Abstract 254: TiMi1 is a novel immune-checkpoint in solid tumors identified via a tumor-infiltrating lymphocyte (TIL)-based RNAi screening
Authors: Michels Tillmann , Hartl Christina A. , Khandelwal Nisit , Breinig Marco , Sorrentino Antonio , Mäder Christina , Umansky Ludmila , Poschke Isabel , Offringa Rienk , Boutros Michael , Eisenberg Galit , Lotem Michal , Beckhove Philipp 
Published: August 2015

Abstract
Immunotherapeutic treatment of melanoma achieved major progress in recent years leading for the first time to improved survival. However, since melanoma cells employ various suppressive mechanisms in order to evade recognition and destruction by immune effector cells many patients still do not benefit from immunotherapy. These mechanisms are far more diverse than reflected by currently used immune modulatory drugs. In this study, we established and utilized a novel high throughput RNAi screening to identify new immune checkpoint molecules in melanoma using antigen-specific patient-derived tumor infiltrating lymphocytes (TILs) in conjunction with primary HLA-matched melanoma cells. Using this approach, we screened a siRNA library targeting more than 1200 surface receptors and kinases to explore novel targets for immunotherapy.
Briefly, HLA-A2 and luciferase positive M579-A2-luc melanoma cells were reversely transfected with the siRNA library and then co-cultured with MART1- and gp100-specific TILs to validate the TIL-mediated tumor lysis. Local regression models (LOESS) were applied to generate a hit list of 48 candidates that negatively regulated CTL cytotoxicity. Interestingly, four candidates of a related breast cancer screen were among the top hits. To streamline the discovery process for large scale molecule libraries, we established a secondary screen assaying multiple T cell activation markers, including effector cytokines.
One of the strongest candidates from our primary and secondary screening is TiMi1 (name altered), a cell surface receptor belonging to the class of GPCRs. We found that knock-down of TiMi1 increased TIL-mediated killing of M579-A2-luc without affecting their viability. TiMi1 knock-down increased TIL activity measured by production of type 1-associated cytokines (e.g. IFN γ and TNF-α), reduced TC apoptosis and increased markers associated with raised activity and cytotoxicity (4-1BB and CD107a). We were able to verify the immune checkpoint function of TiMi1 in melanoma patients using an autologous set of melanoma cells and TILs. Phosphoplex analysis in T cells revealed an involvement of the transcription factor CREB in the mode of action of TiMi1. Preliminary experiments suggest that TiMi1 inhibits anti-tumor immune responses in pancreatic (PDAC) and colorectal (CRC) cancers as well.
In summary, we established a novel antigen-specific screening approach for immune checkpoints expressed in melanoma and were able to identify TiMi1 as a promising candidate. Moreover, TiMi1 inhibits T cell responses in melanoma, PDAC and CRC and might be an interesting target for immunotherapy. Our novel high-throughput screening offers a systematic platform to uncover the “immune-modulatome” of cancer and subsequently discover novel targets for immunotherapy. Since the presented work is considered for patent protection, some gene targets are masked in the presented study.
Citation Format: Tillmann Michels, Christina A. Hartl, Nisit Khandelwal, Marco Breinig, Antonio Sorrentino, Christina Mäder, Ludmila Umansky, Isabel Poschke, Rienk Offringa, Michael Boutros, Galit Eisenberg, Michal Lotem, Philipp Beckhove. TiMi1 is a novel immune-checkpoint in solid tumors identified via a tumor-infiltrating lymphocyte (TIL)-based RNAi screening. [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 254. doi:10.1158/1538-7445.AM2015-254