Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE blockade of PD-1/PD-L1 interaction in coculture cell-based luciferase assay. (A, B) Cytotoxicity assay performed using Cell Counting Kit-8 (CCK) assay. The hPD-1/NFAT Jurkat T cells (A) and hPD-L1/TCR CHO-K1 cells (B) after treatment with SRE for 24 hours. (C, D) The PD-1/PD-L1 blockade bioassay was performed using the Bio-Glo™ luciferase assay system. After addition of hPD-1/NFAT Jurkat T cells and SRE (C) and anti-PD-1 antibodies (αPD-1) (D) , hPD-L1/TCR CHO-K1 cells were seeded for 20 hours. Data are presented as the mean ± SD. * p < 0.05 and *** p < 0.001 compared to the control.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Luciferase, Cytotoxicity Assay, Cell Counting

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE-induced activation of T cells and cytotoxic effect of T cell-mediated cancer cells. (A, B) The cell viability was performed using the CCK-8 assay. Splenocytes were isolated from hPD-L1 MC38 cell-bearing hPD-1 knockin mice. Murine CRC hPD-L1 MC38 cells (A) and hPD-1 mice splenocytes (B) were treated with SRE for 72 hours. (C) Cocultured hPD-L1 MC38 cell viability tested by crystal violet staining; (D) Lactate dehydrogenase (LDH) released by damaged cells, detected via LDH cytotoxicity assay; (E) Relative interleukin-2 (IL-2) level, determined using the mouse IL-2 ELISA set. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Activation Assay, CCK-8 Assay, Isolation, Knock-In, Staining, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE elevated the activation of hPD-1 + CD8 + T cells and the CD8 + T cell-mediated killing effect on hPD-L1 MC38 cancer. (A) Cocultured hPD-L1 MC38 cell viability, tested by crystal violet staining. Cocultured hPD-L1 MC38 cells detected with fluorescence microscopy (× 200) (B) and determined by fluorescent-activated cell sorting analysis (C) . (D) LDH released from damaged cells; (E) Relative perforin 1 (PRF1) level, determined with use of the mouse PRF1 ELISA kit. Data are presented as the mean ± SD. ** p < 0.01 and *** p < 0.001 compared to the vehicle group.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Activation Assay, Staining, Fluorescence, Microscopy, FACS, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: Sanguisorbae Radix extract reduced tumor growth in the hPD-L1 MC38 cell allograft hPD-1 mouse model. (A) Body weight (grams); (B) Tumor volume after 18 days; (C) Tumor weight after 18 days; (D) Images of tumor tissues (bar indicates 5 mm); (E) hPD-L1 MC38 tumor-bearing mice 18 days after treatment; (F) Representative microscopic images (×400) of CD8 and PRF1-positive area of tumor tissues calculated using immunohistochemical analysis. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the vehicle group.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Immunohistochemical staining, Standard Deviation

Journal: Cancer Chemotherapy and Pharmacology

Article Title: Discovery and pharmacological characterization of cetrelimab (JNJ-63723283), an anti–programmed cell death protein-1 (PD-1) antibody, in human cancer models

doi: 10.1007/s00280-022-04415-5

Figure Lengend Snippet: In vitro, cetrelimab binds to PD-1 and blocks PD-1 ligand binding. a Cetrelimab binding to HEK-293 cells (1 × 10 5 cells) overexpressing cyno PD-1. Data are from one experiment. b Cetrelimab binding to endogenous PD-1 expressed in activated human CD4 + and CD8 + T cells. Data are from one experiment. c Inhibition of PD-1 binding to human PD-L1. Error bars represent SD from one representative experiment performed in triplicate. d Inhibition of PD-1 binding to PD-L2. Error bars represent SD from one representative experiment performed in triplicate. IgG4 ITC immunoglobulin G4 isotype control, MSD Meso Scale Discovery, PD-1 programmed cell death protein-1, PD-L1 programmed death-ligand 1, PD-L2 programmed death-ligand 2

Article Snippet: Test antibodies were incubated with biotinylated human PD-1, and subsequent binding to plate-bound human PD-L1 or human PD-L2 ligands was measured using Meso Scale Discovery 6000 platform (Meso Scale Diagnostic, LLC, Rockville, MD).

