Abstract 4155: Identification and characterisation of Affimer Proteins that are able to bind and agonise human co-stimulatory cell surface targets
Authors: Basran Amrik 
Published: August 2019

Abstract
Despite the impressive success of PD-1 pathway and CTLA-4 targeted immunotherapies, there remains a large proportion of cancer patients with many tumor types, that fail to benefit or acquire resistance during therapy. Co-stimulation of the tumor microenvironment may prove to enhance current checkpoint inhibitor therapies and kick start T-cell activity in tumors that have poor immune infiltrate. CD40 is a co-stimulatory protein and Glucocorticoid-induced TNFR-related protein (GITR) is a checkpoint inhibitory protein, both a member of the TNF-receptor superfamily. CD40 is expressed on a range of antigen presenting cells (APCs) binding to its ligand CD40L expressed on CD4+ T-cells, whilst GITR is expressed on T-cells. CD28 is also expressed on T-cells and provides co-stimulatory signals for T-cell activation and survival. CD40, GITR and CD28 are currently being investigated to provide complementary immune-modulation of the tumor micro environment alongside checkpoint inhibitor blockade therapy.
Affimer biotherapeutics are a new protein scaffold with great potential for the generation of biotherapeutics. Based on the human protease inhibitor Stefin A, the scaffold is small (14kDa), lacks any post translational modifications such as disulfide bonds and expresses at high levels recombinantly. The Affimer biotherapeutic platform which is based on the human protein Stefin A, has been used to generate binders to members of the TNFR superfamily (CD40, and GITR) and CD28 using phage display. Selections were carried out with an Affimer phage library of size 6x 1010 made with two random loops each of size nine amino acids. With the Affimers raised against huCD40, we were able to encode those molecules as DNA with a membrane anchor sequence. These molecules were expressed transiently in HEK293 cells in vitro, shown to be correctly folded and anchored on the cell surface. The engineered cells were shown to be able to agonise huCD40 in the HEK Blue reporter gene assay. GITR binding Affimer proteins were able to agonise huGITR in a NF-κB Luciferase assay and huCD28 binding Affimer proteins were able to agonise CD28 in a IL-2 Luciferase gene reporter assay.
We have demonstrated that Affimers can be used to agonise important co-stimulatory cell surface targets, either as recombinant protein or when displayed on a cell surface. Encoding the genes for these Affimers and delivering them directly to a tumour for expression (e.g. using an oncolytic virus) could offer a novel method of safely agonising these important co-stimulatory targets in cancer patients without generating the toxicity seen with using mAbs administered systemically.
Citation Format: Amrik Basran. Identification and characterisation of Affimer Proteins that are able to bind and agonise human co-stimulatory cell surface targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4155.