Identification Of Tumor Necrosis Factor Receptor Ii As A Regulatory T Cell Target For Cancer Immunotherapy Using Designed Ankyrin Repeat Protein Phenotypic Selections

Regulatory T cells (Treg cells) are observed within multiple tumor types and contribute to immune evasion. Effective and specific targeting of Treg cells by antibodies is hampered by the lack of a unique surface marker. With the aim of identifying such a marker, human Treg cells were isolated from peripheral blood, expanded in vitro and used as a source of antigen in phage display selections with a diverse library of designed ankyrin repeat proteins (DARPins). The resulting DARPins were screened for binding to multiple cell-types, and approximately thirty with preferential binding to Treg cells were identified. All of these DARPins were determined to bind to the third and/or fourth cysteine-rich extracellular domains of tumor necrosis factor receptor II (TNFRII, also known as TNFRSF1B or CD120b). Expression of TNFRII has previously been demonstrated on Treg cells, and TNFRII is shown here also to be expressed on CD8+ T cells following exposure to cognate antigen and on the surface of tumor-infiltrating T cells in the 4T1 mouse tumor model. Furthermore, TNFRII mRNA was detected at increased levels in tumor-infiltrating leukocytes isolated from cancer patients. Functional characterisation of the anti-TNFRII DARPins and mAbs revealed an ability to enhance T-cell activation in response to polyclonal and antigen specific stimuli, and in Treg cell suppression assays. These studies demonstrate the utility of DARPins for phenotypic selections to identify drug targets using primary human lymphocytes and highlight TNFRII as a potential therapeutic target enriched on Treg cells. Citation Format: Geoff Williams, Judith Anderton, Vahe Bedian, Jane Coates Ulrichsen, Andrea Gonzalez-Munoz, Sandrine Guillard, Olivia Harris, James Hair, Andrew Leishman, Arthur Lewis, Jacques Moissan, Ralph Minter, Bina Mistry, Julie Parmentier, Edmund Poon, Amy Popple, Steve Rust, Alan Sandercock, Ross Stewart, Viia Valge-Archer, Robert W. Wilkinson. Identification of tumor necrosis factor receptor II as a regulatory T cell target for cancer immunotherapy using designed ankyrin repeat protein phenotypic selections. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4613. doi:10.1158/1538-7445.AM2014-4613