Optimized polyepitope neoantigen DNA vaccines elicit neoantigen-specific immune responses in preclinical models and in clinical translation

Abstract

Background: Preclinical studies and early clinical trials have shown that targeting cancer neoantigens is a promising approach towards the development of personalized cancer immunotherapies. DNA vaccines can be rapidly and efficiently manufactured and can integrate multiple neoantigens simultaneously. We therefore sought to optimize the design of polyepitope DNA vaccines and test optimized polyepitope neoantigen DNA vaccines in preclinical models and in clinical translation.

Methods: We developed and optimized a DNA vaccine platform to target multiple neoantigens. The polyepitope DNA vaccine platform was first optimized using model antigens in vitro and in vivo. We then identified neoantigens in preclinical breast cancer models through genome sequencing and in silico neoantigen prediction pipelines. Optimized polyepitope neoantigen DNA vaccines specific for the murine breast tumor E0771 and 4T1 were designed and their immunogenicity was tested in vivo. We also tested an optimized polyepitope neoantigen DNA vaccine in a patient with metastatic pancreatic neuroendocrine tumor.

Results: Our data support an optimized polyepitope neoantigen DNA vaccine design encoding long (≥20-mer) epitopes with a mutant form of ubiquitin (Ub^mut) fused to the N-terminus for antigen processing and presentation. Optimized polyepitope neoantigen DNA vaccines were immunogenic and generated robust neoantigen-specific immune responses in mice. The magnitude of immune responses generated by optimized polyepitope neoantigen DNA vaccines was similar to that of synthetic long peptide vaccines specific for the same neoantigens. When combined with immune checkpoint blockade therapy, optimized polyepitope neoantigen DNA vaccines were capable of inducing antitumor immunity in preclinical models. Immune monitoring data suggest that optimized polyepitope neoantigen DNA vaccines are capable of inducing neoantigen-specific T cell responses in a patient with metastatic pancreatic neuroendocrine tumor.

Conclusions: We have developed and optimized a novel polyepitope neoantigen DNA vaccine platform that can target multiple neoantigens and induce antitumor immune responses in preclinical models and neoantigen-specific responses in clinical translation.

Keywords: Breast cancer; Neoantigen; Polyepitope DNA vaccine.

Fig. 1

Optimizing the polyepitope DNA vaccine design. a Schematic DNA constructs encoding eight polyepitope model antigens (peptide sequences were listed in Additional file 2, Table S1). Left, polyepitope P20 and M20 differ only in the position of epitopes pp65 and M1. Right, the polyepitope constructs were subcloned into a retroviral vector driven by the MSCV promoter. The HA-tag and IRES-GFP were included to facilitate the in vitro detection of polyepitope protein production. Ub^mut, a mutated (G76V) ubiquitin. b Immunoblot (IB) analysis of the polyepitope proteins. Left, HeLa-A2 cells were transduced with indicated polyepitope constructs. Red arrowheads indicate the ubiquitinated polyepitope proteins. Right, HA/GFP ratio was used to quantify relative levels of polyepitope proteins. Results combined from three independent experiments (mean ± SEM) were shown. c Presentation of antigens by the transduced HeLa-A2 cells. Left, surface staining of the SVG9/HLA-A2 complexes with a TCR-mimic antibody. Mean fluorescence intensity (MFI) of the SVG9/HLA-A2 signal relative to MFI of the co-expressed GFP (mean ± SEM, in triplicates) was shown. Middle, specific lysis of transduced HeLa-A2 cells by SVG9-specific cytotoxic T cells was measured by a ⁵¹Cr-releasing cytotoxicity assay (E:T = 25:1). Right, DNA vaccines induced G209-specific immune response in HHD II mice was measured by an IFN-γ ELISpot assay (mean ± SEM, n = 8). These experiments were repeated at least once and representative results were shown. d Representative dot plots showing SVG9/HLA-A2 tetramer staining of CD8⁺ spleen cells from the vaccinated HHD II mice. Numbers indicate frequencies in each quadrant. *P < 0.05, ***P < 0.001, t-test

