## 2 Materials and Methods
### Gene Construction  
The anti-mouse PD-L1 scFv coding gene (GenBank: KF041825.1) with both an N-terminal Cys-tag and a C-terminal His- and Flag- tags was chemically synthesized and amplified by polymerase chain reaction (PCR) using primers pSQinsertFw55 and pSQin-sertRv57, and KOD-plus Neo DNA polymerase. The product was digested using _Agel_ and _HindIII_, then inserted into _Agel_- and _HindIII_-digested pSQ:aBGPscFv [9] using ligation high ver2, resulting in pSQ:aPDL1 scFv. Afterwards, an N-terminal Strt-tag was added in front of the Cys-tag by PCR using primers CysToStCrCys_Fw and Infusion_His_ToHisFlag-Rv, Hercules II Fusion DNA Polymerase, and pSQ:aPDL1 scFv as a template. The product was inserted into _Agel_- and _BamHI_-digested pSQ:aPDL1 scFv, resulting in pSrTcys::PDL1.  
To construct pSrTcys::aPDL1 VH, VH domain of pSrTcys::aPDL1 scFv was digested using _Ndel_ and _BgIII_, and the extracted DNA was amplified by PCR using primers pSQin-sertFw55 and aPDL1 VH_Rv_infusion, and Hercules II Fusion DNA Polymerase. Vector DNA was amplified by PCR using primers vectorPCR_Ndel_Rv and vectorPCR_BamHI_Fw, and KOD-plus Neo DNA polymerase. The purified PCR products were ligated using an In-Fusion HD cloning kit, resulting in pSrTcys::aPDL1 VH.  
To construct pSrTcys::aPDL1 VL, VH domain was eliminated from pSrTcys::aPDL1 scFv using primers VH_deletion_Fw and VH_deletion_Rv, pSrTcys::aPDL1 scFv as a template, and KOD-One DNA polymerase. The PCR product was incubated at 37 \({}^{\circ}\)C for 2 h with  
Figure 1: Schematic representations of full-sized antibody, antigen-binding fragment (Fab), single chain variable fragment (scFv), variable heavy chain (VH), and variable light chain (VL).  
10 U DpnI, followed by inactivation of the enzyme at 80 \({}^{\circ}\)C for 20 min, resulting in pStrCys::aPDL1 VL.  
The primer sequences are listed in Table 1. The obtained plasmids were prepared using the plasmid Miniprep system, and the entire coding-region sequences were confirmed by sequencing (Table 2).  
saline (PBS, pH 7.4), 0.5 M sodium chloride (NaCl), 10 mM imidazole, and 5% glycerol (pH 7.4)), and resuspended in 15 mL of lysis buffer (50 mM PBS (pH 7.4), 0.5 M NaCl, 10 mM imidazole, 5% glycerol, and 1 mM PMSF), followed by sonication (50% power, 2 sec on/off). After centrifugation (13,000 rpm for 30 min at 4 \({}^{\circ}\)C), the supernatant was purified via gravity purification method using 2 mL of TALON metal-affinity resin-packed column as follows: the supernatant was bound to bead at 4 \({}^{\circ}\)C, and the beads were washed with 15 mL of binding buffer followed by 60 mL of washing buffer (50 mM PBS, 0.5 M NaCl, and 20 mM imidazole). After series addition of 1 mL of elution buffer (50 mM PBS, 0.3 M NaCl, and 250 mM imidazole), each fraction was collected using a disposable gravity column. The eluent was subjected to an ultrafiltration device (3 k), equilibrated with PBS, and concentrated to 250 \(\mu\)L. Protein expression and purification were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and protein concentration was determined on the gel using various concentrations of bovine serum albumin (BSA) as a standard.