Robust anti-tumor T cell response with efficient intratumoral infiltration by nanodisc cancer immunotherapy

Abstract

Potent anti-tumor T cell response and efficient intratumoral T cell infiltration are the major challenges for therapeutic cancer vaccines. To address these issues, a nano-vaccine system has been designed to promote anti-tumor T cell responses, and intratumoral infiltration was examined in various murine tumor models. Subcutaneous vaccination with nanodiscs carrying human papillomavirus (HPV)-16 E7 antigen elicits as high as ~32% E7-specific CD8 α + T cell responses in circulation, representing a 29-fold improvement over the soluble peptide vaccination. Importantly, nanodisc vaccination also promotes robust intratumoral T cell infiltration and eliminates HPV16 E6/E7-expressing TC-1 tumors at mucosal sites, including lungs, inner lip, and intravaginal tissues. In a benchmark study with a live Listeria vaccine combined with anti-PD-1 IgG, nanodiscs plus anti-PD-1 immune checkpoint blockade elicits comparable levels of T cell responses with anti-tumor efficacy. Furthermore, compared with Complete Freund's Adjuvant combined with tetanus toxoid, nanodisc vaccination in HLA-A02 mice generates >200-fold stronger IFN-γ+ T cell responses against a neoantigen from an HLA-A02 melanoma patient. Overall, these results show that the nanodisc system is a promising cancer vaccine platform for inducing anti-tumor T cell responses.

Keywords: cancer vaccine; nanoparticles; neoantigen; papillomavirus.

Figure 1.

Schematic illustration of nanodisc vaccination and immune monitoring in HPV16 mucosal tumor models.

Figure 2.. Subcutaneous nanodisc vaccination induced effective cancer antigen-specific T cell response via efficient lymph node draining.

A-B) C57BL/6 mice were vaccinated on days 0 and 14 with 20 μg E7 peptide and 10 μg CpG in the indicated formulations. Vaccines were given via either the subcutaneous (s.c.) route at tail base or intranasal (i.n.) route. On day 21, the frequency of E7-specific CD8+ T cells among PBMCs was measured by the tetramer staining assay. Shown are A) the representative flow cytometry scatter plots and B) the average values. C) Serial PET images of C57/BL mice at various time points post-injection of ⁶⁴Cu-NOTA-E7 or ⁶⁴Cu-NOTA-nanodisc-E7. D-E) Time−radioactivity curves of Injection site, axillary LNs, inguinal LNs, intestine, liver, blood, and muscle after s.c. injection. F) Biodistribution of ⁶⁴Cu-NOTA-E7 and ⁶⁴Cu-NOTA-nanodisc-E7 at 46 h post-injection. Data are presented as mean ± s.e.m. from a representative experiment from 2 independent experiments (n = 4–5). *p < 0.05, **p < 0.01, ****p < 0.0001 analyzed by (B) one-way ANOVA or (F) two-way ANOVA, with Tukey’s HSD multiple comparison post hoc test.

Figure 3.. Nanodisc vaccination in the TC-1 lung metastasis model.

A-F) To establish a lung metastasis model, C57BL/6 mice were inoculated intravenously with 1×10⁵ TC1-luc cells on day 0. On days 10 and 16, animals were vaccinated with 20 μg E7 peptide and 10 μg CpG formulated as a soluble vaccine or sHDL vaccine. Vaccines were given via either s.c. at the tail base or intranasal (i.n.) route. A) Tumor burden was monitored over time using in vivo whole animal imaging (IVIS). B) Animal survival was measured over 60 days. C-F) Three days after the second vaccination, the frequency of E7-specific CD8a+ T cells was measured among C-D) PBMCs or E-F) lung tissues by the tetramer staining assay. Shown are C, E) the representative flow cytometry scatter plots and D, F) the average values of E7-tetramer+ CD8⍺+ T-cells. Data are presented as mean ± s.e.m. from a representative experiment from 2 independent experiments (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 analyzed by (D, F) one-way ANOVA with Tukey’s HSD multiple comparison post hoc test or by (B) log-rank (Mantel−Cox) test.

Figure 4.. The therapeutic effect of nanodisc vaccination in TC-1 head and neck cancer model.

To establish a head and neck model, C57BL/6 mice were inoculated with 50,000 TC1-luc cells in the inner lip on day 0. On days 6 and 12, animals were vaccinated with 20 μg E7 peptide and 10 μg CpG formulated as a soluble vaccine or sHDL vaccine. The route of vaccination was either s.c. at the tail base or intranasal (i.n.) vaccination as indicated. A) Tumor burden was monitored over time using in vivo whole animal imaging (IVIS). B) Animal survival was measured over 60 days. C-F) Three days after the second vaccination, the frequency of E7-specific CD8⍺+ T cells was measured among C-D) PBMCs or E-F) tumor tissues by the tetramer assay. Shown are C, E) the representative flow cytometry scatter plots and D, F) the average values of E7-tetramer+ CD8⍺+ T-cells. Data are presented as mean ± s.e.m. from a representative experiment from 2 independent experiments (n = 5). *p < 0.05, ****p < 0.0001 analyzed by (D, F) one-way ANOVA with Tukey’s HSD multiple comparison post hoc test or by (B) log-rank (Mantel−Cox) test.

