Journal: mAbs

Article Title: Epitope mapping reveals the binding mechanism of a functional antibody cross-reactive to both human and murine programmed death 1

doi: 10.1080/19420862.2017.1296612

Figure Lengend Snippet: Characterization of antibodies. (A) Cross-reactivity of anti-PD-1 antibodies to human/mouse PD-1. Each antibody was coated on 96-well plate overnight and incubated with hPD-1-His protein (filled shapes) and mPD-1-His protein (open shapes), and then HRP-conjugated anti-His antibody was added for detection. (B-D) Binding titration of the antibodies to hPD-1 expressed on CHO-S cells (B), competition of anti-PD-1 antibodies with hPD-L1 to hPD-1 expressed on CHO-S cells (C) and competition of anti-PD-1 antibody R9 with mPD-L1 to mPD-1 expressed on 293F cells were detected using FACS instrument as described in Materials and Methods. (E-F) Anti-PD-1 antibody R9 can enhance CD4+ T cell proliferation (E) and IFN-γ production (F) in a dose-dependent manner in mixed lymphocyte reaction assay as described in Materials and Methods. (unpaired, 2-tailed t test; * p< 0.05, ** p< 0.01, *** p< 0.001). (G-H) Comparison of R9, pembrolizumab (Pemb) and nivolumab (Nivo) in hPD-1 binding (G) and hPD-L1 competition (H) using FACS method as described in Materials and Methods.

Article Snippet: After interacting with PD-1 mutant protein or with gradient diluted commercial hPD-1_ECD.His (Sino Biological, 10377-H08H) and mPD-1_ECD.His (Sino Biological, 50124-M08H), 0.2 μg/ml of HRP-conjugated anti-His antibody (Rockland Immunochemical, 200–303–382) was added as detection antibody.

Techniques: Incubation, Binding Assay, Titration

Journal: mAbs

Article Title: Epitope mapping reveals the binding mechanism of a functional antibody cross-reactive to both human and murine programmed death 1

doi: 10.1080/19420862.2017.1296612

Figure Lengend Snippet: Hot spot residues mapped on hPD-1 structure. (A) hPD-L1 binding site. Data were obtained from the literature.18 (B-D) Binding sites of antibody R11, R9 and the control antibody R10, respectively. Data were from Table 1. Colors in the pictures are included to help distinguish differences between epitopes. The structure of hPD-1 was taken from the crystal structure (PDB code 4ZQK), whose missing loops (Asp85-Asp92) were remodeled by adopting initial conformations from the NMR structure (PDB code 2M2D). The back β-strands are A′, B, E, D, A and G′. The front sheets are labeled in G, F, C and C′. The C″ strand observed in mPD-1 lost its secondary structure in hPD-1.18, 20

Article Snippet: After interacting with PD-1 mutant protein or with gradient diluted commercial hPD-1_ECD.His (Sino Biological, 10377-H08H) and mPD-1_ECD.His (Sino Biological, 50124-M08H), 0.2 μg/ml of HRP-conjugated anti-His antibody (Rockland Immunochemical, 200–303–382) was added as detection antibody.

Techniques: Binding Assay, Labeling

Journal: mAbs

Article Title: Epitope mapping reveals the binding mechanism of a functional antibody cross-reactive to both human and murine programmed death 1

doi: 10.1080/19420862.2017.1296612

Figure Lengend Snippet: Putative binding modes of R11 (A) and R9 (B) to hPD-1, conducted by in silico modeling. The sticks on hPD-1 structures are the hot-spot residues listed in Table 1. The lines show the neighboring environment near the epitope residues. VH and VL frameworks were colored in green and cyan, respectively. Heavy chain CDRs were colored in salmon and light chain CDRs were colored in wheat. Computational docking results suggested that the Arg100A on CDR H3 of antibody R11 formed a salt bridge (yellow dashes) with Asp85 on hPD-1.

Article Snippet: After interacting with PD-1 mutant protein or with gradient diluted commercial hPD-1_ECD.His (Sino Biological, 10377-H08H) and mPD-1_ECD.His (Sino Biological, 50124-M08H), 0.2 μg/ml of HRP-conjugated anti-His antibody (Rockland Immunochemical, 200–303–382) was added as detection antibody.

Techniques: Binding Assay, In Silico

Journal: mAbs

Article Title: Epitope mapping reveals the binding mechanism of a functional antibody cross-reactive to both human and murine programmed death 1

doi: 10.1080/19420862.2017.1296612

Figure Lengend Snippet: Comparison between human and murine PD-1 structures. (A) Crystal structure of hPD-1 (PDB code 4ZQK). The missing loop (Asp85-Asp92) were remolded. Sticks and lines on the structure are the marks of the identified hot-spot residues in charge of antibody R11 and R9 binding (Table 1). (B) Crystal structure of mPD-1 (PDB code 3BIK). Corresponding to the positions of hot-spot residues on hPD-1, sticks and lines on mPD-1 are the amino acids having different and same residue types, respectively. The structural differences between 2 proteins were marked in orange. (C) Sequence alignment of human and murine PD-1 structures. Arrows are the hot-spot residues identified for R11 binding. Marked box highlighted the amino acid differences in mPD-1.

