Cancer vaccine immunotherapy facilitates the immune system’s recognition of tumorassociated antigens (TAAs), and the biomolecular design of these vaccines using nanoparticles (NPs) is one important approach towards obtaining strong anti-tumor responses. Following activation of dendritic cells (DCs), a robust CD8+ T cell-mediated adaptive immune response is critical for tumor elimination. This work focuses on using the E2 protein NP platform to evaluate: the design of NPs to activate two main DC populations, peptide delivery by NPs to take advantage of DCs’ ability to activate both cytotoxic and T helper cells simultaneously, and breadth of immunotherapeutic efficacy of E2 NPs in a model with immune system T helper cell bias towards humoral rather than cell-mediated immunity.

While the role of efficient antigen-presenting myeloid DCs (mDCs) is conventionally attributed to vaccine efficacy, participation by high cytokine-producing plasmacytoid DCs (pDCs) is less understood and is often overlooked. We observed differential activation of mDCs or pDCs depending on the choice of E2-encapsulated adjuvant, including TLR9 agonist bacterial like DNA (CpGs) or TLR7 agonist viral ssRNA. Transfer of secreted factors from stimulated pDCs enhanced mDC display of the antigen, particularly when stimulated with NPs. These results reveal that pDCs can aid mDCs, highlighting the importance of activating both pDCs and mDCs in designing optimal cancer vaccines, and demonstrate the advantage of using NP-based vaccine delivery.

CD8+ T cell education against major histocompatibility complex (MHC) class I-restricted antigens including TAAs has conventionally been one important goal for NP cancer vaccines. Following uptake and activation of DCs by NPs, DCs can specifically activate CD8+ T cells, mediated by MHC class I proteins. Although eliciting robust antigen-specific CD8+ T cell mediated immunity is critical for anti-tumor efficacy, a growing body of evidence supports the importance of CD4+ T cells activated against MHC class II peptides. However, there have not been studies evaluating whether MHC class I and II TAAs should be delivered on the same NP vehicle or if these TAAs can be delivered separately for an optimal anti-tumor response. In our work, we determined that NPs co-delivering MHC class I and II melanoma TAAs most potently treated B16/F10 melanoma-bearing mice, improving efficacy over single-antigen NPs or a mixture of single-antigen NPs. These studies strongly support that NP vaccines should be designed to simultaneously deliver MHC class I and II antigens on the same vehicle. Developing therapeutics that elicit robust anti-cancer responses over a broad range of biological systems is critical for translation to humans, where there is a breadth of individual responses. We previously observed E2 protein cancer vaccine efficacy in Th1 (T helper class 1)-biased mice, but the efficacy of this platform has not been studied in a Th2-biased model, where eliciting robust Th1 cell-mediated immunity is still important but more challenging to achieve. We developed a therapeutic NP vaccine designed to simultaneously deliver CpG and a MHC class I CT26 colon carcinoma TAA for use in a Th2-biased model. Immunization with these NPs induced a robust antigen-specific IFN-γ response (a prominent Th1 cytokine), even in Th2-biased mice. The NPs treated CT26 tumor growth, a response synergistically strengthened when combined with the immune checkpoint inhibitor therapy anti-PD-1. This study demonstrates the breadth of efficacy across tumor models of a modular NP cancer vaccine platform.