Development of novel APCs-based vaccines for anticancer therapy

The standard treatments of cancer are often limited by a gain of resistance of tumor cells, which can lead to the formation of metastasis in surrounding healthy tissues and difficulties to eliminate the disease. The aim of cancer therapies is the eradication of disseminated tumor cells in the blood circulation and in the various organs and to avoid the occurrence of relapses. Among these, immunotherapy has recently shown interesting improvements and therapeutical benefits. In immunotherapies, the patient’s immune system is instructed to recognize and destroy cancer cells and create a memory effect that can thereby control disease recurrence. Cancer vaccines are a type of immunotherapy able to activate and sustain tumor-specific T cells effector functions with the use of antigen-presenting cells (APCs) stimulated with cancer antigen(s). In the last decades, laboratories focused their attention on dendritic cells (DCs) as a platform to develop cellular-based cancer vaccines, as they are so far the best-known and most efficient APCs for the induction of T cell immunity. However, the clinical benefits of DC vaccines have been so far very limited, despite their demonstrated high safety and tolerance. This observed lack of benefits has been linked to several factors including their limited occurrence in the peripheral blood, their lack of proliferative potential, and their susceptibility to the immunosuppressive tumor microenvironment (TME). To overcome these issues, B cells have been recently investigated as a potential alternative to DCs. Indeed, several studies showed that B cell tumor infiltration often correlates with better prognosis in some cancer types and that, unlike DCs, they can also be easily isolated and expanded from peripheral blood in large numbers. Furthermore, B cells were shown to be less prone to inhibition by the TME. Previous ex vivo studies showed the capacity of B cells to induce tumor-specific T-cell responses. Adoptive transfer of B cells loaded ex vivo with cancer antigens showed protective effects in vivo in mouse and dog models. Based on this collective evidence, B cells constitute a promising alternative for the design of novel cancer therapeutic vaccines. In the first part of this study, we focused on developing an in-house protocol for the isolation and expansion of mouse B cells. Our results showed that incubation of mouse isolated B cells incubated for up to 14 days in the presence of feeder cells expressing CD40 induced an APC phenotype compatible with vaccination approaches. Flow cytometry analysis carried out at different time points (i.e. day 0, day3, day7, day10 and day14) demonstrated in fact the induced increased expression of the CD40, CD80, CD86, MHCI, MHCII APC markers and a low expression of the CD138 marker, usually characterizing terminally differentiated plasma cells. Moreover, we observed that at day 7, cultured B cells presented the highest cell viability and cell number, although still characterized by a high percentage of T cells contamination. On the contrary, B cells at Day 14 presented a more pure population (>90% CD19+) with significantly less occurrence of T cells and still a high viability and cell number compatible with vaccination purposes and were thus selected for subsequent analyses and applications. In the second part of this project, we attempted to further improve the B cell antigen-presenting capability through electroporation of mRNA encoding for co-stimulatory molecules crucial for T cell activation, such as OX40L, 41BBL, IL12p70, and using GFP as positive control. Using this approach, we demonstrated that mRNA electroporation did not affect cell viability and allowed to significantly increase the expression of the 4-1BBL and GFP targets. Based on the results here presented, future tests should focus on testing the antigen-presenting capability of Day 14 B cells in both in vitro and in vivo assays.