Development of a novel B cell-based vaccine for metastatic solid cancers

In the last decades, cancer immunotherapy has made enormous progress and is now considered as a fourth pillar of cancer therapy, together with surgery, chemotherapy and radiotherapy. Several approaches are classified as immunotherapy, whose final aim is to boost the patients’ innate defenses against the malignancy. Among these, cell-based cancer vaccines have been tested in extensive preclinical and clinical studies in the last thirty years, culminating in 2010 with the FDA approval of the first dendritic-cell based cancer vaccine (Sipuleucel-T) for the treatment of prostate cancer. Cell-based cancer vaccines rely on antigen-presenting cells (APCs) to mount a tumor-specific immune response. Dendritic cells (DCs) have been considered for long time as the most potent APC of the immune system and thus the most suitable platform for cancer vaccination approaches. Despite their discrete success and safety profile, DCs present several disadvantages including a low presence in the systemic circulation, low or suboptimal expression of homing molecules and no proliferative potential. On the other hand, a different class of APCs, such as B cells, have recently raised some interest as potential alternative to DCs, for the development of therapeutic vaccines. B cells present several advantages over DCs, such as abundance in the systemic circulation, high proliferative potential and high expression of homing molecules, like CD62L. Based on this evidence, the focus of this project is to investigate whether B cell antigen-presenting capacity can be boosted by increasing the expression of co-stimulatory molecules able to interact with cognate receptors on T cells. To this aim, we choose cell electroporation with encoding mRNA as a method for transient cell engineering to increase the expression of targeted co-stimulatory receptors. Accordingly, we set up a pipeline for the electroporation of CD40-activated murine B cells with mRNAs coding for OX40 ligand (OX40L) and 4-1BB ligand (4-1BBL) co-stimulatory molecules, considered as master regulators and inducers of CD4+ and CD8+ T cell activation, respectively. In order to set up a robust protocol for B cell transfection, we first used green fluorescent protein (GFP) as proof-of-principle target, and evaluated the transfection efficiency by measuring cell viability and protein expression by flow cytometry. From our results, we observed almost 70% of cell viability with a significant increase in target protein expression upon B cell electroporation and concluded that electroporation is safe and efficient, at least in the tested conditions. Based on these results, we then performed OX40L and 4-1BBL mRNA transfection in the same conditions and verified cell viability and target expression, obtaining over 80% of cells expressing OX40L and 4-1BBL 4h after electroporation, with no significant decrease in cell viability. Overall, the collected results showed that electroporation represents a safe and valuable tool for BCs transfection.