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. 2015 Dec 15;21(24):5543-51.
doi: 10.1158/1078-0432.CCR-14-2009. Epub 2015 Jul 17.

Defining Effective Combinations of Immune Checkpoint Blockade and Oncolytic Virotherapy

Juan J Rojas  1 , Padma Sampath  1 , Weizhou Hou  1 , Steve H Thorne  2
Affiliations

Defining Effective Combinations of Immune Checkpoint Blockade and Oncolytic Virotherapy

Juan J Rojas et al. Clin Cancer Res. .

Abstract

Purpose: Recent data from randomized clinical trials with oncolytic viral therapies and with cancer immunotherapies have finally recapitulated the promise these platforms demonstrated in preclinical models. Perhaps the greatest advance with oncolytic virotherapy has been the appreciation of the importance of activation of the immune response in therapeutic activity. Meanwhile, the understanding that blockade of immune checkpoints (with antibodies that block the binding of PD1 to PDL1 or CTLA4 to B7-2) is critical for an effective antitumor immune response has revitalized the field of immunotherapy. The combination of immune activation using an oncolytic virus and blockade of immune checkpoints is therefore a logical next step.

Experimental design: Here, we explore such combinations and demonstrate their potential to produce enhanced responses in mouse tumor models. Different combinations and regimens were explored in immunocompetent mouse models of renal and colorectal cancer. Bioluminescence imaging and immune assays were used to determine the mechanisms mediating synergistic or antagonistic combinations.

Results: Interaction between immune checkpoint inhibitors and oncolytic virotherapy was found to be complex, with correct selection of viral strain, antibody, and timing of the combination being critical for synergistic effects. Indeed, some combinations produced antagonistic effects and loss of therapeutic activity. A period of oncolytic viral replication and directed targeting of the immune response against the tumor were required for the most beneficial effects, with CD8(+) and NK, but not CD4(+) cells mediating the effects.

Conclusions: These considerations will be critical in the design of the inevitable clinical translation of these combination approaches. Clin Cancer Res; 21(24); 5543-51. ©2015 AACR.See related commentary by Slaney and Darcy, p. 5417.

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Conflict of interest statement

Conflict of Interest: Dr. Steve Thorne holds equity in Western Oncolytics Ltd. None of the other authors have any conflict of interest.

