Radiation therapy (RT) and chemotherapy have traditionally focused on a cell-intrinsic mode of action such as genetic alteration of tumor cells (ie, DNA double-stranded breaks, DNA cross-linking, mitotic catastrophe, and other chromosome abnormalities) leading to cell cycle arrest and cell death. However, recent antitumor strategies have been increasingly focused on tumor cell-extrinsic factors such as induction of systemic antitumor immunity. Radiation-induced immunogenic cell death (ICD) results in stimulation of dendritic cells (DC),1 that efficiently engulf tumors and cross-presents tumor-associated antigens (TAAs) to T cells, eliciting an antitumor response2 capable of recognizing distant non-irradiated tumors, known as the abscopal effect.3–5 Another important antitumor cell-extrinsic effect induced by RT is radiation-induced damage of the tumor vasculature.6 7 Tumor-associated endothelium is significantly altered in response to a single large dose of radiation (<10 Gy), which in turn makes the tumor microenvironment (TME) hypoxic, acidic, and nutritionally deprived, thereby indirectly inducing tumor cell death.8 9 Because of the potential to damage the tumor-associated endothelium and induce ICD, combination treatment of RT together with immunotherapy and/or anti-angiogenic agents can often result in improved therapeutic efficacy.10

Active immunotherapies, such as cancer vaccines, aim to reprogram the patient’s immune system to recognize and eliminate its own cancer cells via recognition of TAAs.11 Immunological targeting of antigens expressed in the tumor-associated endothelial and/or stromal cells, rather than in the tumor cells themselves, is an alternative vaccination approach and has several advantages compared with targeting tumor-based TAAs and include reduced probability of tumor antigen escape variant generation due to the increased genetic stability of the tumor vasculature compared with tumor cells, and improved accessibility by immune cells.12 In our previously published work,13 we showed that prophylactic immunization with plasmid-DNA encoding the Tem1 cDNA fused to the minimal domain of the C fragment of tetanus toxin (TT), used as an immunoenhancer,14–16 resulted in complete tumor rejection. When used therapeutically, this approach reduced tumor progression in the CT26 colorectal and TC1 lung cancer models in a T cell-dependent manner. Immunization with the tumor endothelial marker 1 (TEM1) plasmid-DNA construct reduced tumor microvascular density (MVD), decreased tumor blood perfusion, increased hypoxia, and induced potent epitope spreading.13

Despite showing encouraging results, therapeutic vaccination with the TEM1 plasmid-DNA did not however result in complete tumor regression. Several strategies to improve vaccination efficacy have been investigated.11 17 18 Combinations of heterologous modalities of immunization (ie, vaccinating with different vectors encoding the same immunogen) have shown enhanced immune responses to the target antigen.19 The rationale behind this strategy is that by using different vectors as boosters, it is possible to bypass the immune response elicited against the primer and also strengthen the immune response against the target antigen.19–21

In the current study, we develop a novel recombinant Adenovirus 5 (Ad5) vaccine expressing TEM1-TT fusion protein (TEM1 Ad5) and demonstrate that heterologous priming with TEM1 plasmid-DNA vaccine followed by TEM1 Ad5 vaccine significantly improved TEM1-specific immune responses and antitumor effects compared with either vector alone.20 22 In vivo, dual treatment with RT and heterologous TEM1 vaccination disrupted the functional vasculature of the irradiated tumor, increased the systemic adaptive immune response, and significantly inhibited the growth of irradiated and non-irradiated (abscopal) tumor as compared with monotherapy. Characterization of the tumor stroma of treated animals reveals that RT promotes immune recognition of tumor-associated vasculature by anti-TEM1 T cells. Interestingly, blocking the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) interaction during dual therapy augmented epitope spreading toward the dominant gp70 viral-antigen while paradoxically reducing the frequency of vaccine-induced responses against self TEM1 antigen.