Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: Characterization of hPD1(25-167)-His and hPD1(25-167)-3S-IG

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques:

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–c Characterization of the assay reference standard-recombinant hPD1(25-167)-His protein. a SDS-PAGE analysis of the purified hPD1(25-167)-His protein expressed in HEK293 cells. Lane 1: molecular weight marker, lane 2: blank, lane 3: 1 μg of the purified hPD1(25-167)-His protein. The gel was stained with Simply Blue SafeStain. b Concentration series of hPDL-1 (0.125–2 μM) binding to captured hPD1(25-167)-His protein. c Equilibrium analysis of hPDL-1 binding to hPD1(25-167)-His yields a K D of 2.8 μM (average value over multiple surfaces)

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques: Recombinant, SDS Page, Purification, Molecular Weight, Marker, Staining, Concentration Assay, Binding Assay

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: Binding Affinity of Anti-PD-1 Antibody MIH4 for Recombinant Human sPD-1 Proteins

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques: Binding Assay, Recombinant

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–b Detection of endogenous sPD-1 protein and spike recovery in sera from normal and cancer individuals. a Levels of sPD-1 in serum samples from normal and cancer individuals: normal, n = 15; melanoma, n = 15; renal cell carcinoma, n = 11; squamous lung cancer, n = 7; non-squamous lung cancer, n = 8; multiple myeloma, n = 10; Hodgkin’s lymphoma, n = 7. b Spike and recovery study: 500 pg/mL hPD1(25-167)-His protein was spiked into serum samples from normal and cancer individuals: normal, n = 10; melanoma n = 8; renal cell carcinoma, n = 11; squamous lung cancer, n = 7; non-squamous lung cancer, n = 8; multiple myeloma, n = 10; Hodgkin’s lymphoma, n = 7. % Spike recovery = 100*(sample value post-spike − sample value pre-spike)/spiked value. Results are shown with box and whiskers graphs. The box included data within 5 to 95 percentiles with the median line in the middle and extended values in the whiskers. The dotted lines depict the nominal spiked value ± 25%

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques:

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–b Dilution linearity studies in melanoma and spiked normal sera. a Three melanoma serum samples (M1, M2, M3) were each diluted 1.5–8-fold with assay buffer; recombinant hPD1(25-167)-His protein standard in a SeraSub solution was diluted in same concentration range. b Three normal human serum samples (N1, N2, N3) were spiked with 5000 pg/mL of human hPD1(25-167)-His and were each diluted 2–256-fold with assay buffer. The back-calculated sPD-1 concentrations (observed concentrations times the dilution factor) were plotted against dilution factors. The dotted lines depict 75–125% range of the nominal spiked value

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques: Recombinant, Concentration Assay

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–b Effect of PDL-1, PDL-2, and nivolumab on QC sample performance. a sPD-1 in a melanoma serum sample measured in the absence or presence of 20 nM (1 μg/mL) of human PDL-1 or PDL-2 proteins. b QC performance from analytical runs in the absence or presence of 685 nM (100 μg/mL) nivolumab. Results are shown as mean and SD of each group (n = 2–4)

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques:

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 1. Target cell–bound PD-L1 ImmTAAI molecules inhibit TCR complex signaling. (A) Schematic of the HEK293T-A2–Jurkat NFL PD-1 reporter assay. (B) PD-L1 ImmTAAI titrations were incubated with PPI15–24 peptide–pulsed or nonpulsed HEK293T-A2 target cells. ImmTAAI binding was quantified by flow cytometry, and dose-response curves were plotted (n = 3 and representative of 3 independent experiments). (C) Dose responses of the PD-L1 ImmTAAIs and PD-L1 Fc were tested in the HEK293T-A2–Jurkat NFL PD-1 reporter assay. Normalized NFAT activity was plotted against ImmTAAI con- centration to calculate IC50 values (n = 3 and representative of 3 independent experiments). (D) Relative NFAT activity at 100 nM ImmTAAI was plotted for pulsed (+ PPI15–24) versus nonpulsed (no peptide) target cells (n = 3 and representative of 3 independent experiments). All data are plotted as mean ± SD and were compared by 2-way ANOVA with repeated measures and Tukey’s or Sidak’s multiple-comparison test. ***P ≤ 0.001, ****P ≤ 0.0001. IgV, immunoglobulin-like variable domain; fl, full-length.

