Journal: Cell Reports Methods

Article Title: Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV

doi: 10.1016/j.crmeth.2022.100236

Figure Lengend Snippet:

Article Snippet: 155Gd_PD-1 , Fluidigm , Cat# 3155009B, RRID: AB_2811087.

Techniques: Virus, Recombinant, Electroporation, Antibody Labeling, Labeling, Software

Journal: Acta Pharmaceutica Sinica. B

Article Title: YPD-30, a prodrug of YPD-29B, is an oral small-molecule inhibitor targeting PD-L1 for the treatment of human cancer

doi: 10.1016/j.apsb.2022.02.031

Figure Lengend Snippet: The chemical structure of YPD-29B and its activity in vitro . (A) Chemical structure of YPD-29B. (B) YPD-29B disturbs the hPD-1/hPD-L1 interaction as shown by the HTRF assay. The PD-L1 neutralizing antibody acts as the positive control. The assays were performed in duplicate at each concentration. (C) The specific K D value of YPD-29B by the SPR method. k a , association rate constant; k d , dissociation rate constant; K D , equilibrium dissociation constant ( k d / k a ). (D) YPD-29B blocks the hB7-1/hPD-L1 interaction as determined by ELISA. hPD-L1 neutralizing antibody acts as the positive control. The assays were performed in duplicate at each concentration. (E) YPD-29B has no effect on hPD-1/hPD-L2 interaction by ELISA. hPD-1 neutralizing antibody is a positive control. The assays were performed in duplicate at each concentration.

Article Snippet: Lectin from Phaseolus vulgaris (PHA), hPD-L1 protein (Sino Biological, Beijing, China), and PD-L1 antibody (Bio X Cell, Lebanon, NH, USA), or compounds were added to primary human T cell cultures (HemaCare, Los Angeles, CA, USA) for 3 days.

Techniques: Activity Assay, In Vitro, HTRF Assay, Positive Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: YPD-30, a prodrug of YPD-29B, is an oral small-molecule inhibitor targeting PD-L1 for the treatment of human cancer

doi: 10.1016/j.apsb.2022.02.031

Figure Lengend Snippet: The selectivity of YPD-29B on different immune checkpoints.

Article Snippet: Lectin from Phaseolus vulgaris (PHA), hPD-L1 protein (Sino Biological, Beijing, China), and PD-L1 antibody (Bio X Cell, Lebanon, NH, USA), or compounds were added to primary human T cell cultures (HemaCare, Los Angeles, CA, USA) for 3 days.

Techniques: Positive Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: YPD-30, a prodrug of YPD-29B, is an oral small-molecule inhibitor targeting PD-L1 for the treatment of human cancer

doi: 10.1016/j.apsb.2022.02.031

Figure Lengend Snippet: The effects of YPD-29B on T lymphocyte activation. (A) YPD-29B activates the PD-1/NFAT reporter-Jurkat cells. TCR Activator/PD-L1 CHO cells were seeded first, then next day the compounds or antibody were added to the cells. After 30-min incubation, the PD-1/NFAT reporter-Jurkat cells were added to the CHO cells. After 5 h incubation, luciferase assay was determined by ONE-Step luciferase assay system in triplicate at each concentration. (B) YPD-29B rescues IFN- γ expression in human PBMC cells from PD-L1 inhibition. 3 × 10 5 /well PBMC cells were stimulated by 5 μg/mL PHA and then exposed to 1 μg/mL hPD-L1 protein. The IFN- γ concentration was determined by ELISA assay. The assays were performed in triplicate at each concentration. Data are presented as mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs . indicated, data is analyzed using Student's t -test.

Article Snippet: Lectin from Phaseolus vulgaris (PHA), hPD-L1 protein (Sino Biological, Beijing, China), and PD-L1 antibody (Bio X Cell, Lebanon, NH, USA), or compounds were added to primary human T cell cultures (HemaCare, Los Angeles, CA, USA) for 3 days.

Techniques: Activation Assay, Incubation, Luciferase, Concentration Assay, Expressing, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: YPD-30, a prodrug of YPD-29B, is an oral small-molecule inhibitor targeting PD-L1 for the treatment of human cancer

