Pd1 h5229

SPR analysis was performed in HBS-EP+ (BR-1006-69, GE Healthcare) running buffer using the GE Biacore T200. First, human PD-L1 (PD1-H5229, Acro Biosystems Inc.) and human LAG-3 (LA3-H5222, Acro Biosystems) were immobilized onto a CM5 sensor chip (29–1496-03, GE Healthcare) at 300 RU and 180 RU, respectively. For PD-L1 affinity detection, serial two-fold dilutions of each antibody (with a starting concentration of 10 nM) as well as blank running buffer were injected and flowed over the sensor surface with an association time of 180 s and a dissociation time of 600 s in each running cycle. A mixture of 10 mM glycine (pH 1.5, BR-1003-54, GE Healthcare) and 10 mM glycine (pH 2.0, BR-1003-55, GE Healthcare) (v/v = 1:2) was injected for sensor regeneration. For LAG-3 affinity detection, serial two-fold dilutions of each antibody (with a starting concentration of 40 nM) were flowed over the LAG-3 sensor surface with an association time of 180 s and a dissociation time of 600 s. At the end of each cycle, a pulse injection of 3 M MgCl₂ (BR-1008-39, GE Healthcare) was used for sensor regeneration. Raw data were processed using a 1:1 binding model in the Biacore T200 evaluation software version 3.1.

Biacore running buffer was 1×HBS-EP+ Buffer prepared from 10×HBS-EP+ Buffer (GE Healthcare, BR-1006-69). An anti-human IgG chip was prepared by immobilizing antihuman IgG Fc antibody via amine coupling (GE Healthcare, BR-1003-50) on 1–8 flow cells a CM5 chip (GE Healthcare, 29-1496-03). The sensor chip surfaces were then flushed with HBS-EP+ buffer to stabilize the baseline. Then 25 nmol/L monomeric hPD-L1 (ACROBiosystems, PD1-H5229) solution was 2-fold serially diluted and 50 nmol/L monomeric h41-BB (ACROBiosystems, 41B-H5227) solution was 2-fold serially diluted. The serially diluted hPD-L1 and h4-1BB were injected into flow cells 3 and 4 sequentially. The association and dissociation time were 180 and 400 seconds, respectively. The surface was regenerated with 10 mmol/L glycine pH 1.5 (GE Healthcare, BR-1003-54) by injecting into flow cells 3 and 4 at 30 μL/minute for 30 seconds followed by a stabilization period of 60 seconds. Binding kinetics was calculated using Biacore Insight Evaluation Software (Version 2.0.15.12933) and a 1:1 binding model for curve fitting.

The affinity of RW102, RW103, and ABDRW102 binding to human PD-L1 protein (PD1-H5229, ACROBiosystems) was tested by surface plasmon resonance (SPR). All measurements were performed on a Biacore T200 device (GE HealthCare) at 25°C with HEPES-buffered saline (0.01 M HEPES pH 7.4, 0.15 M NaCl, three mM ethylenediaminetetraacetic acid, 0.005% Tween 20) as the running buffer. Briefly, different dilutions of samples to be tested were run at 50 µL/min on a CM5 sensor chip with a high density of human PD-L1 protein, and specific binding signals (response units) were recorded. Samples were allowed to bind with the target protein for 300 s, and dissociation was monitored for 180 s. The equilibrium dissociation constant K_D was calculated by fitting the obtained sensor grams to theoretical curves using Biacore Evaluation software. Also, the binding affinity of NOTA-RW102, NOTA-ABDRW102, and DFO-ABDRW102 to human PD-L1 protein, the binding affinity of ABDRW102 to human serum albumin (HSA) and murine serum albumin (MSA), the binding affinity of RW102 and NOTA-RW102 to murine PD-L1 (PD1-M5220, ACROBiosystems) were determined similarly.