A212 EXPRESSION AND CHARACTERIZATION OF SOLUBLE PEKIN DUCK PROGRAMMED DEATH-1

Abstract Background Programmed death 1 (PD-1, CD279) negatively regulates TCR complex-initiated signaling by interacting with its cognate ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD 273). Recently, therapeutic targeting aimed at abrogation of co-inhibition through PD-1 has led to breakthroughs in the treatment of cancer and has shown potential for the treatment of chronic viral infections. We have identified the duck PD-1, PD-L1 and PD-L2 proteins that have a sequence identity of 31%, 45% and 36% with human PD-1, PD-L1 and PD-L2, respectively. Aims The aim of this study was to express soluble duck PD-1 (duPD-1) for functional characterization in the duck hepatitis B model. Methods The duPD-1 ectodomain (501 bp) was inserted into pZeo2B in frame with a polyHis tag and expressed in 293T cells. Culture supernatants were assessed immunoblot and soluble duPD-1 was affinity purified and conjugated with RPE. Full-length PD-L1 and PD-L2 fused in frame with eGFP were inserted in pZeo2B and expressed in 293T cells, respectively. Binding of soluble duPD-1 to duPD-L1 and duPD-L2 were assessed by FACS in PD-L1-eGFP-, PD-L2-eGFP- and eGFP-transfected 293T cells. Results Soluble duPD-1 could be expressed as a secreted protein in 293T cells. Affinity purified soluble duPD-1 conjugated with RPE could bind to duPD-L1 and duPD-L2 expressed on 293T cells. Conclusions Soluble duPD-1 can be expressed as a secreted protein and retains binding affinity to duPD-L1 and duPD-L2 that can be used to study the role of PD-1 co-inhibition in the duck hepatitis B infection model. Funding Agencies CIHRCanadian Liver Foundation (CLF) and the Canadian Foundation for Innovation (CFI).