Figure 5.. The therapeutic effect of nanodisc vaccination in TC-1 cervical cancer model.

To establish an HPV-associated cervical cancer model, C57BL/6 mice were inoculated with 4×10⁴ TC1-luc cells in the vaginal tract on day 0. On days 6 and 12, animals were vaccinated with 20 μg E7 peptide and 10 μg CpG formulated as a soluble vaccine or sHDL vaccine. The route of vaccination was either s.c. at the tail base or intranasal (i.n.) vaccination as indicated. A) Tumor burden was monitored over time using in vivo whole animal imaging (IVIS). B) Animal survival was measured over 60 days. C-F) Three days after the second vaccination, the frequency of E7-specific CD8a+ T cells was measured among C-D) PBMCs or E-F) tumor tissues by the tetramer staining assay. Shown are C, E) the representative flow cytometry scatter plots and D, F) the average values of E7-tetramer+ CD8a+ T-cells. Data are presented as mean ± s.e.m. from a representative experiment from 2 independent experiments (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 analyzed by (D, F) one-way ANOVA with Tukey’s HSD multiple comparison post hoc test or by (B) log-rank (Mantel−Cox) test.

Figure 6.. CD8+ T cell responses induced by nanodiscs and Listeria vaccine.

A) C57BL/6 mice were vaccinated on days 0 and 30 with 10⁷/100 μl/mouse Listeria-Gp33 (i.v. route) or sHDL nanodiscs carrying Gp33 and CpG (s.c. route). On days 7 and 35, splenocytes were re-stimulated with Gp33 for intracellular cytokine staining (ICS) for IFN-γ and TNF-⍺. B) C57BL/6 mice were vaccinated on days 0 and 30 with 10⁷/100 μl/mouse Listeria-Adpgk (i.v. route) or sHDL nanodiscs carrying Adpgk and CpG (s.c. route). Anti-PD-1 IgG was given i.p. on days 4 and 34. On days 7 and 35, splenocytes were re-stimulated with Adpgk for ICS. Data are presented as mean ± s.e.m. from a representative experiment from 2 independent experiments (n = 7). *p < 0.05, **p < 0.01, analyzed by one-way ANOVA with Tukey’s HSD multiple comparison post hoc test.

Figure 7.. Comparison of therapeutic effect and T cell infiltration in TME after Nanodiscs and Listeria vaccine.

A) C57BL/6 mice were inoculated at s.c. flank with MC38 cancer cells on day 0. On days 10 and 17, animals were vaccinated with 10⁷/100 μl/mouse Listeria-Adpgk intravenously (i.v.) or nanodisc-Adpgk/CpG (s.c.). Anti-PD-1 IgG was injected i.p. on days 11, 14, 18, and 21. For antitumor immune evaluation, the spleens were harvested on day 28 and processed for peptide stimulation and intracellular cytokine staining (ICS). B) Tumor growth was monitored. C) Adpgk-specific CD8a+ T cells were quantified by the tetramer staining among PBMCs, spleen, and tumor tissues. D) PBMCs, spleen, and tumor tissues were isolated and re-stimulated with Adpgk neoepitope, followed by intracellular cytokine staining. Data are presented as mean ± s.e.m. n = 10 for B) and n = 4–7 for C-D).

Figure 8.

A) HLA-A02 transgenic mice were vaccinated on days 0 and 14 with 10 μg/dose of neoantigen peptide from a HLA-A02 melanoma patient in Complete Freund's Adjuvant (CFA) containing 2 μg/dose of tetanus toxoid. On day 28, the animals were boosted with either the same CFA +TT formulation or nanodiscs containing 15 μg/dose of CpG. B) On day 35, antigen-specific T cell responses were evaluated by ELISPOT after restimulating splenocytes with 0.1, 1, or 10 μg/mL of the antigen peptide. C) HLA-A02 transgenic mice were vaccinated on days 0 and 21 with nanodiscs containing 15 μg/dose of CpG and either neoantigen peptide from an HLA-A02 melanoma patient (Mel-Ag) or HLA-A02-restricted flu antigen peptide, M1_58–66. D-E) On day 28, PBMCs were analyzed for antigen-specific, IFN-γ+ T-cell responses by intracellular cytokine staining after ex vivo restimulation with 10 μg/mL of each peptide. Data are presented as mean ± s.e.m. from a representative experiment from 2 independent experiments (n = 3). ****p < 0.0001 analyzed by (b) two-way ANOVA with Tukey’s HSD multiple comparison post hoc test.