Boosting IL-12-induced anti-glioblastoma activity via immunotherapeutic extracellular vesicles.

PROJECT SUMMARY The objective of this application is to develop the career of Dr. Breyne, facilitating his transition to a stable, independent phase and establishing his research program. He will pursue a rigorous career development plan to further his training in (i) extracellular vesicle (EV) biology, (ii) immune-oncology, and (iii) professional

development. Progress in these domains will be accomplished through coursework, attendance and speaking at conferences and workshops, writing last author papers, mentoring others, and leading team efforts. The training environment is exceptional, and the collaborators are world-class. The K22 Award will give the PI protected time

to generate data for an R01 proposal while launching his independent faculty position. Once glioblastoma (GB) progresses after first-line therapy, treatment options are limited, and managing recurrent GB remains challenging. While newly diagnosed GB patients have a life expectancy of ~15 months, it is reduced

to 6 to 9 months after relapse. Recently, localized delivery of interleukin-12 (IL12) has had promising anti-tumoral effects in patients with high-grade, recurrent GB extending the survival to ~17 months. Our preliminary data show that intratumoral IL12, together with local expression of a costimulatory factor for cytotoxic T lymphocytes (CTLs)

- 4-1BBL, can induce sustained anti-glioma responses. The objective of our studies is to develop a clinically relevant strategy combining these factors while expanding our understanding of immune-suppressive resistance against IL12-induced anti-glioma activity. In Aim 1, we will establish disruptive technology presenting

combinations of immuno-modulatory factors on a single nanocarrier. In essence, dendritic cell (DC)-derived EVs will be designed to display IL12, 4-1BBL, and a neoantigen:MHC complex. In line with our findings of GB-treated mice with IL12/4-1BBL combo- compared to IL12 monotherapy, we anticipate that immunotherapeutic EVs will

translate to ~68% improvement in the survival of GB-bearing mice. In Aim 2, the resistance mechanisms induced by GB against IL12 treatment will be explored. We have observed that myeloid cell subsets sense antitumor immunity activated by IL12. By combining single-cell RNA sequencing analysis with multiparametric flow

cytometry in IL12-treated GB mouse models, we will uncover the pro-oncogenic signaling resulting in a PD-L1 increase and 4-1BBL decrease. In turn, these targets will be monitored in response to intratumoral EV treatment anticipating the attenuation of GB resistance. Aim 3 will investigate CTL:DC crosstalk driving IL12 treatment.

CTLs are responsible for IL12-induced anti-glioma activity, as shown by our CD8 depletion experiments. However, little is known about DCs in an IL12-stimulated GB brain. Transgenic mouse models will aid us by marking or depleting relevant DCs in the tumor and studying the anti-tumor activity of our engineered EVs.

This unique proposal addresses a crucial and timely unmet need in the field. We plan to engage the therapeutic potential of EVs to arrest GB progression. This work will undoubtedly provide a strong foundation for the candidate's independent leadership in brain tumor research.