Ph.D. Candidate University of Illinois at Urbana Champaign Champaign, Illinois, United States
Introduction:: Current cancer vaccines typically rely on the use of antigens and adjuvants to stimulate an immune response against tumor cells. However, the development of safe and effective cancer vaccines remains a major challenge due to limitations in antigen selection and the potential for adverse side effects associated with adjuvant use. In this study, we explored the use of alpha helical peptides as a potential adjuvant-free approach for delivering cancer-encoding mRNA to dendritic cells (DCs). We engineered a cationic polypeptide containing a cell-penetrating peptide that enables the peptide to create pores in the DC membrane, facilitating the delivery of mRNA to the cytosol. We hypothesized that the pore-forming activity of the peptide would also serve as a "danger signal," activating the DCs and allowing them to function as an adjuvant. Our results demonstrate that the alpha helical peptide-mediated delivery of cancer mRNA to DCs is an efficient and effective approach for inducing an immune response against cancer cells. This approach has the potential to eliminate the need for traditional adjuvants in cancer vaccines, simplifying the vaccine design and improving patient outcomes.
Materials and Methods:: To evaluate the efficacy of our alpha-helical peptide-mediated delivery approach, we first synthesized a cationic polymer containing a cell-penetrating peptide. The synthesis involved the polymerization of propargyloxybenzyl N-carboxyanhydride (POB-NCA), followed by conjugation with azido guanidines through click chemistry. We then used the polypeptide to form polyplexes with mRNA encoding ovalbumin (OVA) or neoantigen peptides (4T1-26, 31, 32, and 35) at different N/P ratios. We analyzed the activation and antigen presentation of bone marrow-derived dendritic cells (BMDCs) after incubation with the different N/P ratio polyplexes using FACS assays. In vivo studies were performed using C57BL/6 mice injected with EG.7-OVA cells and Balb/c mice injected with 4T1 cells as tumor models. We compared the tumor efficacy of our polyplex group with that of commercially available gene transfection agent (Lipofectamine 2000) and bare mRNA.
Results, Conclusions, and Discussions:: In this study, we aimed to develop a novel cancer vaccine strategy utilizing mRNA delivery via cationic polymers and a cell-penetrating alpha-helical peptide. We first synthesized cationic polymers with the alpha-helical peptide and evaluated their effectiveness in activating bone marrow-derived dendritic cells (BMDCs). Our results demonstrated that the alpha-helical peptide was capable of inducing BMDC activation to a level comparable to that achieved with the commonly used adjuvant CpG, suggesting that this approach could be a promising alternative to adjuvant-based cancer vaccines.
Next, we generated polyplexes with varying nitrogen-to-phosphorus (N/P) ratios containing mRNA encoding ovalbumin (OVA) or neoantigen peptides (4T1-26, 31, 32, and 35). Our results showed that polyplexes with higher N/P ratios led to increased activation and antigen processing of DCs, suggesting that the alpha-helical peptide-mediated delivery could enhance immune responses. Subsequently, we administered the mRNA vaccines subcutaneously and observed a superior cytotoxic T lymphocyte (CTL) response compared to the Lipofectamine 2000-treated group and the bare mRNA-treated group. This led to enhanced tumor suppression and prolonged survival of mice upon subcutaneous injection of tumor cells.
Following the vaccination and prophylactic study of our mRNA vaccine, we evaluated its therapeutic effect. Surprisingly, our mRNA vaccine showed improved tumor suppression and prolonged survival of mice in not only the EG.7-OVA tumor model but also in the challenging triple-negative breast cancer (4T1) tumor. Notably, a mixture of neoantigen peptides specific to the 4T1 tumor was employed in this study. These results suggest that the alpha-helical peptide-mediated mRNA delivery could be a promising approach for cancer vaccination, potentially eliminating the need for additional adjuvants.
Our study highlights the potential of utilizing mRNA vaccines for cancer treatment and the importance of optimizing mRNA delivery strategies for effective immune activation. This approach holds the potential for developing a straightforward and effective cancer vaccine strategy that can elicit robust and long-lasting anti-tumor immune responses.
