Self-Replicating RNAs Drive Protective Anti-tumor T Cell Responses to Neoantigen Vaccine Targets in a Combinatorial Approach

Abstract

Historically poor clinical results of tumor vaccines have been attributed to weakly immunogenic antigen targets, limited specificity, and vaccine platforms that fail to induce high-quality polyfunctional T cells, central to mediating cellular immunity. We show here that the combination of antigen selection, construct design, and a robust vaccine platform based on the Synthetically Modified Alpha Replicon RNA Technology (SMARRT), a self-replicating RNA, leads to control of tumor growth in mice. Therapeutic immunization with SMARRT replicon-based vaccines expressing tumor-specific neoantigens or tumor-associated antigen were able to generate polyfunctional CD4⁺ and CD8⁺ T cell responses in mice. Additionally, checkpoint inhibitors, or co-administration of cytokine also expressed from the SMARRT platform, synergized to enhance responses further. Lastly, SMARRT-based immunization of non-human primates was able to elicit high-quality T cell responses, demonstrating translatability and clinical feasibility of synthetic replicon technology for therapeutic oncology vaccines.

Keywords: RNA; T cell; immuno-oncology; neoantigen; replicon; self-replicating; synthetic; tumor; vaccine.

Graphical abstract

Figure 1

Ancer-Designed Polytope Neoantigen Constructs Expressed in the SMARRT Platform Generate Polyfunctional CD4 and CD8 T Cell Responses upon Vaccination (A) Schematic showing polytope design considerations following neoantigen identification. SMARRT replicons expressing neoantigen cassettes designed with Ancer were injected into BALB/c mice at a single dose of 10 μg, and spleens were removed 14 days later and restimulated with a peptide pool containing the top 50 Ancer predicted neoantigens from CT26. T cell function was measured by IFN-γ ELISpot (B), and intracellular cytokine staining is shown for selected replicons (C). Graphs show mean with standard deviation, n = 5 mice per group. Statistical testing was carried out with ordinary one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001.

Figure 2

SMARRT.Ancer Primes a Limited Number of Dominant T Cell Epitopes SMARRT.Ancer (containing the top 20 CT26 neoantigens) was used to immunize BALB/c mice with varying prime/boost interval lengths. Splenocytes were analyzed by intracellular cytokine staining (on the indicated days post-final injection) by restimulating individually with all 20 neoantigens encoded in the cassette. (A) and (B) show the percentage of IFN-γ⁺ CD8 and CD4 T cells specific for peptides stimulating a response above mock (data not shown). (C) and (D) show the polyfunctionality of CD8 and CD4 T cells in response to the dominant neoantigen 20 and 4, respectively. Graphs show mean with standard deviation, n = 5 mice per group. Statistical analysis was performed using one-way ANOVA with Tukeys multiple comparison test. For (C) and (D), the triple functional subsets were compared. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001

Figure 3

SMARRT Can Prime Polyfunctional T Cell Responses Specific to the Shared Neoantigen KRAS G12V HLA-A∗1101 transgenic mice were vaccinated with SMARRT replicons encoding an epitope from either WT or G12V mutant KRAS and boosted on days 21 and 41. Spleens were analyzed 7 days post final boost by intracellular cytokine staining (A) and IFN-γ ELISpot (B) following restimulation ex vivo with the indicated peptides. Graphs show mean with standard deviation, n = 3 mice per group. Statistical analysis was performed using Mann-Whitney U Test, panel B compared double cytokine positive T cells. ^∗p<0.05; ^∗∗∗∗p<0.0001.

Figure 4

Therapeutic Vaccination with SMARRT.Ancer Inhibits Tumor Growth and Enhances Survival Mice were implanted with 3 × 10⁵ CT26 cells s.c. at day 0. SMARRT was injected i.m. on days 3 and 17. Tumor growth (A) and overall survival (B) were measured (n = 18/group). In a separate study, mice (n = 6/group) were injected with CT26 and SMARRT constructs as described above; additionally, some groups received anti-PD-1 blocking mAbs on days 8, 11, 15, and 18. (C) Shows tumor growth until day 23. Tumors were excised on day 23 and T cells were analyzed by flow cytometry for cytokine production following ex vivo peptide re-stimulation. (D) Shows CD8 T cells and (E) shows CD4 T cells. Tumor growth curves show median tumor volume, and statistical testing was done with two-way ANOVA. Survival curve statistical testing was done using log-rank (Mantel Cox) test. Bar graphs show mean with standard deviation. ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 5

SMARRT Can Be Used to Express Cytokines, Resulting in Epitope Spreading to Weakly Immunogenic Tumor-Associated Antigens SMARRT expressing hIL-7/15 fusion, mIL-7/15 fusion, mIL-7, mIL-15, mGM-CSF, and IL-12 were used to transfect BHK cells. (A) Supernatants were harvested 24 h post-electroporation and cytokine concentrations were measured using ELISA. SMARRT.Trp2 was used to immunize C57BL/6 mice on days 0 and 14. Spleens were harvested on day 21 and restimulated with the dominant CD8 epitope TRP2_180–188. (B and C) T cell function was measured by IFN-γ ELISpot (B) and intracellular cytokine staining (C). (D) Mice were immunized as described above with SMARRT.Trp2; in addition, mice were immunized with SMARRT.IL-12 at prime only, as indicated in the figure. The graph shows intracellular cytokine staining following stimulation with an overlapping peptide pool of TRP2 (Sub.) or TRP2_180–188 (Dom.). Graphs in (B)–(D) show mean with standard deviation, n = 5 mice per group. Statistical testing for (B) and (C) was done using Mann-Whitney U test. Statistical testing in (D) was done using ordinary one-way ANOVA to compare the total IFN-γ⁺ CD8 T cells, ∗p < 0.05; ∗∗p < 0.01.

Figure 6

SMARRT Primes Polyfunctional T Cell Responses in Non-human Primates SMARRT replicons expressing HA from the H1N1 or H5N1 strains of influenza virus were used to immunize rhesus macaques on day 0 and 56 at the indicated doses. Saline immunization was used as a control. The animals were bled on day 77 and PBMCs were re-stimulated with peptide pools encoding either H1 or H5 HA sequences. (A) and (B) show activated (OX40⁺ CD25⁺) CD8 and CD4 T cells found in the PBMCs as measured by flow cytometry (solid circles, LNP formulated 100 μg; open circles, unformulated 100 μg; squares, formulated 10 μg; triangles, formulated 1 μg). (C) and (D) show cytokine production of CD8 and CD4 T cells measured by flow cytometry. Each bar/symbol corresponds to one animal. Statistical testing was carried out with one-tailed Mann-Whitney U test, ∗p < 0.05.