Advent Life Sciences (United Kingdom)

Abstract Affimer® biotherapeutics are novel 15kDa scaffold proteins based on the human protease inhibitor Stefin A. Affimer® proteins lack disulfide bonds and post translational modifications including glycosylation. Large, diverse phage display libraries have been created which incorporate peptide loops into the scaffold backbone. We have previously identified high-affinity competitive binders to human programmed death-ligand 1 (PD-L1) using phage display which block interaction with programmed death 1 (PD-1). Lead anti-PD-L1 Affimer® proteins were formatted by fusing to human Fc or as in-line-fusions to a serum albumin binding Affimer® to extend pharmacokinetic half-life. Here we describe novel immunocytokines that consist of half-life extended Affimer® antagonist to PD-L1 fused to human Interleukin 15 (IL-15) receptor alpha sushi domain and human IL-15 (PDL1-IL15 Affimer® fusion proteins). PD-L1 is upregulated on tumor cells, macrophages, and dendritic cells in the tumor microenvironment. PD-L1 engagement with PD-1 on T cells inhibits T cell proliferation, transcriptional activation, and cytokine release, resulting in suppressed anti-tumor response. IL-15 signals through the IL-2 receptor beta chain and the common gamma chain (IL-2Rβ/γ) and induces differentiation and proliferation of CD8+ T cells and Natural Killer (NK) cells; it does not stimulate regulatory T cells (Tregs). Immunostimulatory cytokines have therapeutic potential but are often associated with toxicity when administered systemically. PDL1-IL15 Affimer® fusion proteins have the potential to bring the immunomodulatory cytokine directly to the tumor and reverse the immunosuppressive tumor microenvironment by enhancing T cell activation, while also introducing a checkpoint inhibitor to augment the anti-tumor immune response. We characterized binding and activity of PDL1-IL15 Affimer® fusion proteins in in vitro assays. PDL1-IL15 Affimer® fusion proteins bind both PD-L1 and IL-2Rβ/γ and exhibit PD-1:PD-L1 blocking activity and IL-2Rβ/γ activation. PDL1-IL15 Affimer® fusion proteins reversed T cell exhaustion in a human mixed lymphocyte reaction assay: PDL1-IL15 Affimer® fusion proteins showed greater activity than either the PD-L1 or the IL-15 component alone. Furthermore, PDL1-IL15 Affimer® fusion proteins enhanced immune cell killing of tumor cells in co-culture assays of human peripheral blood mononuclear cells and tumor cells. In summary, data suggests that PDL1-IL15 Affimer® fusion proteins can reverse T cell exhaustion and induce immune cell activation to elicit an anti-tumor effect. Ongoing in vivo work aims to investigate the efficacy and tolerability profile of PDL1-IL15 Affimer® fusion proteins to direct future development of this therapeutic. In addition, the data will support wider utility of the Affimer® platform to develop new ways of targeting the tumor while reducing systemic toxicity. Citation Format: Victoria Juskaite, Deepa Avisetti, David H Jones, Ahmad Sobri, Gregory Billenness, Hanna Buist, Tik Nga Tong, Norhakim Yahya, Fiona McLaughlin. A novel, tumor-targeted immunocytokine comprising an anti-PD-L1 Affimer® fused to IL-15 exhibits potent anti-tumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A077.