Abscopal Effect in Non-injected Tumors Achieved with Cytokine-Armed Oncolytic Adenovirus

Abstract

Cancer treatment with local administration of armed oncolytic viruses could potentially induce systemic antitumor effects, or the abscopal effect, as they self-amplify in tumors, induce danger signaling, and promote tumor-associated antigen presentation. In this study, oncolytic adenovirus coding for human tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 (also known as [a.k.a.] TILT-123) provoked antitumor efficacy in tumors that were injected with Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 and those that were left non-injected in the same animal. Importantly, the virus was able to travel to distant tumors. To dissect the effects of oncolysis and cytokines, we studied replication-incompetent viruses in mice. Systemic antitumor effects were similar in both models, highlighting the importance of the arming device. The cytokines induced positive changes in immune cell infiltrates and induced the expression of several immune-reaction-related genes in tumors. In addition, Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 was able to increase homing of adoptively transferred tumor-infiltrating lymphocytes into both injected and non-injected tumors, possibly mediated through chemokine expression. In summary, local treatment with Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 resulted in systemic antitumor efficacy by inducing immune cell infiltration and trafficking into both treated and untreated tumors. Moreover, the oncolytic adenovirus platform had superior systemic effects over replication-deficient vector through spreading into distant tumors.

Keywords: abscopal effect; adenovirus; immunotherapy.

Figure 1

Treatment with Oncolytic Virus Controls the Growth of Both Injected and Non-injected Tumors Hamsters were treated on days 1, 8, 15, 22, and 29 with 1 × 10⁸ VPs intratumorally (i.t.) and with 5 × 10⁷ TILs on day 1 intraperitoneally (i.p.). The growth of injected (A) and non-injected (B) hamster tumors (n = 5–6) was measured every 2–3 days until day 33. During the follow-up period, two animals were sacrificed from the mock group (day 24), two animals from the group receiving TILs only (day 22), and one animal from groups treated with Ad5/3-E2F-d24-hTNF-α (day 29) and Ad5/3-E2F-d24-hIL-2 (day 29). Small amounts of viral DNA were detectable also in non-injected tumors on day 16 (C). There were no differences between the injected and non-injected tumor sizes on day 33 (D). The graphs show mean plus SEM. Statistical differences were evaluated with mixed model analysis; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 2

Replication-Incompetent Virus Induces Growth Control Also in the Non-injected Tumor at an Early Time Point The sizes of injected (A and C) and non-injected (B and D) B16-OVA tumors in mice on day 8 (n = 3–8) without T cell transfer (A and B) or with OT-I cells (C and D) were compared with average mock tumor size on the same time point. Similar results were obtained for injected (E) and non-injected (F) tumors when the experiment was repeated. Viral DNA was detected only in the injected tumors (G). The bars show mean plus SEM. Statistical significance was evaluated with 2-way ANOVA: ****p < 0.0001; ***p < 0.001; *p < 0.05.

Figure 3

Treatment with Armed Viruses Induces Positive Changes in Immune Cells in the Tumor Microenvironment Tumor samples were collected on day 8, and the presence of natural killer cells (A), mature dendritic cells (B), macrophages (C), and M2-like macrophages (D) in tumors was detected with flow cytometry. Melanoma-specific T cells were detected with pentamers and the results with OVA⁺, Trp2⁺, and gp100⁺ cells pooled into one graph (E). Statistical differences were analyzed against corresponding mock tumor. The bars show mean plus SEM. Black bar indicates mock control, white bars unarmed virus or OT-I controls, and gray bars treatment groups with armed viruses. Unpaired t test was performed to analyze statistical differences; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 4

Armed Virus Induces TIL and TCR Graft Trafficking to and Persistence in Both Tumors TILs were labeled with radioactive indium, and the trafficking into (A) injected and (B) non-injected tumors was followed with SPECT/CT imaging over time. On day 0, the hamsters received the labeled TILs intraperitoneally and viruses or PBS control intratumorally into one of the two tumors (n = 2/group). The difference between the groups was most prominent 96 hr after the administration of the cells (C). TCR-modified OT-I cells were labeled with fluorescent-labeled nanoparticles, and their presence in injected and non-injected tumors was investigated after 5 days (n = 6) (D). Tumors injected with viruses coding for TNF-α and IL-2 had significantly higher numbers of infused OT-I cells compared with vehicle-treated tumors (Kruskal-Wallis test, *p < 0.05).

Figure 5

List of Genes Upregulated over 2-Fold Compared with Corresponding Mock Group Genes in cluster 1 are upregulated only in non-injected tumors, genes in cluster 2 are upregulated in both tumors, genes in cluster 3 are upregulated with Ad5/3-E2F-d24 injection, genes in cluster 4 are upregulated with either virus, genes in cluster 5 are upregulated with Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 only, and genes in cluster 6 with any injection. Black box indicates over 2-fold upregulation.

Figure 6

List of Genes Downregulated over 2-Fold Compared with Corresponding Mock Group Genes in cluster 1 are downregulated in TIL group, genes in cluster 2 are downregulated with a virus injection, and genes in cluster 3 are downregulated only in non-injected tumors. Black box indicates over 2-fold downregulation.