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Reporter Assay, Incubation, Binding Assay, Flow Cytometry, Activity Assay, Comparison

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 2. Target cell–bound PD-1 antibody ImmTAAI molecules exhibit superior inhibition of TCR complex signaling over PD-L1 ImmTAAI molecules. (A and B) PD-1 antibody ImmTAAI molecules constructed using a panel of VHH PD-1 antibodies and an scFv PD-1 antibody (CA949) were tested in the HEK293T-A2/anti-CD3: Jurkat NFL PD-1 reporter assay as described in Figure 1 (n = 3 and representative of 3 independent experiments). (C and D) Rep- resentative VHH-based (H5) and scFv-based (CA949) ImmTAAI molecules were tested alongside the IgV–PD-L1 ImmTAAI in the HEK293T-A2/anti-CD3: Jurkat NFL PD-1 reporter assay, and data are plotted as described above (n = 3 and representative of 3 independent experiments). (E and F) PD-1 ago- nist ImmTAAI molecules were generated by fusing CA949 scFV antibody and IgV–PD-L1 to different TCRs against either gp100280–288 pHLA-A2 or PPI15–24 pHLA-A2. The ImmTAAI molecules were tested in the HEK293T-A2/anti-CD3: Jurkat NFL PD-1 reporter assay, using HEK293T-A2 target cells pulsed with the appropriate targeting peptide (n = 6 and representative of 2 independent experiments). All data are plotted as mean ± SD and were compared by 2-way ANOVA with repeated measures and Tukey’s or Sidak’s multiple-comparison test. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Inhibition, Construct, Reporter Assay, Generated, Comparison

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 3. β Cell–targeted PD-1 agonist ImmTAAI molecules inhibit TCR complex signaling. (A) Schematic of the ECN90 β cell line: Jurkat NFL Mel5 PD-1 reporter assay. ECN90 cells were pulsed with Melan-A activating peptide, and titrations of PD-L1 or PD-1 Ab ImmTAAI molecules, PD-L1 Fc, or PPI TCR alone added. (B–D) Either ECN90 cells were incubated for 3 hours and analyzed for PD-L1 ImmTAAI binding (n = 3 and representative of 3 independent experiments) (B), or Jurkat NFL Mel5 PD-1 cells were immediately added to test activity in the PD-1 reporter assay (n = 3 and representative of 3 indepen- dent experiments) (C and D). All data are plotted as mean ± SD. Abbreviations: IgV, Immunoglobulin-like variable domain; fl, full length

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Reporter Assay, Incubation, Binding Assay, Activity Assay

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 5. H5 ImmTAAI localizes at the target cell–T cell interface and inhibits proximal TCR signaling. (A) Schematic of the cell assay used to visualize H5 ImmTAAI localization. (B) Phase contrast and confocal fluorescence images of H5 ImmTAAI localization on ECN90 β cell target cells cocultured with either PD-1+ or PD-1– Jurkat cells. Upper panel: phase contrast. Lower panel: fluorescence images, a white arrow indicates ImmTAAI accumulation (representative images from 3 independent studies). Total original magnification, ×100. (C) The numbers of ECN90 β cell: Jurkat conju- gates with ImmTAAI accumulation (white arrow) were counted and plotted as a percentage of the total number of cell conjugates (n = 100 conjugates for conditions using Jurkat Mel5 PD-1 cells and n = 96 for Jurkat Mel5 cells, plotted as mean data from 3 independent studies). (D) Jurkat Mel5 PD-1 cells were stimulated with Melan-A–pulsed ECN90 or ECN90 PD-L1 cells for the indicated times. Whole cell lysates for each condition and time point were resolved by SDS-PAGE, transferred to PVDF membranes, and probed by Western blotting for phospho–SLP-76 (pSLP-76), total SLP-76 (SLP-76), phospho-PLCγ (pPLCγ), total PLCγ (PLCγ), phospho–ZAP-70 (pZAP-70), and total ZAP-70 (representative blots from 3 independent experiments). (E) Jurkat Mel5 PD-1 cells were stimulated with Melan-A–pulsed ECN90 cells, in the presence or absence of H5 ImmTAAI, and Western blots were performed as above (representative blots from 3 independent experiments).

