Invest. 2018;128(5):1971-1984. https://doi.org/10.1172/JCI96791.
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Categories: Research Article Immunology Oncology

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 1

OVA-Clec9A-TNE targets cross-presenting DCs and promotes antigen-specific T cell responses.

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OVA-Clec9A-TNE targets cross-presenting DCs and promotes antigen-specifi...
(A) Images of C57BL/6 mice 1 day after i.v. injection of DiR-labeled Clec9A-TNE, isotype-TNE, or free DiR solution as indicated. (B and C) Images of C57BL/6 mice 7 days after i.v. injection of DiR-labeled Clec9A-TNE (B) or DiR-labeled isotype-TNE (C). p/s/cm/sr/μW/cm2, radiant efficiency, total photons per second per square centimeter per steradian in the irradiance range (microwatts per square centimeter); p, photons; sr, steradian. (D) Binding/uptake of TNE by CD8+ cDCs, CD8– cDCs, and pDCs in C57BL/6 mouse spleen 1 day after i.v. injection (n = 3). (E) CD11c+ DCs sorted from naive C57BL/6 mice were incubated with DiI-labeled Clec9A-TNE or isotype-TNE (red) for 3 hours. Cells were then washed and stained with anti-EEA1 or anti-LAMP1 (green) and DAPI (nuclei, blue). (F and G) Proliferation of CD8+ OT-I (F) and CD4+ OT-II (G) cells in inguinal lymph nodes (LN) and spleen 6 days after i.v. injection of 5 μg of soluble OVA, or 200 μl of Clec9A-TNE, OVA-Clec9A-TNE, or OVA-isotype-TNE (formulated with 5 μg of OVA). (H) OT-I T cell proliferation in spleens of mice 5 days after i.v. injection of OVA-Clec9A-TNE, OVA-isotype-TNE (each formulated with 200 ng of OVA), or 1 μg of OVA anti-clec9A^(10B4)-OVA or isotype^(GL117)-OVA fusion protein conjugates without or with CpG (n = 4 from 2 individual experiments). Serum IFN-α level at 2, 4, and 24 hours after vaccination was measured by ELISA. (I) Percent in vivo OVA-specific killing 5 days after i.v. injection with OVA-Clec9A-TNE or OVA-isotype-TNE (both formulated with 5 μg of OVA), Clec9A-TNE, or 5 μg soluble OVA (n = 10–14 from 3 individual experiments). (J) C57BL/6 mice were injected i.v. with 5 μg of Clec9A-OVA, OVA-Clec9A-TNE, isotype-OVA, or OVA-isotype-TNE without additional adjuvant. Serum anti-OVA Ig titer was quantified 1, 2, and 3 weeks later by ELISA (n = 10 from 2 separate experiments). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by Tukey’s multiple-comparisons test.