Techniques: In Vitro, Ligand Binding Assay, Binding Assay, Inhibition

Journal: Cancer Chemotherapy and Pharmacology

Article Title: Discovery and pharmacological characterization of cetrelimab (JNJ-63723283), an anti–programmed cell death protein-1 (PD-1) antibody, in human cancer models

doi: 10.1007/s00280-022-04415-5

Figure Lengend Snippet: Cetrelimab promoted T cell activation and reversed PD-1-mediated suppression of T cell receptor signaling in vitro. a MLR assay. Purified human T cells (1.5 × 10 5 cells) were activated by stimulation with allogeneic, major histocompatibility complex–mismatched, dendritic cells (5 × 10 3 cells) for 5 days in the presence of cetrelimab, pembrolizumab analog, nivolumab analog, or isotype-control antibody. Data are from one representative experiment. Error bars represent SEM from one donor with one experiment in quintuplicate. b SEB assay. Isolated PBMCs (2 × 10 5 cells) were stimulated with SEB and incubated with cetrelimab or isotype-control antibody for 3 to 6 days. Error bars represent the estimate of variability from the linear mixed-effect model for two donors, with each treatment in quadruplicate. *Significantly different from cells + SEB, P < 0.0001. c CMV recall. CMV-reactive T cells (1.5 × 10 5 cells) were stimulated with CMV antigen for 6 days in the presence of cetrelimab or isotype-control antibody. Error bars represent SEM. d NFAT assay. Jurkat T cells (7.5 × 10 4 cells) were incubated with PD-L1 positive Chinese hamster ovary-K1 cells (4 × 10 4 cells) in the presence of cetrelimab, pembrolizumab analog, nivolumab analog, or isotype-control antibody for 24 h. Data are from one experiment. No error bars are shown as replicates were averaged and divided by the cells-only control. CMV cytomegalovirus, IFN interferon, IL- interleukin-, MLR mixed lymphocyte reaction, NFAT nuclear factor of activated T cells, PBMC peripheral blood mononuclear cell, PD-1 programmed cell death protein-1, SEB staphylococcal enterotoxin B, SEM standard error of the mean, TNF tumor necrosis factor

Article Snippet: Test antibodies were incubated with biotinylated human PD-1, and subsequent binding to plate-bound human PD-L1 or human PD-L2 ligands was measured using Meso Scale Discovery 6000 platform (Meso Scale Diagnostic, LLC, Rockville, MD).

Techniques: Activation Assay, In Vitro, Mlr Assay, Purification, Isolation, Incubation

Journal: Cancer Chemotherapy and Pharmacology

Article Title: Discovery and pharmacological characterization of cetrelimab (JNJ-63723283), an anti–programmed cell death protein-1 (PD-1) antibody, in human cancer models

doi: 10.1007/s00280-022-04415-5

Figure Lengend Snippet: Cetrelimab showed antitumor efficacy in two in vivo models. a MC38 tumor growth inhibition in PD-1 knock-in mice. Error bars represent SEM, n = 10. b Survival of PD-1 knock-in mice bearing MC38 tumors following cetrelimab and pembrolizumab analog treatment, n = 10. c Patient-derived LG1306 lung tumor growth inhibition in huCD34 + NSG mice by cetrelimab and commercially available pembrolizumab. Error bars represent SEM, n = 10–11 from two donors. d Cetrelimab and commercially available pembrolizumab–enhanced peripheral blood T cells from in huCD34 + -humanized NSG mice bearing established LG1306 PDX tumors. Error bars represent SEM, n = 10–11 from two donors. * P < 0.05 versus isotype-control–treated mice. PBS phosphate buffer saline, PD-1 programmed cell death protein-1, PDX patient-derived xenograft, SEM standard error of the mean

Article Snippet: Test antibodies were incubated with biotinylated human PD-1, and subsequent binding to plate-bound human PD-L1 or human PD-L2 ligands was measured using Meso Scale Discovery 6000 platform (Meso Scale Diagnostic, LLC, Rockville, MD).

Techniques: In Vivo, Inhibition, Knock-In, Derivative Assay