Figures

Figure 1
Figure 1. Combining Oncolytic Vaccinia Virus and anti-CTLA4 antibody therapies
(a) Anti-CTLA4 antibody injection reduces Vaccinia Virus replication in the tumor in vivo. Balb/c mice with subcutaneous Renca tumors (renal adenocarcinoma) were randomized and injected with a single intravenous dose of 2×108 plaque-forming units (pfu) per mouse of oncolytic B18R- Vaccinia Virus (VV). In the combination group, 100 μg of mouse anti-CTLA4 antibody were injected intraperitoneally on days 0, 3 and 6 post-virus administration. Bioluminescence imaging was used to follow viral luciferase transgene expression from within the tumor. Mean values of 9–10 animals +SD are plotted. Representative luciferase signals from at day 3 post-injection are also depicted (tumors are circled). (b) Viral/anti-CTLA4 combiunation results in increased levels of Vaccinia-specific cytotoxic T cells (CTLs). Mice were treated as in (a), adding PBS and single therapy with anti-CTLA4 antibody as additional controls. At day 3 and 8 post-virus injection, spleens were harvested and quantified by IFN-γ ELISpot assay for Vaccinia-reacting T cells. Values of individual mice and means ± SEM of the different treatments are plotted. (c) Alternative schedule for Vaccinia Virus and anti-CTLA4 antibody combination. Anti-CTLA4 antibody doses were administrated at days 4, 7 and 10 after virus injection, in an approach designed to permit an initial period of viral replication. (d) Injection of anti-CTLA4 antibody after Vaccinia Virus replication improves therapeutic activity of combination therapy. Mice (Balb/c bearing Renca tumors) were treated as before or in combination with anti-CTLA4 antibody as depicted in (c). Relative tumor growth and (e) Kaplan-Meier survival curves are plotted. For survival curves, the end point was established at a tumor volume of ≥750 mm3. Mean values of 7–8 mice/group +SE are plotted. (* P<0.05 compared with VV group; φ P<0.05 compared with PBS group; ψ P<0.05 compared with anti-CTLA4 group; # P<0.05 compared with VV+anti-CTLA4 day 0 group).
Figure 2
Figure 2. Combination of Vaccinia Virus with anti-CD25 antibody did not provide any therapeutic advantage
(a) Anti-CD25 antibody therapy effect on Vaccinia Virus replication. Balb/c mice bearing Renca tumors were injected intravenously with 2×108 PFU/mouse of oncolytic Vaccinia Virus (VV, strain B18R-). For the combination group, a dose of 200 μg of mouse anti-CD25 antibody was also injected intraperitoneally at days 0, 3 and 6 post-virus administration. Viral luciferase expression from within the tumor was quantified at indicated time points by bioluminescence imaging. Mean values of 7–8 animals +SD are plotted. Bioluminescence signals from one representative animal of each group at day 3 post-administration are also shown (tumors are circled). (b) Mice (Balb/c with subcutaneous Renca tumors) were treated IV with 2×108 pfu of VV (n=10–12 per group). For combination groups, anti-CTLA4 or anti-CD25 antibodies were injected with 100 or 200 μg/mouse, respectively, at days 4, 7 and 10 post-virus injection. PBS was injected intraperitoneally as a control. Tumor growth was followed by caliper measurements. Means +SE are plotted. (*P<0.05 compared with PBS group; # P<0.05 compared with VV group; φ P<0.05 compared with VV+anti-CD25 day 4 group).
Figure 3
Figure 3. Therapeutic activity of oncolytic vaccinia in combination with anti-CTLA4 antibody is viral strain dependent
2×108 pfu of oncolytic Vaccinia Virus (B18R- or vvDD) were administrated intravenously to Balb/c mice bearing subcutaneous Renca tumors. At days 4, 7 and 10 after virus injection, a dose of 100 μg of anti-CTLA4 antibody was injected intraperitoneally. B18R- displayed greater inhibition of tumor growth relative to vvDD. Relative tumor volume after virus administration is plotted (n=12–15 mice/group +SE). (* P<0.05 compared with PBS group; φ P<0.05 compared with vvDD+anti-CTLA4 day 4 group).
Figure 4
Figure 4. Optimized combination therapy results in synergistic anticancer activity
(a) Renca (left) or MC38 (right) tumors were implanted into Balb/c or C57/Bl6 mice respectively. Mice were injected with PBS or 2×108 pfu of B18R- oncolytic Vaccinia Virus (VV) through the tail vein. For the anti-CTLA4 group, 100 μg of anti-CTLA4 antibody was injected intraperitoneally at days 0, 3 and 6. For the combination group, anti-CTLA4 antibody doses were administrated at days 4, 7 and 10 after virus injection. Tumor volumes were measured and relative tumor volume +SE of the 12–15 mice/group is plotted. (b) Combination therapy increases cytotoxic T cells recognizing tumor antigens. Cellular immune responses to tumor cells was evaluated by IFN-γ ELISpot assay. At day 11 post-virus administration spleens were harvested from Balb/c mice bearing Renca tumors and treated as in (a). Splenocytes were evaluated for CTLs recognizing Renca cells. Values of individual mice and means ± SEM are depicted. (*P<0.05 compared with PBS group; φ P<0.05 compared with VV group; # P<0.05 compared with anti-CTLA4 group)
Figure 5
Figure 5. Altered T-cell repertoire in the tumor after Vaccinia/anti-CTLA4 combination therapy
Balb/c mice with subcutaneous Renca tumors were treated as before (Fig 4) and tumors were harvested at day 11 post-virus injection and evaluated for lymphocyte populations by flow cytometry. Number of (a) CD3+CD4+ and (b) CD3+CD8+ cells per 200000 total cells are plotted. (c) Representative distributions of CD4+ and CD8+ populations within CD3+ population within the tumor. (d) Percentage of regulatory T-cells (CD25+Foxp3+) within the CD3+CD4+ population of the tumor. Values for individual tumors and means +SEM are plotted. (e) Numbers of NK cells (NKp46+NKg2D+CD3−) and NK-T cells (NKp46+NKg2D+CD3+) per 200 000 events within the tumor. (* P<0.05 compared with PBS group; # P<0.05 compared with anti-CTLA4 group; φ P<0.05 compared with B18R- group).
Figure 6
Figure 6. Depletion of different immune cell subsets alters Vaccinia Virus replication and anti-tumor activity of combination therapy
(a) Replication of Vaccinia Virus (VV strain B18R-) is increased by depletion of CD4+, CD8+, and NK cells. Balb/C mice injected with CD4, CD8, and NK depleting antibodies were challenged with Renca tumors and treated as before (Fig 4). Viral luciferase expression from within the tumor was quantified at indicated time points by bioluminescence imaging. Mean values of 12–13 animals +SD are depicted. (b) CD8+ and NK cells are essential for the anti-tumor activity of Vaccinia Virus/anti-CTLA4 combination therapy. Mice were treated as in (a) and tumor growth was monitored (+SE, 12–15 mice/group) (*P<0.05 compared with PBS group; # P<0.05 compared with No depletion group).
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