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Fluorescence, SDS Page, Western Blot

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 7. PD-1 antibody ImmTAAI molecules inhibit autoreactive human CD8+ T cells and protect target cells from T cell killing. (A) Schematic of the EndoC-β Red cell–CD8+ T cell clone killing assay. (B and D) PPI6–14-HLA-A2-specific autoreactive T cell clones 4b (B) or 12b (D) were added to EndoC-β Red cells in the presence of PPI antibody or gp100 PD-1 antibody ImmTAAI titrations or TCR only controls. PD-L1–transduced EndoC-β Red target cells with or without anti–PD-L1 blocking antibody were used as additional controls. For each sample EndoC-β Red target cell number, rela- tive to cells at (t = 0) was measured over time by imaging and growth curves generated. AUC was calculated for each curve and EC50 data generated by plotting AUC over ImmTAAI concentration (representative data from 3 independent experiments). (C and E) Supernatants were collected from the killing assays at 24 hours. IFN-γ levels were measured by MSD ELISA, and dose-response curves were plotted to generate EC50 values. Nonstim- ulated T cells alone were assessed as additional controls (n = 2, data are plotted as mean ± SD and representative of 3 independent experiments).

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Clone Assay, Blocking Assay, Imaging, Generated, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 1. Target cell–bound PD-L1 ImmTAAI molecules inhibit TCR complex signaling. (A) Schematic of the HEK293T-A2–Jurkat NFL PD-1 reporter assay. (B) PD-L1 ImmTAAI titrations were incubated with PPI15–24 peptide–pulsed or nonpulsed HEK293T-A2 target cells. ImmTAAI binding was quantified by flow cytometry, and dose-response curves were plotted (n = 3 and representative of 3 independent experiments). (C) Dose responses of the PD-L1 ImmTAAIs and PD-L1 Fc were tested in the HEK293T-A2–Jurkat NFL PD-1 reporter assay. Normalized NFAT activity was plotted against ImmTAAI con- centration to calculate IC50 values (n = 3 and representative of 3 independent experiments). (D) Relative NFAT activity at 100 nM ImmTAAI was plotted for pulsed (+ PPI15–24) versus nonpulsed (no peptide) target cells (n = 3 and representative of 3 independent experiments). All data are plotted as mean ± SD and were compared by 2-way ANOVA with repeated measures and Tukey’s or Sidak’s multiple-comparison test. ***P ≤ 0.001, ****P ≤ 0.0001. IgV, immunoglobulin-like variable domain; fl, full-length.

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Reporter Assay, Incubation, Binding Assay, Flow Cytometry, Activity Assay, Comparison