doi: 10.1016/j.apsb.2022.02.031

Figure Lengend Snippet: YPD-29B binds to hPD-L1 and the hPD-L1 is then internalized. (A) The PD-L1 protein is bound by YPD-29B on the cell surface. The hPD-L1 MC38 cells were treated with anti-PDL1 antibody (10 nmol/L), BMS202 (10 μmol/L), and YPD-29B (0.1, 1, 10 μmol/L) for 24 h. The cells were collected and stained for PE-marked-hPD-L1. The cell surface expression of PD-L1 was determined by flow cytometry. The combined data at least triplicates were shown. Occupancy ratio = (MFI of Control − MFI of Treated group)/(MFI of Control − MFI of blank group) × 100%, MFI (Median Fluorescence Intensity). Data are presented as mean ± SD, ( n = 3); ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. indicated, data is analyzed using Student's t -test. (B) YPD-29B induces PD-L1 protein internalization. After a 24-h treatment, the PD-L1 protein present in the cytoplasm (Cyto) and membrane (Mem) fractions of the cells were determined by Western blotting and a representative image is shown. Na/K-ATPase and β -actin are loading as controls, respectively. (C) Statistical analysis of the Western blotting findings in (B). The densitometric analysis of the bands was determined by Image J. Statistical analysis was used to compare each YPD-29B treated group and the control group. (D) The internalization and localization of hPD-L1 is determined by immunostaining. The hPD-L1 MC38 cells were treatment with 1 and 10 μmol/L YPD-29B for 24 h. Then the cells were immunostained with hPD-L1 antibody (green) and the nuclei were stained with DAPI (Blue). Scale bar = 20 μm. (E) YPD-29B induces total PD-L1 degradation. hPD-L1 MC38 cells were treated with 0.1, 1, and 10 μmol/L of YDP-29B for 24 h. Then the whole cells are collected for Western blotting. (F) YPD-29B induces PD-L1 degradation through the lysosome pathway. The hPD-L1 MC38 cells were treated with 5 μmol/L MG132 or 100 μmol/L chloroquine for 1 h, then followed by treatment with 10 μmol/L YPD-29B for 24 h. Then the whole cells are collected for Western blotting.

Article Snippet: Lectin from Phaseolus vulgaris (PHA), hPD-L1 protein (Sino Biological, Beijing, China), and PD-L1 antibody (Bio X Cell, Lebanon, NH, USA), or compounds were added to primary human T cell cultures (HemaCare, Los Angeles, CA, USA) for 3 days.

Techniques: Staining, Expressing, Flow Cytometry, Fluorescence, Western Blot, Immunostaining

Journal: Acta Pharmaceutica Sinica. B

Article Title: YPD-30, a prodrug of YPD-29B, is an oral small-molecule inhibitor targeting PD-L1 for the treatment of human cancer

doi: 10.1016/j.apsb.2022.02.031

Figure Lengend Snippet: Predicted binding modality of YPD-29B with hPD-L1. (A) YPD-29B in the PD-L1 dimer binding pocket. (B) The binding conformations of compound YPD-29B in the active site of protein. Compound YPD-29B is shown as a cyan stick structure and the surrounding residues are gray. π – π stacking interactions are in yellow dotted lines and anion– π interactions are in purple dotted lines, and the salt bridge is in orange dotted line.

Article Snippet: Lectin from Phaseolus vulgaris (PHA), hPD-L1 protein (Sino Biological, Beijing, China), and PD-L1 antibody (Bio X Cell, Lebanon, NH, USA), or compounds were added to primary human T cell cultures (HemaCare, Los Angeles, CA, USA) for 3 days.

Techniques: Binding Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: YPD-30, a prodrug of YPD-29B, is an oral small-molecule inhibitor targeting PD-L1 for the treatment of human cancer

doi: 10.1016/j.apsb.2022.02.031

Figure Lengend Snippet: The antitumor activity of YPD-30 in vivo . (A) Chemical structure of YPD-30. (B) YPD-30 interrupts the hPD-1/hPD-L1 interaction as shown by the HTRF assay. (C) YPD-30 weakly activates T cells. The PBMC cells were treated with or without PHA (1 μg/mL) and PD-L1 (5 μg/mL). Then different concentrations of YPD-29B or YPD-30 were used to treat the cells for 72 h, and the IFN- γ in the supernatant was determined by ELISA ( n = 3). Data are presented as mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs . the group only with PHA and PD-L1 (in black). (D) Diagram of double humanization mouse model. In this model, the expression of mouse PD-L1 in the mouse colon MC38 cancer cells is knocked out and then human PD-L1 is knocked in. In addition, the PD-1 protein in the tumor bearing mouse is humanized. (E) Images of the stripped tumor ( n = 7). On Day 21, the mice were euthanized and the tumor tissue was excised and then photographed. (F) The growth curve of tumor volume ( n = 7). The tumor volume is measured on Days 1, 3, 7, 9, 13, 16, 20. Quantitative data are presented as mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01; ∗∗∗ P < 0.001 vs . vehicle. (G) The inhibitory effect of YPD-30 on tumor weight ( n = 7). Quantitative data was presented as mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs . vehicle. (H) The body weight of each group ( n = 7). Mice weight was measured every day and the quantitative data are presented as mean ± SD.

Article Snippet: Lectin from Phaseolus vulgaris (PHA), hPD-L1 protein (Sino Biological, Beijing, China), and PD-L1 antibody (Bio X Cell, Lebanon, NH, USA), or compounds were added to primary human T cell cultures (HemaCare, Los Angeles, CA, USA) for 3 days.