Article Snippet: After interacting with PD-1 mutant protein or with gradient diluted commercial hPD-1_ECD.His (Sino Biological, 10377-H08H) and mPD-1_ECD.His (Sino Biological, 50124-M08H), 0.2 μg/ml of HRP-conjugated anti-His antibody (Rockland Immunochemical, 200–303–382) was added as detection antibody.

Techniques: Binding Assay, Sequencing

Journal: mAbs

Article Title: Epitope mapping reveals the binding mechanism of a functional antibody cross-reactive to both human and murine programmed death 1

doi: 10.1080/19420862.2017.1296612

Figure Lengend Snippet: The influences of some epitope hot spots to the capability of antibody cross-reactivity. The hot spots located on hPD-1 were changed to the corresponding mPD-1 amino acids and the binding activity of (A) R11 and (B) R9 were tested by ELISA. M1: V64M; M2: 83PEDRSQPGQDC93 to 83CNGLSQPVQDA93; M3: A129H, Q133K, K135E. 2 μg/ml of each antibody was coated at 96-well plate overnight and incubated with (A) 10 ng/ml and (B) 150 ng/ml mutants, then HRP-anti-His antibody were added for detection. The relative binding changes of the PD-1 mutants were normalized to the original binding of antibody and hPD-1 (unpaired, 2-tailed t test; * p< 0.05, ** p< 0.01, *** p< 0.001).

Article Snippet: After interacting with PD-1 mutant protein or with gradient diluted commercial hPD-1_ECD.His (Sino Biological, 10377-H08H) and mPD-1_ECD.His (Sino Biological, 50124-M08H), 0.2 μg/ml of HRP-conjugated anti-His antibody (Rockland Immunochemical, 200–303–382) was added as detection antibody.

Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet:

Article Snippet: The plate was then washed with PBST (PBS, 0.05% Tween 20) (MA0015, Meilunbio) (T8220, Solarbio) and blocked with 3% BSA (97061-420, VWR) at 37°C for 90 min. After repeated washing, hFc-tagged recombinant proteins, including CD3ε (10977-H02H; Sino Biological), CTLA-4 (CT4-H5255; Acro Biosystems), CD28 (CD8-H525a; Acro Biosystems), CD96 (TAE-H5252; Acro Biosystems), LAG-3 (LA3-H5255; Acro Biosystems), TIM-3 (TM3-H5258; Acro Biosystems), CD40 (CD0-5253; Acro Biosystems), ICOS (ICS-H5258; Acro biosystems), OX40 (OX0-H5255; Acro Biosystems), TIGHT (TIT-H5254; Acro Biosystems), LY86 (10242-H02H; Sino Biological), LILRB4 (16742-H02H; Sino Biological), CD27 (CD7-H5254; Acro biosystems), PD-1 (10377-H02H; Sino Biological), and CD8b (11031-HCCH; Sino Biological), were added into each well.

Techniques: Recombinant, Derivative Assay, Microarray, Enzyme-linked Immunosorbent Assay, Lysis, Blocking Assay, Staining, Transfection, Marker, Flow Cytometry, shRNA, Plasmid Preparation, Negative Control, Silver Staining, Bicinchoninic Acid Protein Assay, Selection, Extraction, Labeling, RNA Expression, Cell Culture, Software

Journal: bioRxiv

Article Title: The natural stilbenoid (–)-hopeaphenol inhibits cellular entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7 and B.1.351 variants

doi: 10.1101/2021.04.29.442010

Figure Lengend Snippet: Identification of stilbenoids as Spike-ACE2 inhibitors by AlphaScreen. A, Demonstration of AlphaScreen-based fluorescence due to interactions of His-tagged Spike RBD (from USA-WA1/2020) and Fc-tagged ACE2 peptides. B, Dose-response curves of stilbenoids and resveratrol on fluorescence inhibition due to disruption of RBD/ACE2 interactions. C, Demonstration of AlphaScreen-based fluorescence due to interactions of His-tagged PD-1 and Fc-tagged PD-L1 peptides. D, Dose-response curves of stilbenoids and control inhibitor BMS-1166 on fluorescence inhibition due to disruption of PD-1/PD-L1 interactions.

Article Snippet: 0.5 nM of human PD-L1-Fc (Sino Biological) was incubated with 5 nM HIS-tagged human PD-1 (Sino Biological) in the presence of 5 μg/mL protein A AlphaScreen acceptor bead and 5 μg/mL nickel chelate donor bead in a total volume of 10 μL of 20 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% Tween in white, opaque low-volume 384-well plates.

Techniques: Amplified Luminescent Proximity Homogenous Assay, Fluorescence, Inhibition