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 2. Target cell–bound PD-1 antibody ImmTAAI molecules exhibit superior inhibition of TCR complex signaling over PD-L1 ImmTAAI molecules. (A and B) PD-1 antibody ImmTAAI molecules constructed using a panel of VHH PD-1 antibodies and an scFv PD-1 antibody (CA949) were tested in the HEK293T-A2/anti-CD3: Jurkat NFL PD-1 reporter assay as described in Figure 1 (n = 3 and representative of 3 independent experiments). (C and D) Rep- resentative VHH-based (H5) and scFv-based (CA949) ImmTAAI molecules were tested alongside the IgV–PD-L1 ImmTAAI in the HEK293T-A2/anti-CD3: Jurkat NFL PD-1 reporter assay, and data are plotted as described above (n = 3 and representative of 3 independent experiments). (E and F) PD-1 ago- nist ImmTAAI molecules were generated by fusing CA949 scFV antibody and IgV–PD-L1 to different TCRs against either gp100280–288 pHLA-A2 or PPI15–24 pHLA-A2. The ImmTAAI molecules were tested in the HEK293T-A2/anti-CD3: Jurkat NFL PD-1 reporter assay, using HEK293T-A2 target cells pulsed with the appropriate targeting peptide (n = 6 and representative of 2 independent experiments). All data are plotted as mean ± SD and were compared by 2-way ANOVA with repeated measures and Tukey’s or Sidak’s multiple-comparison test. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Inhibition, Construct, Reporter Assay, Generated, Comparison

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 3. β Cell–targeted PD-1 agonist ImmTAAI molecules inhibit TCR complex signaling. (A) Schematic of the ECN90 β cell line: Jurkat NFL Mel5 PD-1 reporter assay. ECN90 cells were pulsed with Melan-A activating peptide, and titrations of PD-L1 or PD-1 Ab ImmTAAI molecules, PD-L1 Fc, or PPI TCR alone added. (B–D) Either ECN90 cells were incubated for 3 hours and analyzed for PD-L1 ImmTAAI binding (n = 3 and representative of 3 independent experiments) (B), or Jurkat NFL Mel5 PD-1 cells were immediately added to test activity in the PD-1 reporter assay (n = 3 and representative of 3 indepen- dent experiments) (C and D). All data are plotted as mean ± SD. Abbreviations: IgV, Immunoglobulin-like variable domain; fl, full length

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Reporter Assay, Incubation, Binding Assay, Activity Assay

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 5. H5 ImmTAAI localizes at the target cell–T cell interface and inhibits proximal TCR signaling. (A) Schematic of the cell assay used to visualize H5 ImmTAAI localization. (B) Phase contrast and confocal fluorescence images of H5 ImmTAAI localization on ECN90 β cell target cells cocultured with either PD-1+ or PD-1– Jurkat cells. Upper panel: phase contrast. Lower panel: fluorescence images, a white arrow indicates ImmTAAI accumulation (representative images from 3 independent studies). Total original magnification, ×100. (C) The numbers of ECN90 β cell: Jurkat conju- gates with ImmTAAI accumulation (white arrow) were counted and plotted as a percentage of the total number of cell conjugates (n = 100 conjugates for conditions using Jurkat Mel5 PD-1 cells and n = 96 for Jurkat Mel5 cells, plotted as mean data from 3 independent studies). (D) Jurkat Mel5 PD-1 cells were stimulated with Melan-A–pulsed ECN90 or ECN90 PD-L1 cells for the indicated times. Whole cell lysates for each condition and time point were resolved by SDS-PAGE, transferred to PVDF membranes, and probed by Western blotting for phospho–SLP-76 (pSLP-76), total SLP-76 (SLP-76), phospho-PLCγ (pPLCγ), total PLCγ (PLCγ), phospho–ZAP-70 (pZAP-70), and total ZAP-70 (representative blots from 3 independent experiments). (E) Jurkat Mel5 PD-1 cells were stimulated with Melan-A–pulsed ECN90 cells, in the presence or absence of H5 ImmTAAI, and Western blots were performed as above (representative blots from 3 independent experiments).