Techniques: Activity Assay, In Vivo, HTRF Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: YPD-30, a prodrug of YPD-29B, is an oral small-molecule inhibitor targeting PD-L1 for the treatment of human cancer

doi: 10.1016/j.apsb.2022.02.031

Figure Lengend Snippet: The affinity of YPD-29B in different species of PD-L1.

Article Snippet: Lectin from Phaseolus vulgaris (PHA), hPD-L1 protein (Sino Biological, Beijing, China), and PD-L1 antibody (Bio X Cell, Lebanon, NH, USA), or compounds were added to primary human T cell cultures (HemaCare, Los Angeles, CA, USA) for 3 days.

Techniques:

Journal: Acta Pharmaceutica Sinica. B

Article Title: YPD-30, a prodrug of YPD-29B, is an oral small-molecule inhibitor targeting PD-L1 for the treatment of human cancer

doi: 10.1016/j.apsb.2022.02.031

Figure Lengend Snippet: The antitumor mechanism of YPD-30 in vivo by immunohistochemical staining. (A) CD3 + , CD8 + and hPD-L1 in hPD-L1 MC38 xenograft tumor tissue was collected and subjected to immunohistochemical staining with specific antibodies. Scale bar = 10 μm. (B) Quantification of CD3 + , CD8 + and hPD-L1 expression in tumor tissue ( n = 8). ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs . indicated.

Article Snippet: Lectin from Phaseolus vulgaris (PHA), hPD-L1 protein (Sino Biological, Beijing, China), and PD-L1 antibody (Bio X Cell, Lebanon, NH, USA), or compounds were added to primary human T cell cultures (HemaCare, Los Angeles, CA, USA) for 3 days.

Techniques: In Vivo, Immunohistochemical staining, Staining, Expressing

Journal: Journal of Thoracic Disease

Article Title: Membrane PD-L1 expression and soluble PD-L1 plasma levels in idiopathic pulmonary fibrosis—a pilot study

doi: 10.21037/jtd.2018.11.16

Figure Lengend Snippet: Clinical characteristics of IPF patients undergone lung biopsy whose FFPE IPF lung tissue samples were tested for PD-L1 expression

Article Snippet: For the PD-L1 (B7-H1/CD274) determination in human plasma was used DuoSet ELISA system (R&D systems Europe, Ltd. Abingdon, UK), as a sandwich enzyme-linked immunosorbent assay (ELISA) specific for the human B7-H1.

Techniques: Expressing

Journal: Journal of Thoracic Disease

Article Title: Membrane PD-L1 expression and soluble PD-L1 plasma levels in idiopathic pulmonary fibrosis—a pilot study

doi: 10.21037/jtd.2018.11.16

Figure Lengend Snippet: Programmed cell death-ligand-1 (PD-L1) expression in alveolar macrophages in different histological stages of usual interstitial pneumonia pattern (idiopathic pulmonary fibrosis). (A) Cystic remodeling of lung parenchyma (“honeycomb changes”) (H&E, ×20); (B) PD-L1 expression: >1% positivity in alveolar macrophages; intensity of expression +1 (×20); (C) fibroblastic foci and moderate interstitial inflammation (H&E, ×20); (D) PD-L1 expression: >50% positivity in alveolar macrophages; intensity of expression +2 (×20); (E) fibroblastic foci and moderate interstitial inflammation (H&E, ×20); (F) PD-L1 expression: >50% positivity in alveolar macrophages; intensity of expression +2 (×20); (G) cystic remodeling of lung parenchyma (H&E, ×20); (H) PD-L1 expression: >1% positivity in alveolar macrophages; intensity of expression +1 (×20); (I) fibroblastic foci, smooth muscle proliferation and moderate interstitial inflammation (H&E, ×20); (J) PD-L1 expression: >50% positivity in alveolar macrophages; intensity of expression +2 (×20).

Article Snippet: For the PD-L1 (B7-H1/CD274) determination in human plasma was used DuoSet ELISA system (R&D systems Europe, Ltd. Abingdon, UK), as a sandwich enzyme-linked immunosorbent assay (ELISA) specific for the human B7-H1.

Techniques: Expressing

Journal: Journal of Thoracic Disease

Article Title: Membrane PD-L1 expression and soluble PD-L1 plasma levels in idiopathic pulmonary fibrosis—a pilot study

doi: 10.21037/jtd.2018.11.16

Figure Lengend Snippet: sPD-L1 concentration in sub-groups according to IPF stage

Article Snippet: For the PD-L1 (B7-H1/CD274) determination in human plasma was used DuoSet ELISA system (R&D systems Europe, Ltd. Abingdon, UK), as a sandwich enzyme-linked immunosorbent assay (ELISA) specific for the human B7-H1.

Techniques: Concentration Assay