Fig. 2

Polyepitope neoantigen DNA vaccine elicit neoantigen-specific T cell responses in vivo. Neoantigens were identified for E0771 and 4T1.2 breast cancer models. Polyepitope neoantigen DNA vaccines were created for each and were used to immunize mice by gene gun. Spleen cells from mice vaccinated with polyepitope DNA vaccines (red) and control empty vector DNA (black) were harvested and used in IFN-γ ELISpot assay. T cell responses to selected neoantigens were shown (mean ± SEM) for Ub^mut-E0771 (a) and Ub^mut-4T1.2 (b). Of note, 8- to 10-mer minimal peptides were used in the assays for Ub^mut-E0771 (a), but 29-mer long peptides were used for Ub^mut-4T1.2 (b). Experiments were repeated at least two more times for panel a, and similar results were obtained. **P < 0.01, ***P < 0.001, t-test

Fig. 3

Polyepitope E0771 neoantigen DNA vaccines combined with anti-PD-L1 immunotherapy suppressed tumor growth in vivo. a Scheduling of DNA vaccination and anti-PD-L1 treatment. Wildtype female C57BL/6 mice (n = 15 per group) were vaccinated by gene gun on days − 4, − 1, and 2 and challenged with 10⁶ E0771 cells on day 0. Anti-PD-L1 or control antibodies were administered every 3–4 days. b Tumors were measured with electronic calipers of the longest (L) and perpendicular (W) diagonals. Tumor sizes (mean ± SEM) were calculated as (L × W²)/2. Results from one of the three independent experiments were shown. c In a parallel experiment, tumors were harvested and dissociated to prepare single cell suspension on day 14. TILs were analyzed by Lrrc27/D^b dextramer staining and flow cytometry. P = 0.0381, one-way ANOVA. d Tumor-draining lymph nodes (LN) were harvested on day 14. LN cells were used in an IFN-γ ELISpot assay and stimulated with selected MT peptides (8- to 10-mer). e Spleen cells were harvested from treated tumor-bearing mice on day 26 and used in an IFN-γ ELISpot assay. The studies were repeated once and similar results were obtained. Error bars, SEM. *P < 0.05, **P < 0.01, ***P < 0.001, t-test

Fig. 4

Polyepitope DNA vaccine generated similar magnitude of immune responses as synthetic long peptide vaccines. a Comparison of IFN-γ ELISpot results (mean ± SEM) induced by polyepitope Ub^mut-E0771 DNA vaccine and SLP vaccine. Wildtype C57BL/6 mice were vaccinated with Ub^mut-E0771 vaccine or a mixture of three SLPs. The schedule for both platforms was optimized independently. The IFN-γ ELISpot assay was performed on the same day when immune responses are at peak level. The experiment was repeated once and similar results were obtained. b Specificity of DNA vaccine-generated immune response towards neoantigens (MT) over corresponding WT peptides. An IFN-γ ELISpot assay was performed by using 8- to 10-mer MT and WT peptides at different concentrations. Results shown were from one of the two independent experiments. Results generated with high (2.5 μg/ml) and low (10 pg/ml) MT/WT Lrrc27 peptides were also shown. *P < 0.05, paired t-test

Fig. 5

An optimized polyepitope neoantigen DNA vaccine is capable of inducing neoantigen-specific T cell responses in a patient with metastatic pancreatic neuroendocrine cancer. PBMC from patient GTB16 were obtained before (pre-vaccine) and after (post-vaccine) vaccination with an optimized polyepitope neoantigen DNA vaccine. PBMC were stimulated in vitro for 12 days with peptides corresponding to the indicated neoantigens and then an IFNγ ELISpot assay was performed. The number of spot forming cells (SFC) specific for each neoantigen is indicated. Nonspecific background counts, assessed by incubating cells without peptide during the ELISpot assay, were subtracted. The assays were repeated twice and similar results were obtained. Please note that the vaccine incorporated 13 neoantigens. A robust response was observed to 3/13 neoantigens. The other neoantigens did not induce a response