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Fluorescence, SDS Page, Western Blot

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 6. PD-1 antibody ImmTAAI molecules bound to antigen-presenting cells inhibit primary human CD4+ T cells. (A) PD-1 agonist ImmTAAI binding to Raji-A2 target cells was analyzed by flow cytometry (n = 2 and representative of 3 independent experiments). (B and C) PD-1 agonist ImmTAAI titrations were preincubated for 1 hour with PPI15–24 peptide–pulsed or nonpulsed SEB-loaded Raji-A2 cells. Preactivated T cells were add- ed to the Raji-A2 cells and incubated for 48 hours. Supernatants were collected and IL-2 levels measured by ELISA. Dose response curves plotted to obtain IC50 values and relative inhibition of IL-2 release at 100 nM ImmTAAI was plotted for pulsed (+ PPI15–24) versus nonpulsed target cells (n = 3 and representative of 3 independent experiments). Data are plotted as mean ± SD and were compared by 2-way ANOVA with repeated measures and Tukey’s or Sidak’s multiple-comparison test. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Binding Assay, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay, Inhibition, Comparison

Journal: JCI insight

Article Title: Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

doi: 10.1172/jci.insight.152468

Figure Lengend Snippet: Figure 7. PD-1 antibody ImmTAAI molecules inhibit autoreactive human CD8+ T cells and protect target cells from T cell killing. (A) Schematic of the EndoC-β Red cell–CD8+ T cell clone killing assay. (B and D) PPI6–14-HLA-A2-specific autoreactive T cell clones 4b (B) or 12b (D) were added to EndoC-β Red cells in the presence of PPI antibody or gp100 PD-1 antibody ImmTAAI titrations or TCR only controls. PD-L1–transduced EndoC-β Red target cells with or without anti–PD-L1 blocking antibody were used as additional controls. For each sample EndoC-β Red target cell number, rela- tive to cells at (t = 0) was measured over time by imaging and growth curves generated. AUC was calculated for each curve and EC50 data generated by plotting AUC over ImmTAAI concentration (representative data from 3 independent experiments). (C and E) Supernatants were collected from the killing assays at 24 hours. IFN-γ levels were measured by MSD ELISA, and dose-response curves were plotted to generate EC50 values. Nonstim- ulated T cells alone were assessed as additional controls (n = 2, data are plotted as mean ± SD and representative of 3 independent experiments).

Article Snippet: Lentiviral vectors containing PDCD1 (PD-1) and CD274 (PDL1) were obtained from OriGene (RC210364L1 and RC213071L1, respectively).

Techniques: Clone Assay, Blocking Assay, Imaging, Generated, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models

doi: 10.18632/oncotarget.26785

Figure Lengend Snippet: Wells in a 96-well microtiter plate were coated with ( A ) recombinant human or ( B ) mouse PD-L1-Fc followed by co-incubation with His-tagged recombinant PD-1-Fc in competition with 25, 50 or 100 µL of supernatant of undefined scFv PD-L1 (scFv) and scFvFc PD-L1 (scFvFc) protein concentration collected from CT26 cells maximally infected with RRV-scFv-PDL1 and RRV-scFvFc-PDL1, respectively. Anti-human or mouse PD-L1 antibody was included as positive control (indicated as α-hPD-L1 and α-mPD-L1). Anti- His tag antibody was used to detect bound His-tagged PD-1-Fc. Optical density was measured at 450 nm. The percentage of inhibition was calculated with respect to the supernatant from CT26 maximally infected with RRV-GFP (non-scFv-PD-L1) used in the competition. Error bars indicate the standard deviation of the dataset. ( C ) Wells were coated with recombinant human PDL1-Fc followed by co-incubation with His-tagged recombinant PD-1-Fc in competition with purified scFv PD-L1 or anti-human PD-L1 antibody range from 0.01–100 nM. IC50 values were determined using best-fit values from non-linear three-parameters logistic.

Article Snippet: Subsequently, PD-L1-Fc coated wells were co-incubated with 1 µg/mL His-tagged recombinant human PD-1 (SinoBiological, # 10377-H08H) or His-tagged recombinant mouse PD-1 (abcam, # ab180051) in competition with supernatant of undefined protein concentration collected from CT26 cells maximally infected with RRV-scFv-PDL1 or RRV-scFvFc-PDL1 (100, 50 and 25 μL), anti-PDL1 antibody (anti-human PD-L1, ebioscience # 14-5983-82; anti-mouse PD-L1 BioLegend # 124302) or purified scFv PDL1 protein (Accelagen Inc.) for 2 hours at room temperature.

