Enhancing Efficacy of Anticancer Vaccines by Targeted Delivery to Tumor-Draining Lymph Nodes

Nanoparticle conjugation enhances cell targeting and therapeutic efficacy of OVA + CpG cancer vaccines. Responses of B16-F10 (A–D) and E.G7-OVA (E–K) tumor-bearing mice to therapeutic vaccination are shown. Vaccination schedule (A), B16-F10 tumor volumes (B), overall survival rates (C), and circulating TRP-2_180–188–specific CD8⁺ T cells (D) 11 days after tumor inoculation (as determined by TRP-2_180–188–MHCI pentamer staining). Vaccination schedule (E), E.G7-OVA tumor volumes (F), percentage of mice with tumor shrinkage after vaccination (G), overall survival rates (H), and circulating OVA_257–264–specific CD8⁺ T cells (I) on days 11 and 18. J and K, on day 7 after tumor inoculation, E.G7-OVA tumor-bearing mice were injected intradermally with Alexa Fluor 488–labeled OVA and DY633-labeled CpG, either free or nanoparticle conjugated, in the front footpad either ipsi or contra to the tumor, and brachial LNs were analyzed 24 hours later. Nanoparticle conjugation leads to better targeting of OVA and CpG by mature macrophages (MΦ, MHCII⁺ CD11b⁺) and DCs (MHCII⁺ CD11c⁺) in the tdLN after ipsi injection (J) and in the non-tdLN after contra injection (K). Data reflect two independent experiments with 8 mice per group. *, P < 0.05; **, P < 0.01.

The tdLN is a more effective vaccine target site than the non-tdLN for a tumor antigen, but a less effective site for a nontumor antigen. Responses of EL-4 and E.G7-OVA tumor-bearing mice to therapeutic vaccination ipsi or contra to the tumor. A, vaccination schedule. B, day 11 tumor volumes, and C, circulating OVA_257–264–specific CD8⁺ T cells 11 days after tumor inoculation in EL-4 versus E.G7-OVA tumor-bearing mice. Data reflect two independent experiments with 5 to 10 mice per group. **, P < 0.01.

Therapeutic vaccines targeting the tdLN are more effective than those targeting the non-tdLN, but only after tumor growth has begun. Response of E.G7-OVA tumor-bearing mice to therapeutic vaccination with 10 μg NP-OVA + 1 μg NP-CpG ipsi or contra to the tumor 4 days (A), 7 days (B), or 11 days (C) after tumor inoculation: tumor volumes (top), survival (middle), and proportions of circulating OVA_257–264–specific CD8⁺ T cells (bottom) 7, 14, and 21 days after vaccination of immunized and control mice (n.d., no data; statistics, ipsi vs. contra). A, at 4 days after inoculation, before tumors are visible, there is very little difference between vaccinating the tdLN versus the non-tdLN in terms of tumor response. B, in contrast, when vaccinated after 7 days, when tumors are visible and have presumably affected the tdLN, large differences are seen between contra versus ipsi vaccination, and only the ipsi-vaccinated group survives with no tumor regrowth. C, when mice are vaccinated at day 11 of tumor growth, which is close to the size limit before death or sacrifice is necessary, only the tdLN-targeted group can be rescued, while the non–tdLN-targeted group show no response to the vaccine. Response of B16-F10 melanoma-bearing mice after vaccination with 10 μg NP-TRP-2_180–188 + 1 μg NP-CpG ipsi or contra to the tumor: tumor volumes (D), survival (E), and proportions (F) of circulating TRP-2_180–188–specific CD8⁺ T cells 11 days after tumor inoculation of immunized and control B16-F10 tumor-bearing mice. Data reflect several independent experiments with 5 to 10 mice per group. *, P < 0.05; **, P < 0.01; n.s., not significant P > 0.05.

Targeting a nanoparticle vaccine to the tdLN enhances the effector CD8⁺ T-cell response locally and systemically. CD8⁺ T-cell responses 7 days after vaccination in E.G7-OVA tumor-bearing mice immunized with 10 μg NP-OVA + 1 μg NP-CpG ipsi or contra to the tumor. A, vaccination schedule. B, representative flow cytometry plots of SIINFEKL–MHCI pentamer staining of tumor-infiltrating CTLs (CD44⁺ CD62L⁻ effector CD8⁺ T cells). C, proportion of OVA_257–264–specific CTLs (CD44⁺ CD62L⁻ effector CD8⁺ T cells) in the tumor, spleen, tdLN, and non-tdLN as determined by SIINFEKL–MHCI pentamer staining. D, tumor-infiltrating OVA_257–264–specific CD8⁺ T cells with an effector (CD44⁺ CD62L⁻) versus an exhausted (PD-1⁺) phenotype. E–H, cells from spleens and LNs were restimulated ex vivo with SIINFEKL (1 μg/mL) for 6 hours before staining for intracellular cytokines and analysis by flow cytometry. E, representative flow cytometry plots of spleen CD8⁺ T cells stained for IFN-γ⁺ and TNF-α⁺. Values in the dot plots represent the percentage of CD8⁺ T cells in each gate. Proportion of IFN-γ⁺, IFN-γ⁺ TNF-α⁺, and IFN-γ⁺ TNF-α⁺ IL-2⁺ cytotoxic CD8⁺ T cells in the spleen (F), tdLN (G), and non-tdLN (H) of ipsi, contra, and control mice after SIINFEKL restimulation. Data reflect two independent experiments with 8 mice per group. *, P < 0.05; **, P < 0.01.