Soluble production of a biologically active single-chain antibody against murine PD-L1 in Escherichia coli.
Programmed death ligand 1 (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 (αPD-L1 scFv). The scFv was expressed as soluble protein in the periplasm and culture supernatant of recombinant Escherichia coli and purified with a 6×-His tag using immobile metal affinity chromatography. The dissociation constant of αPD-L1 scFv was determined to be 7.11×10(-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.
Department of Surgery, University of Minnesota, 420 Delaware St SE, MMC195, Minneapolis, MN 55455, USA. Electronic address: dree0036@umn.edu.
Jeremy J
JJ
Drees
Department of Surgery, University of Minnesota, 420 Delaware St SE, MMC195, Minneapolis, MN 55455, USA.
Lance B
LB
Augustin
Department of Surgery, University of Minnesota, 420 Delaware St SE, MMC195, Minneapolis, MN 55455, USA.
Michael J
MJ
Mertensotto
Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, 321 Church St SE, Minneapolis, MN 55455, USA.
Janet L
JL
Schottel
Department of Surgery, University of Minnesota, 420 Delaware St SE, MMC195, Minneapolis, MN 55455, USA.
Arnold S
AS
Leonard
Department of Surgery, University of Minnesota, 420 Delaware St SE, MMC195, Minneapolis, MN 55455, USA.
Daniel A
DA
Saltzman
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D060890:B7-H1 Antigen / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D006801:Humans; D051379:Mice; D019151:Peptide Library; D012333:RNA, Messenger / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D012995:Solubility
D000911:Antibodies, Monoclonal; D060890:B7-H1 Antigen; C498919:Cd274 protein, mouse; D019151:Peptide Library; D012333:RNA, Messenger; D057127:Single-Chain Antibodies
Affinity; Antibody; Avian library; PD-1; PD-L1; Phage display; scFv