Techniques: Recombinant, Incubation, Protein Concentration, Infection, Positive Control, Inhibition, Standard Deviation, Purification

Journal: Oncotarget

Article Title: PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models

doi: 10.18632/oncotarget.26785

Figure Lengend Snippet: Mixtures of SEE loaded, SEE + anti-human PD-L1 (10 µg/mL) or SEE + purified scFv-PD-L1 (10 µg/mL) Raji B cells or Raji B cells overexpressing human PD-L1 (with rested Jurkat T cells overexpressing human PD-1) were co-cultured in RPMI-1640 medium containing 10% human AB serum for stimulation for 20–22 hours followed by IL-2 measurement by ELISA.

Article Snippet: Subsequently, PD-L1-Fc coated wells were co-incubated with 1 µg/mL His-tagged recombinant human PD-1 (SinoBiological, # 10377-H08H) or His-tagged recombinant mouse PD-1 (abcam, # ab180051) in competition with supernatant of undefined protein concentration collected from CT26 cells maximally infected with RRV-scFv-PDL1 or RRV-scFvFc-PDL1 (100, 50 and 25 μL), anti-PDL1 antibody (anti-human PD-L1, ebioscience # 14-5983-82; anti-mouse PD-L1 BioLegend # 124302) or purified scFv PDL1 protein (Accelagen Inc.) for 2 hours at room temperature.

Techniques: Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models

doi: 10.18632/oncotarget.26785

Figure Lengend Snippet: ( A ) Subcutaneous tumor model using Tu-2449SC cells maximally infected with RRV-scFv PDL1 or RRV-GFP at indicated ratios were subcutaneously implanted on the right flank of 8-week-old female B6C3F1 mice and assigned to indicated groups ( n = 10 per group). Control groups are mice implanted with 100% Tu-2449SC/RRVGFP cells that received PBS (0% scFv) or anti-mouse PD-1 antibody (0% scFv + α-PD-1). Anti-PD-1 antibody (Clone J43) was i. p. administered on day 0 (300 µg per mouse), day 3, day 6 and day 9 (200 µg per mouse). Statistical significance was determined by 2-way ANOVA of the following data sets: * p < 0.0001 for 0% vs 30% scFv; ** p < 0.0001 for 0% vs 100% scFv. ( B ) Naïve mice ( n = 5) and mice that cleared their initial tumor implant from RRV-scFv-PDL1 treated group ( n = 15) were challenged with 2 × 10 6 Tu-2449SC on the left side of the flank. Tumor growth and measurement were monitored over time. * p < 0.0001 (0% scFv vs. 2%, 30% and 100% scFv). Error bars indicate the standard error measurement (SEM) of the dataset. ( C ) and ( D ) Frequency of tumor-infiltrating CD4 + (C) and CD8 + (D) lymphocytes among CD45 + cells by flow cytometry, 39 days post-tumor implantation. Statistical analyses were performed using Mann-Whitney test at 95% CI ( * P < 0.05, ** P < 0.01). Values represent means ± SD of the dataset.

Article Snippet: Subsequently, PD-L1-Fc coated wells were co-incubated with 1 µg/mL His-tagged recombinant human PD-1 (SinoBiological, # 10377-H08H) or His-tagged recombinant mouse PD-1 (abcam, # ab180051) in competition with supernatant of undefined protein concentration collected from CT26 cells maximally infected with RRV-scFv-PDL1 or RRV-scFvFc-PDL1 (100, 50 and 25 μL), anti-PDL1 antibody (anti-human PD-L1, ebioscience # 14-5983-82; anti-mouse PD-L1 BioLegend # 124302) or purified scFv PDL1 protein (Accelagen Inc.) for 2 hours at room temperature.

