Abstract A56: Identification and characterization of an alternative cancer-derived PDL1 isoform
Authors: Hassounah Nadia B. , Freeman Samuel , Beauchamp Ellen M. , Pedamallu Chandra S. , Koyama Shohei , Souders Nicholas , Martin Sunil , Akbay Esra A. , Freeman Gordon J. , Dranoff Glenn , Wong Kwok-Kin , Hammerman Peter S. 
Published: March 2017

Abstract
Cancer immunotherapy has been demonstrated to be an effective strategy for the treatment of numerous tumor types. Antibodies targeting the PD1 receptor and its ligands, PDL1 and PDL2, have recently been FDA approved for multiple tumor types. Although there has been considerable success in treating patients using anti-PD1/PDL1 therapies, only a subset of patients respond to this treatment, highlighting a gap in the understanding of the mechanisms driving immune evasion in cancer. We previously identified a novel potential mechanism of immune evasion in a pan-cancer computational analysis of HPV integration sites in the human genome (Parfenov et al. PNAS. 2014), where we detected a case in which HPV integration into the PDL1 locus was predicted to drive expression of a novel truncated PDL1 isoform (tPDL1) lacking the transmembrane domain. We aimed to characterize the localization and function of tPDL1 with the hypothesis that tPDL1 functions as an immunomodulatory factor which will act as a soluble negative regulator of T cell function. We extended our observations into a large cohort of human cancers (Cancer Cell Line Encyclopedia and TCGA Network) and demonstrated that this novel form of PDL1 is expressed in multiple cancer types including bladder, breast, lung, ovarian and other non-HPV-driven cancers. In human cancer cell lines predicted to have high secreted PDL1 levels (RKO, CAL62), we were able to measure a secreted form of PDL1 protein by western blotting of protein precipitation of cell supernatants and/or by ELISA. In contrast, we were unable to detect secreted PDL1 in the media from negative control cells HEK293T and RERF-LC-Ad1 (predicted to have high wildtype (WT) PDL1 but low secreted PDL1). We expressed tPDL1 in HEK293T cells and found that a higher proportion of tPDL1 is secreted compared to WT PDL1, as measured by ELISA and western blotting of protein precipitation from cell supernatants and cell lysates. Surface staining for tPDL1 also verified that tPDL1 is not membrane-bound, as measured by flow cytometry. The ability of tPDL1 to inhibit T cell function was assessed by measuring IL2 and IFNγ secretion. We noted a reduction in both IL2 and IFNγ secretion in primary human T cell blasts which were incubated with recombinant FC-tagged tPDL1 or FC-tagged PDL1 (R&D, positive control), compared to FC-tagged IgG1 (R&D, negative control), in the presence of anti-CD3 antibody stimulation. The data support the hypothesis that tPDL1 acts as a soluble negative regulator of T cell function. Future directions will include more extensive characterization of inhibition of T cell function by tPDL1.
Citation Format: Nadia B. Hassounah, Samuel Freeman, Ellen M. Beauchamp, Chandra S. Pedamallu, Shohei Koyama, Nicholas Souders, Sunil Martin, Esra A. Akbay, Gordon J. Freeman, Glenn Dranoff, Kwok-Kin Wong, Peter S. Hammerman. Identification and characterization of an alternative cancer-derived PDL1 isoform. [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 A56.