Abstract B36: Modulating the tumor microenvironment to enhance cancer immunotherapy by inducing phosphatidylserine expression on the tumor surface
Authors: Kaneko Kensuke , Osada Takuya , Freimark Bruce D. , Lyerly Herbert Kim 
Published: March 2017

Abstract
Background and Hypothesis: While immune checkpoint blockade, in particular PD-1 or PD-L1 inhibition, has revolutionized cancer immunotherapy, a significant number of patients with solid tumors fail to respond to therapy. An emerging consensus is the need for a local infiltration of immune effector T cells, in combination with PD-1 or PD-L1, for anticancer responses. We are exploring an improved strategy to modulate the tumor microenvironment by targeting phosphatidylserine (PS), which is a phospholipid membrane component maintained on the cytosolic side of the cell membrane in viable cells. When a cell undergoes apoptosis or stress induced by the microenvironment, PS is no longer restricted to the cytosolic domain and gets transposed to the extracellular surface where it serves a variety of functions, including as a ligand for another immune checkpoint, TIM-3. In fact, PS-targeting antibodies induce immunity against weakly immunogenic tumor cells (He at al, 2009), and may serve to enhance anti-cancer immunotherapy. Chemotherapy and radiation therapy are often used to treat cancers. In addition, photodynamic therapy using a photosensitizer conjugated with a target specific molecule has been recognized as an emerging therapeutic strategy. Therefore, we sought to determine the expression of PS following treatment with chemotherapy, radiation, or photodynamic therapy, and to determine if the addition of a PS-targeting antibody could augment antitumor response, especially in combination with PD-1 inhibition.
Methods and Results: We first confirmed the upregulation of PS on malignant human cells following treatment with chemotherapy by FACS analysis using PS-targeting antibody mch1N11. PS expression was increased (50.2% positive compared to 5.0% in untreated cells) after 18 h of incubation with etoposide (25µM). We then tested PS expression on murine triple negative breast cancer cell lines (E0771-OVA, 4T1) at days 1, 2, and 4 after a single dose of X-ray (30, 40, or 50 Gy). E0771-OVA cells showed increased PS expression at day 2, while 4T1 cell lines increased PS expression at day 4. E0771-OVA cells were also irradiated using a more conventional schedule, specifically daily administration (6 Gy/day 5 days), leading to increase PS expression. Additionally, we tested PS expression on E0771-OVA cells after photodynamic therapy using HS201, which is a synthesized photosensitizer compound constructed of verteporfin and HSP 90 inhibitor. PS expression increased at day 1 when the cells were treated with a single dose of laser (10 or 60J) after 30 minutes conjugation of HS201 (1 or 10 μM).
After confirming upregulation of PS by chemotherapy, radiotherapy, and photodynamic therapy, we next tested the combination of PS-targeting mAb mch1N11 with chemotherapy in vivo. We treated C57BL/6 mice bearing E0771-OVA tumors with paclitaxel (PTX), PS-targeting mAb mch1N11, anti-PD-L1 Ab, or combination. As single agents, both mch1N11 and anti-PD-L1 mAbs showed tumor growth suppression; however, combination of those two and 3 combination showed stronger effect. On the other hand, PTX didn't seem to add any additional effect.
Conclusions: PS expression is upregulated by chemotherapy, radiotherapy, and photodynamic therapy in vitro. A combination therapy blocking PS by PS-targeting mAb and immune checkpoint blockade with anti-PD-L1 mAb appears superior to single agent therapy. Our future plan is to investigate the effect of local irradiation or photodynamic therapy combined with PS-targeting mAb and anti-PD-L1 Ab.
Citation Format: Kensuke Kaneko, Takuya Osada, Bruce D. Freimark, Herbert Kim Lyerly. Modulating the tumor microenvironment to enhance cancer immunotherapy by inducing phosphatidylserine expression on the tumor surface. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B36.