Lara Stein

For successful therapeutic interventions in cancer immunotherapy, strong antigen-specific immune responses are required. To this end, immunostimulating cues must be combined with antigens to simultaneously arrive at antigen-presenting cells and initiate cellular immune responses. Recently, imidazoquinolines have shown their vast potential as small molecular Toll-like receptor 7/8 (TLR7/8) agonists for immunostimulation when delivered by nanocarriers. At the same time, peptide antigens are promising antigen candidates but require combination with immune-stimulating adjuvants to boost their immunogenicity and exploit their full potential. Consequently, we herein present biodegradable polycarbonate nanogels as versatile delivery system for adjuvants within the particles’ core as well as for peptide antigens by surface decoration. For that purpose, orthogonally addressable multifunctional polycarbonate block copolymers were synthesized, enabling adjuvant conjugation through reactive ester chemistry and peptide decoration by strain-promoted alkyne-azide cycloaddition (SPAAC). In preparation for SPAAC, CD4⁺-specific peptide sequences of the model protein antigen ovalbumin were equipped with DBCO-moieties by site-selective modification at their N-terminal cysteine. With their azide groups exposed on their surface, the adjuvant-loaded nanogels were then efficiently decorated with DBCO-functional CD4⁺-peptides by SPAAC. In vitro evaluation of the adjuvant-loaded peptide-decorated gels then confirmed their strong immunostimulating properties as well as their high biocompatibility. Despite their covalent conjugation, the CD4⁺-peptide-decorated nanogels led to maturation of primary antigen-presenting cells and the downstream priming of CD4⁺-T cells. Subsequently, the peptide-decorated nanogels loaded with TLR7/8 agonist were successfully processed by antigen-presenting cells, enabling potent immune responses for future application in antigen-specific cancer immunotherapy.

The cellular composition of the tumor microenvironment, including tumor, immune, stromal, and endothelial cells, significantly influences responses to cancer therapies. In this study, we analyzed the impact of oxidative stress, induced by cold atmospheric plasma (CAP), on tumor cells, T cells, and macrophages, which comprise part of the melanoma microenvironment. To accomplish this, cells were grown in different in vitro cell culture models and were treated with varying amounts of CAP. Subsequent alterations in viability, proliferation, and phenotype were analyzed via flow cytometry and metabolic alterations by Seahorse Cell Mito Stress Tests. It was found that cells generally exhibited reduced viability and proliferation, stemming from CAP induced G2/M cell cycle arrest and subsequent apoptosis, as well as increased mitochondrial stress following CAP treatment. Overall, sensitivity to CAP treatment was found to be cell type dependent with T cells being the most affected. Interestingly, CAP influenced the polarization of M0 macrophages to a “M0/M2-like” phenotype, and M1 macrophages were found to display a heightened sensitivity to CAP induced mitochondrial stress. CAP also inhibited the growth and killed melanoma cells in 2D and 3D in vitro cell culture models in a dose-dependent manner. Improving our understanding of oxidative stress, mechanisms to manipulate it, and its implications for the tumor microenvironment may help in the discovery of new therapeutic targets.

The generation of specific humoral and cellular immune responses plays a pivotal role in the development of effective vaccines against tumors. Especially the presence of antigen-specific, cytotoxic T cells influences the outcome of therapeutic cancer vaccinations. Different strategies, ranging from delivering antigen-encoding mRNAs to peptides or full antigens, are accessible but often suffer from insufficient immunogenicity and require immune-boosting adjuvants as well as carrier platforms to ensure stability and adequate retention. Here, we introduce a pH-responsive nanogel platform as a two-component antitumor vaccine that is safe for intravenous application and elicits robust immune responses in vitro and in vivo. The underlying chemical design allows for straightforward covalent attachment of a model antigen (ovalbumin) and an immune adjuvant (imidazoquinoline-type TLR7/8 agonist) onto the same nanocarrier system. In addition to eliciting antigen-specific T and B cell responses that outperform mixtures of individual components, our two-component nanovaccine leads in prophylactic and therapeutic studies to an antigen-specific growth reduction of different tumors expressing ovalbumin intracellularly or on their surface. Regarding the versatile opportunities for functionalization, our nanogels are promising for the development of highly customized and potent nanovaccines.