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

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 (Ubmut) 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.

EP4 mediates PGE2 dependent cell survival through the PI3 kinase/AKT pathway.

The anti-apoptotic effect of PGE(2) was examined in Jurkat cells (human T-cell leukemia) by incubation with PGE(2) (5 nM) prior to treatment with the cancer chemotherapeutic agent camptothecin. Apoptosis was evaluated by caspase-3 activity in cell extracts and flow cytometry of propidium iodide-labeled cells. Pre-incubation with PGE(2) reduced camptothecin-induced caspase activity by 30% and apoptosis by 35%, respectively. Pharmacological data demonstrate that the EP4 receptor is responsible for mediating the protection from camptothecin-induced apoptosis. Pre-treatment of the cells with the EP4 antagonist (EP4A) prior to PGE(2) and camptothecin abolished the increased survival effect of PGE(2). Specific inhibition of the downstream of PI3 kinase or AKT/protein kinase but not protein kinase A prevents the observed increase in cell survival elicited by PGE(2). These findings have critical implications regarding the mechanism and potential application of PGE(2) receptor specific inhibition in cancer therapy.

Loss of p21Waf1/Cip1/Sdi1 enhances intestinal stem cell survival following radiation injury.

The microcolony assay following gamma irradiation (IR) is a functional assay of intestinal stem cell fate. The cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1/Sdi1) (p21) regulates cell cycle arrest following DNA damage. To explore the role of p21 on stem cell fate, we examined the effects of p21 deletion on intestinal crypt survival following IR and expression of the stem/progenitor cell marker Musashi-1 (Msi-1) and the antiapoptotic gene survivin. Intestinal stem cell survival in adult wild-type (WT) and p21(-/-) mice was measured using the microcolony assay. Msi-1, p21, and survivin mRNA were measured using real-time PCR and immunohistochemical analysis. Laser capture microdissection (LCM) was used to isolate mRNA from the crypt stem cell zone. No differences in radiation-induced apoptosis were observed between WT and p21(-/-) mice. However, increased crypt survival (3.0-fold) was observed in p21(-/-) compared with WT mice 3.5 days after 13 Gy. Msi-1 and survivin mRNA were elevated 12- and 7.5-fold, respectively, in LCM-dissected crypts of p21(-/-) compared with WT mice. In conclusion, deletion of p21 results in protection of crypt stem/progenitor cells from IR-induced cell death. Furthermore, the increase in crypt survival is associated with increased numbers of Msi-1- and survivin-expressing cells in regenerative crypts.

Azoxymethane protects intestinal stem cells and reduces crypt epithelial mitosis through a COX-1-dependent mechanism.

Azoxymethane (AOM) is a potent DNA-damaging agent and carcinogen that induces intestinal and colonic tumors in rodents. Evaluation of the stem cell population by colony formation assay reveals that, within 8 h after treatment, AOM (10 mg/kg) elicited a prosurvival response. In wild-type (WT) mice, AOM treatment induced a 2.5-fold increase in intestinal crypt stem cell survival. AOM treatment increased stem cell survival in cyclooxygenase (COX)-2(-/-) but not COX-1(-/-) mice, confirming a role of COX-1 in the AOM-induced increase in stem cell survival. COX-1 mRNA and protein expression as well as COX-1-derived PGE(2) synthesis were increased 8 h after AOM treatment. Immunohistochemical staining of COX-1 demonstrated expression of the enzyme in the crypt epithelial cells, especially in the columnar epithelial cells between the Paneth cells adjacent to the stem cell zone. WT mice receiving AOM exhibited increased intestinal apoptosis and a simultaneous reduction in crypt mitotic figures within 8 h of injection. There were no significant differences in baseline or AOM-induced intestinal epithelial apoptosis between WT and COX-1(-/-) mice, but there was a complete reversal of the AOM-mediated reduction in mitosis in COX-1(-/-) mice. This suggests that COX-1-derived PGE(2) may play a key role in the early phase of intestinal tumorigenesis in response to DNA damage and suggests that COX-1 may be a potential therapeutic target in this model of colon cancer.

Prosurvival and antiapoptotic effects of PGE2 in radiation injury are mediated by EP2 receptor in intestine.

The biological activities of PGE(2) are mediated through EP receptors (EP(1)-EP(4)), plasma membrane G protein-coupled receptors that differ in ligand binding and signal-transduction pathways. We investigated gastrointestinal EP(2) receptor expression in adult mice before and after radiation injury and evaluated intestinal stem cell survival and crypt epithelial apoptosis after radiation injury in EP(2) null mice. EP(2) was expressed throughout the gut. Intestinal EP(2) mRNA increased fivefold after gamma-irradiation. Crypt survival was diminished in EP(2)-/- mice (4.06 crypts/cross section) compared with wild-type littermates (8.15 crypts/cross section). Radiation-induced apoptosis was significantly increased in EP(2)-/- mice compared with wild-type littermates. Apoptosis was 1.6-fold higher in EP(2) (-/-) mice (5.9 apoptotic cells/crypt) than in wild-type mice (3.5 apoptotic cells/crypt). The EP(2) receptor is expressed in mouse gastrointestinal epithelial cells and is upregulated following radiation injury. The effects of PGE(2) on both crypt epithelial apoptosis and intestinal crypt stem cell survival are mediated through the EP(2) receptor.