1. Enhancement Of T-Cell Immunity To Osteosarcoma By Modulation Of Programmed Death Receptor Pathway Pooja Hingorani, Danielle Lussier, Joseph Blattman

2. Overview T cell tolerance and immune inhibitory pathways in osteosarcoma Review our data

3. Immune tolerance in osteosarcoma Loss of Class I HLA expression in osteosarcoma tumors related to worse overall survival* CTLA4 polymorphisms are associated with higher risk of developing osteosarcoma** Metastatic osteosarcoma expresses T cell Ig/mucin molecule 3 (TIM3), which in other tumor settings inhibits the function of infiltrating CTL, leading to tumor progression # B7-H3 expression, a co-inhibitory protein involved in tumor immune escape from T cells, inversely correlates with CTL infiltration in human osteosarcoma, and is indicative of poor prognosis in osteosarcoma patients @ *Tshukahara et al., Cancer Sci 2006; **Liu et al, DNA Cell Bio 2011; #Shang et al., Oncol Lett 2013; @ Wang et al., Plos One 2013

4. Relative expression of PDL1 in osteosarcoma Shen J K et al. Cancer Immunol Res 2014;2:690-698 ©2014 by American Association for Cancer Research

5. Overall survival of 37 patients with osteosarcoma in relation to PDL1 gene expression. Shen J K et al. Cancer Immunol Res 2014;2:690-698 ©2014 by American Association for Cancer Research

6. Characterization of the origins of metastases. Shen J K et al. Cancer Immunol Res 2014;2:690-698 ©2014 by American Association for Cancer Research

7. Specific Aims Evaluate expression of PD-L1 on tumor cells and PD-1 on TILs in patient samples and metastatic osteosarcoma cell line (in vitro and in vivo) Evaluate the functional ability of TILs infiltrating metastatic tumors Evaluate the effect of PD-L1 blockade on development and progression of pulmonary metastases in vivo Evaluate the effect of combinational therapies (PD-L1 antibody + chemotherapy; PD-L1 antibody + ipilimumab)

8. PD-L1 is expressed in human metastatic osteosarcoma 50um Isotype Metastatic Osteosarcoma PD-L1 Primary Osteosarcoma 50um 12/ 16 (75% ) of metastatic tumors had some PD-L1 expression

9. PD-1 is expressed on human metastatic TILs 50um Isotype PD-1 50um Metastatic Osteosarcoma Primary Osteosarcoma -13/16 (81%) of metastatic tumors had PD-1+ TILs -In 11/16 (70%) metastatic tumors, PD-1 expression on TILs correlated with PD-L1 expression on tumor cells

10. PD-L1 expression is increased in K7M2 osteosarcoma cells PD-L1 % of Maximum 4073 1848 Pre Implantation Post Tumor 30%

11. TILs from lung metastases are PD-1+ %PD1+ SPLEEN CD8TIL CD8 * CD8 PD-1 SPLEENTIL 94% 6% 87% 13% Isotype PD1 Healthy Lung Late Disease Lung Early Disease Lung 50 µm

12. TILs are functionally impaired in the presence of tumor cells * * %IFNγ+ * * * -+-+ Ag PD-L1+ K7M2 PD-L1- 4T1 %TNF+ * * * -+-+ Ag PD-L1+ K7M2 PD-L1- 4T1 %IL-2+ * * -+-+ Ag PD-L1+ K7M2 PD-L1- 4T1

13. %IFNγ+ Control PD-L1 Ab * * %TNF+ * * * Control PD-L1 Ab %IL-2+ * * * Control PD-L1 Ab A. B. C. PD-L1 blockade restores function to TILs

14. Blockade of PD-1:PD-L1 significantly enhances survival in metastatic osteosarcoma model Days post implant Percent Survival P=0.0005

15. 30 day PD-L1 antibody treatment does not significantly improve survival compared to 15 day treatment Is immune escape occurring?

16. PD-L1 antibody treatment changes inhibitory receptor expression * *

17. PD-L1 antibody treated osteosarcoma is resistant to additional treatment when injected into naïve mice. PD-L1 % of Maximum *

18. Combinational PD-L1 antibody treatment coupled with conventional chemotherapy The combination improves survival as compared to chemotherapy alone but is similar to PD-L1 antibody alone.

19. Combinational PD-L1 antibody coupled with CTLA-4 antibody treatment improves survival

20. In conclusion PD-1/PD-L1 interaction induces immune tolerance in metastatic osteosarcoma PD-L1 blockade improves survival but not 100% Resistance to therapy develops by up-regulation of alternative immune escape pathways Combinational immune therapies may have the highest impact on disease control in metastatic osteosarcoma

21. Joseph Blattman Danielle Lussier John Johnson Lauren O’Neill Lizbeth Nieves Megan McAfee Susan Holechek Paul Dickman Patients and families Acknowledgements