1. Oncolytic Vaccinia Virus Expressing IL15/IL15Rα Fusion Protein Induces Immune Cell Infiltration into Tumor and Improves Anti-tumor Efficacy
Stacy J. Kowalsky MD, Z. Sheng Guo PhD,
Roshni Ravindranathan MS, David L. Bartlett MD
University of Pittsburgh, Department of Surgery/Division of Surgical Oncology
University of Pittsburgh Cancer Institute

2. Disclosures
All authors deny conflicts of interest

3. Background Vaccinia Virus
DNA virus, transcribed in cytoplasm using viral-derived proteins
vvDD = tumor-selective Vaccinia virus
Attenuated
Oncolytic activity1
Accepts 25 kb foreign DNA2
Novel virus design (GM-CSF3)
1-McCart et al. 2001, Cancer Research; 2-Smith and Moss 1983, Gene; 3-Heo et al. 2013, Nat Med

4. Interleukin 15
Proliferation/activation of CD4+/CD8+ T cells and NK cells
Memory CD8+ T cells
Shared β and γ receptor chains with IL2
Unique α chain
Transpresentation to NK and memory T cells
Fusion protein improved activity over IL15 alone4
Tosic and others5 (University of Illinois-Urbana Champaign)
Myxoma expressing functional IL15/IL15Rα
B16-F10 melanoma model
CD8+ T and NK cells infiltration
Improved survival
4-Dubois et al. 2008, J Immunol; 5-Tosic et al. 2014, PLoS One

5. Hypothesis
A novel Vaccinia virus expressing IL15/IL15Rα fusion protein (vvDDIL15Rα) will induce CD8+ T and NK cell infiltration to the tumor microenvironment (TME), enhancing anti-tumor efficacy over vvDD

6. Experimental Designs MC38 murine colorectal cancer model
Examine TME of transplantable tumor following IV administration of vvDDIL15Rα or vvDD
Monitor tumor progression/survival in carcinomatosis model with locoregional virus
Combine virus with immunotherapy (PD1 blockade)
In vitro replication analysis

7. TME Analysis Control Tumor Tissue 1e8 pfu vvDD IV Day 2, 4, 6
1e8 pfu vvDDIL15Rα IV

8. A34R IL15/IL15Rα
*** p=0.0001
* p<0.05, ** p<0.005

9. CD8 D2 D4

10. NK cell Markers D2
NKG2D
NKp46 D4 D4 D6 D6
* p< *** p<0.001

11. GZMB TGFβ D4 D6
** p=0.001
*** p=0.0001

12. Carcinomatosis Model Day 0 Day 5 Tumor Progression Survival MC38luc
Control
Day 0
Tumor Progression
Survival
MC38luc
2e8 pfu vvDD
Day 5
2e8 pfu vvDDIL15Rα

13. Control
vvDD
vvDDIL15Rα D4 D9

14. Tumor Progression

15. *** p<0.0010

16. MC38 Tumor Rechallenge IP MC38-inoculated mice “cured” with vvDDIL15Rα
MC38luc
IP MC38-inoculated mice “cured” with vvDDIL15Rα
(Day 132 after previous IP MC38)
MC38luc
Naïve WT mice

17. Tumor Growth

18. PD1 blockade T cell Tumor cell
PDL-1
Response to PD1 inhibitors correlates with PDL-1 expression in TME6
Chronic viral infection associated with PD-1/PDL-1 immune escape7
vvDD treatment → enhanced PDL-1 expression in TME
PD-1 blockade
6-Topalian et al. 2012, NEJM; 7-Shin and Wherry 2007, Curr Opion Immunol

19. Carcinomatosis Model with PD1 Blockade
Control
Anti-PD1 Ab
7 days
2e8 pfu vvDD IP
Anti-PD1 Ab
IP MC38luc
2e8 pfu vvDDIL15Rα IP
Anti-PD1 Ab

20. Tumor progression

22. *** **
*** p=0.0003, ** p=0.002
*** p=0.0007, **** p<0.0001

23. In vitro MC38 Replication Assays
MOI 0.1
MOI 1

24. Conclusions vvDDIL15Rα Improved anti-tumor efficacy over vvDD
Replicates in MC38 in vitro and in vivo
Induces changes in TME, with ↑ expression of CD8 and activating NK cell receptors
Improved anti-tumor efficacy over vvDD
“Cured” mice show anti-tumor immune memory
Combination with PD1 blockade dramatically improves effect

25. Questions?

26. Sources
McCart JA, Ward JM, Lee J, Hu Y, Alexander HR, Libutti SK et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Research. 2001;61:
Smith GL and Moss B. Infectious poxvirus vectors have capacity for at least base pairs of foreign DNA. Gene. 1983;25:21-8.
Heo J, Reid T, Ruo L, Breitbach CJ, Rose S, Bloomston M et al. Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer. Nature Medicine. 2013;19(3):
Dubois S, Patel HJ, Zhang M, Waldmann TA, Müller JR. Preassociation of IL-15 with IL-15Rα-IgG1-Fc enhances its activity on proliferation of NK and CD8+/CD44highT cells and its antitumor action. Journal of Immunology. 2008;180:
Tosic V, Thomas DL, Kranz DM, Liu J, McFadden G, Shisler JL, et al. Myxoma virus expressing a fusion protein of interleukin-15 (IL15) and IL15 receptor alpha has enhanced antitumor activity. PLoS One. 2014;9(10):e
Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF et al. Safety, activity, and immune correlates of anti–PD-1 antibody in cancer. New England Journal of Medicine. 2012;366(26):2443–54
Shin H and Wherry EJ. CD8 T cell dysfunction during chronic viral infection. Current Opinion in Immunology. 2007;19: