Abstract T‐cells specific for foreign ( e.g. , viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen‐specific T‐cells are thought to be less powerful. However, synthetic T‐cell vaccines composed of Melan‐A/MART‐1 peptide, CpG and IFA can induce high frequencies of tumor‐specific CD8 T‐cells in PBMC of melanoma patients. Here we analyzed the functionality of these T‐cells directly ex vivo , by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL‐2) and upregulation of LAMP‐1 (CD107a) by tumor (Melan‐A/MART‐1) specific T‐cells was comparable to virus (EBV‐BMLF1) specific CD8 T‐cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor‐ and virus‐specific T‐cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T‐cells were induced irrespective of patient's age or gender. Finally, CD8 T‐cell function correlated with disease‐free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor‐specific CD8 T‐cells can reach functional competence in vivo , encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.
Abstract Background The activation of dendritic cells (DCs) is pivotal for generating antigen-specific T-cell responses to eradicate tumor cells. Hence, immunotherapies targeting this interplay are especially intriguing. Moreover, it is of interest to modulate the tumor microenvironment (TME), as this harsh milieu often impairs adaptive immune responses. Oncolytic viral therapy presents an opportunity to overcome the immunosuppression in tumors by destroying tumor cells and thereby releasing antigens and immunostimulatory factors. These effects can be further amplified by the introduction of transgenes expressed by the virus. Methods Lokon oncolytic adenoviruses (LOAd) belong to a platform of chimeric serotype Ad5/35 viruses that have their replication restricted to tumor cells, but the expression of transgenes is permitted in all infected cells. LOAd732 is a novel oncolytic adenovirus that expresses three essential immunostimulatory transgenes: trimerized membrane-bound CD40L, 4-1BBL and IL-2. Transgene expression was determined with flow cytometry and ELISA and the oncolytic function was evaluated with viability assays and xenograft models. The activation profiles of DCs were investigated in co-cultures with tumor cells or in an autologous antigen-specific T cell model by flow cytometry and multiplex proteomic analysis. Statistical differences were analyzed with Kruskal–Wallis test followed by Dunn’s multiple comparison test. Results All three transgenes were expressed in infected melanoma cells and DCs and transgene expression did not impair the oncolytic activity in tumor cells. DCs were matured post LOAd732 infection and expressed a multitude of co-stimulatory molecules and pro-inflammatory cytokines crucial for T-cell responses. Furthermore, these DCs were capable of expanding and stimulating antigen-specific T cells in addition to natural killer (NK) cells. Strikingly, the addition of immunosuppressive cytokines TGF-β1 and IL-10 did not affect the ability of LOAd732-matured DCs to expand antigen-specific T cells and these cells retained an enhanced activation profile. Conclusions LOAd732 is a novel immunostimulatory gene therapy based on an oncolytic adenovirus that expresses three transgenes, which are essential for mediating an anti-tumor immune response by activating DCs and stimulating T and NK cells even under imunosuppressive conditions commonly present in the TME. These qualities make LOAd732 an appealing new immunotherapy approach.
