Vaccines to immunologically target cancers: Androgen receptor splice variants as antigens in prostate cancer vaccines & vector directed responses in self-replicating RNA based HER2 breast cancer vaccines

Abstract

Cancer vaccines encoding tumor associated antigens are a promising, albeit challenging, immunotherapeutic strategy being explored in many cancers. The immunosuppressive characteristics of many tumors make inducing a robust response to tumor associated antigens reliant on very immunogenic vaccine vectors and careful selection and evaluation of the tumor associated antigen to target with vaccination. Here we present work on both cancer vaccine target antigen selection and cancer vaccine vector optimization utilizing IFNγ ELISpots, ELISAs, intracellular cytokine staining flow cytometry, multiple mouse cancer cell lines, including a novel murine prostate cancer cell line, P3CA, which we additionally characterized by bulk RNA sequencing. Alphavirus like replicon particle (VRP)-specific neutralizing antibodies were determined using an immunofluorescence microscopy-based protocol. While one prostate cancer vaccine has been approved by the FDA, all Phase III clinical trials on prostate cancer vaccines conducted after the introduction of next-generation antiandrogens have not improved clinical outcomes. We hypothesized that vaccination targeting androgen receptor (AR) and AR RNA splice variant 7 (AR-V7) would induce anti-tumor immunity in the context of standard of care therapies. Using an adenoviral vaccine vector, we demonstrated robust AR specific T cell responses capable of anti-tumor immunity in the immunosuppressive microenvironment of a novel murine prostate cancer cell line. Vaccines were most effective when combined with anti-PD-1 immune checkpoint inhibitors, and our results indicate that AR and AR-V7 targeting vaccines may be useful as prostate cancer immunotherapies. The induction of significant neutralizing antibodies against adenoviral vectors prevents the use of these vectors in homologous prime-boosting vaccine regimens. We hypothesized that alphaviral self-replicating RNA vectors would also include significant vector directed responses, limiting their immunogenicity toward the target antigen, and demonstrated that VRP vaccine vectors produce significant vector directed adaptive immune responses in the form of neutralizing antibodies and T cell specific responses in samples from a previously conducted Phase I trial of a HER2-VRP breast cancer vaccine. Data generated from clinical samples and mechanistic preclinical mouse studies indicated that vector directed adaptive immune responses specific to NSPs are detectable and likely compete for immunodominance with less immunogenic or tolerized target antigen specific responses. We found that VRP vaccination produced significant neutralizing antibody titers after two doses of VRP vaccines in clinical trial participants and mouse models. To further explore the impact of vector directed immunity, we conducted a preclinical mouse study utilizing a “heterologous-prime”, where after receiving two doses of a VRP vaccine encoding carcinoembryonic antigen (CEA), mice were given a single dose of HER2-VRP. HER2 specific priming was significantly impaired in mice previously vaccinated with a VRP vector, resulting in significantly lower IgG titers than mice receiving a single dose of HER2-VRP alone and HER2-sepcific responses by IFNγ ELISpot that were not statistically different from unvaccinated controls. We conclude that AR and AR-V7 targeted vaccination is a promising prostate cancer immunotherapeutic strategy, inducing antigen specific T cell responses. Improved vaccine strategies will be required before these results could be translated to the clinic. Alphaviral vectors known for their immunogenicity, including LNP formulated self-replicating alphaviral vectors, may outperform the adenoviral vectors used in our preclinical AR vaccine studies, but were also shown to induce significant vector specific T cell responses which compete for immunodominance with target antigen specific T cells. This competition for immunodominance is especially relevant when targeting tolerized tumor associated antigens like AR or AR-V7.