Programmed death ligand I (PD-L1), is an important regulator of T-cell activation and has emerged as an important target for cancer immunotherapy. Single chain variable fragments (scFvs) have several desirable characteristics and are an attractive alternative to monoclonal antibodies for experimental or therapeutic purposes. Three chickens were immunized against murine PD-L1, and mRNA isolated from their spleens was used to generate an immunized immunoglobulin variable region library. Using splice-overlap extension PCR, variable region cDNAs were combined to generate full-length scFvs. M13 phage display of the resulting scFv library identified a functional scFv against PD-L1 (alpha PD-L1 scFv). The scFv was expressed as soluble protein in the periplasm and culture supernatant of recombinant Escherichia coli and purified with a 6x-His tag using immobile metal affinity chromatography. The dissociation constant of alpha PD-L1 scFv was determined to be 7.11 x 10(-10) M, and the scFv demonstrated inhibitory biological activity comparable to an antagonistic monoclonal antibody, providing an alternative agent for blocking PD-1/PD-L1 signaling. (C) 2013 Elsevier Inc. All rights reserved.