Abstract 4706: TCR engineered adoptive T-cell therapy for lung cancer is augmented by combined PD1 and TIM3 antibody blockade
Authors: Moon Edmund K. , Ranganathan Raghuveer , Liu Xiaojun , Verona Raluca , Snyder Linda , June Carl H. , Zhao Yangbing , Albelda Steven M. 
Published: August 2015

Abstract
INTRODUCTION:
ACT has shown great promise in melanoma and hematologic malignancies; however one major limitation of engineered T cells targeting solid tumors is likely to be tumor microenvironment-induced hypofunction of the T cells. To study and limit this problem, we have developed a model in which human T cells engineered to target the antigen NYESO1 using a high-affinity engineered TCR (Ly95) are injected into mice bearing human A549 lung cancer cells. Using this model we demonstrate upregulation of PD1 and TIM3 on Ly95 TILs. We were able to augment T cell anti-tumor activity by combining Ly95 T cell therapy with anti-hPD1 and anti-hTIM3 antibodies.
Materials and Methods:
In vitro: Human T cells activated by anti-CD3/CD28 Dynabeads and transduced with lentivirus had 50% expression of Ly95 TCR as measured by flow cytometry. They were cocultured with marked tumor cells to measure IFNγ release and antigen-specific killing. In vivo: Immunodeficient mice with 200mm3 flank A549-A2-ESO (AAE) tumors received 107 T cells via tail vein. Three weeks later, tumors were harvested/digested, and human TILs were isolated/assessed for tumor killing/IFNγ secretion. This was repeated after the TILs were rested for 24hrs at 37°C/5%CO2. The number of TILs and PD1/TIM3 expression on the isolated TILs were assessed by flow cytometry at fresh harvest and post rest. The in vivo experiment was repeated comparing Ly95 T cells alone vs. Ly95 T cells plus either/both intraperitoneal (IP) anti-hPD1 or/and IP anti-hTIM3 at 10mg/kg every 5 days.
Results:
Ly95 TCR T cells were able to kill AAE tumor cells and secrete high amounts of IFNγ in an antigen-specific/dose dependent fashion after 18hr coculture. 107 IV Ly95 T cells were able to slow AAE flank tumor growth as compared to control tumors (498mm3 vs. 1009mm3, p&lt;0.05.) Flow cytometric analysis of harvested/digested tumors revealed that 5.2% of the tumor digest was human TILs. Freshly isolated TILs were hypofunctional in their ability to kill tumor cells and release IFNγ when compared to cryopreserved Ly95 T cells (p&lt;0.05.) After overnight rest away from tumor, TILs improved in function. Further analysis revealed that Ly95 TILs had upregulated their expression of PD1 and TIM3 (increase from 5 to 40% in PD1 and from 17 to 50% in TIM3.) Combining a single Ly95 T cell IV injection with multiple IP anti-hPD1 and anti-hTIM3 injections resulted in 43% reduction in flank tumor size compared to Ly95 T cell injection alone (189mm3 vs. 332mm3, p&lt;0.05.)
Conclusions:
The PD1 and TIM3 pathways are involved in tumor-induced hypofunction of TCR engineered TILs. Combining anti-hPD1 and anti-hTIM3 antibodies with TCR T cells, and likely CAR T cells, will likely enhance the efficacy of these approaches in lung cancer and other solid tumors.
Citation Format: Edmund K. Moon, Raghuveer Ranganathan, Xiaojun Liu, Raluca Verona, Linda Snyder, Carl H. June, Yangbing Zhao, Steven M. Albelda. TCR engineered adoptive T-cell therapy for lung cancer is augmented by combined PD1 and TIM3 antibody blockade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4706. doi:10.1158/1538-7445.AM2015-4706