Abstract 1689: Exosomes derived from tumor cells treated with immunostimulatory oncolytic virotherapy exert immune activation instead of immunosuppression

Abstract

Abstract Immunostimulatory gene therapy using oncolytic viruses - OV - for combined gene transfer and tumor-specific oncolysis is a promising immunotherapy for cancer. Due to neutralizing antibodies in the blood, OVs are commonly administered by intratumoral delivery. The immune activation is both local and systemic as activated dendritic cells in the injected lesion travel to lymph nodes to activate tumor-reactive T cells that can in turn act on both injected and non-injected lesions. In the present project, we demonstrate that OV-infected tumor cells released exosomes that cargo mRNA and protein coded by the human immunostimulatory genes expressed by the OV. Tumor cell-derived exosomes are commonly immunosuppressive because they cargo immunosuppressive molecules expressed by the tumor. We hypothesized that exosomes derived from OV-infected tumor cells may instead be immunostimulatory. To investigate the role of exosomes derived from OV-treated tumors, human melanoma Mel526 cells were infected by delolimogene mupadenorepvec -LOAd703 - which is an adenoviral serotype 5/35 OV armed with costimulatory molecules CD40L and 4-1BBL. LOAd703 could induce expression of CD40L and 4-1BBL on the melanoma cell surface and kill the cells by replication-induced oncolysis after 48-72 hours culture as evaluated by MTS. Exosomes were purified prior oncolysis and investigated for the presence of CD40L and 4-1BBL mRNA and protein, and for their capacity to stimulate immune responses. The results demonstrated that the exosomes can indeed cargo CD40L and 4-1BBL both as mRNA as shown by PCR, and protein as shown by ELISA, western blot, flow cytometry and electron microscopy post LOAd703 tumor cell infection. Furthermore, the exosomes from CD40L and 4-1BBL-expressing tumor cells could stimulate robust dendritic cell activation with enhanced levels of MHC class I and II as well as costimulatory molecules CD80, CD86 and CD83. In contrast, MHC class I and the costimulatory molecules were reduced on dendritic cells stimulated with exosomes derived from non-infected tumor cells, which is in line with previous knowledge of exosomes as immunosuppressants in the tumor. However, PD-L1 was increased on dendritic cells stimulated with the exosomes from CD40L and 4-1BBL-expressing tumor cells. Hence, LOAd703 therapy may benefit from combination with PD-L1 or PD-1 checkpoint blockade. In conclusion, tumor-derived exosomes released by OV-infected tumor cells can cargo the immunostimulatory transgenes expressed by the OVs and are then converted from immunosuppressive to immune activating exosomes as shown by their capacity to activate immune cells such as dendritic cells. Exosomes that cargo immunostimulatory transgenes from OV-infected tumor cells may support the systemic immunity gained by OVs after local intratumoral delivery. Citation Format: Alireza Labani-Motlagh, Sedigheh Naseri, Jessica Wenthe, Emma Eriksson, Angelica Loskog. Exosomes derived from tumor cells treated with immunostimulatory oncolytic virotherapy exert immune activation instead of immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1689.