Techniques: Infection, Flow Cytometry, Tumor Implantation, MANN-WHITNEY

Journal: Oncotarget

Article Title: PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models

doi: 10.18632/oncotarget.26785

Figure Lengend Snippet: ( A ) Female B6C3F1 mice (8-week-old; n = 10 per group) were i.c. implanted with 1 × 10 4 of Tu-2449 cells. Survival analysis was monitored for 90 days. Mice in the experimental groups were injected with 1E5 or 1E6 TU of purified RRV-scFv-PDL1 vector on day 4 post tumor implant. Control groups are mice bearing 100% pre-transduced scFv PD-L1 expressing tumor cells (100% scFv) and mice treated anti-PD-1 antibody (Naive + αPD-1) or isotype control (Naive + Isotype), (300 µg per mouse i.p. induction on day 4; 200 µg per mouse maintenance dose on day 10, 14 and 17 indicated by black arrows). Survival data were plotted by the Kaplan–Meier method. Statistical significance of survival between mice treated with anti-PD-1 antibody and injection-treated RRVscFvPDL1 mice was determined by the Log-rank (Mantel-Cox) test, * p = 0.0045. ( B ) Mice which had survived from initial tumor implant from RRV-scFv-PDL1 pre-transduced or injectiontreated groups were challenged with 2 × 10 6 Tu-2449SC cells on the right flank on day 80 (red arrow) post primary tumor implant. Tumor growth and measurement were monitored over time. Statistical significance was determined by 2-way ANOVA of the following data sets: * p < 0.0001 for Naive vs 1E6 TU RRV-scFv PD-L1. Error bars indicate the SEM of the dataset.

Article Snippet: Subsequently, PD-L1-Fc coated wells were co-incubated with 1 µg/mL His-tagged recombinant human PD-1 (SinoBiological, # 10377-H08H) or His-tagged recombinant mouse PD-1 (abcam, # ab180051) in competition with supernatant of undefined protein concentration collected from CT26 cells maximally infected with RRV-scFv-PDL1 or RRV-scFvFc-PDL1 (100, 50 and 25 μL), anti-PDL1 antibody (anti-human PD-L1, ebioscience # 14-5983-82; anti-mouse PD-L1 BioLegend # 124302) or purified scFv PDL1 protein (Accelagen Inc.) for 2 hours at room temperature.

Techniques: Injection, Purification, Plasmid Preparation, Expressing

Journal: EMBO Molecular Medicine

Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

doi: 10.15252/emmm.201911571

Figure Lengend Snippet: A Schematic diagram for compounds screening on Jurkat and Raji stable lines. B Scatterplot of relative luciferase activity of JP‐luc stimulated with SEE‐loaded Raji‐L1 in the presence of 1 μM chemicals. Y axis denotes the ratio of luciferase of chemical treated well against that of DMSO treated well. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. C FACS analysis of the cytotoxic efficiency of OT‐I CTLs against EG7‐L1 in the presence of MB. aPD1 antibody served as positive control. Splenocytes from OT‐I mice in culture were stimulated with 10 nM of SINFEEKL peptide for 3 days to generate mature CTLs. CTLs were incubated with CFSE‐labeled EG7‐L1 cells in the presence of MB at indicated concentrations. Cytotoxicity was determined by flow cytometry. Data are representative of three independent experiments (effector‐to‐target ratio = 10:1, 5:1, 2:1, unpaired t ‐test, killing time: 5 h). EG7‐L1: EG7 overexpressing PD‐L1. D Statistical results of (Fig C). E MB enhanced cytotoxicity of OT‐I CTLs against IFNγ‐treated B16‐F10‐OVA. Cytotoxicity was determined by the relative area unoccupied by crystal violet stained cells examined under microphotograph. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Digested hPD‐1 and hPD‐L1 were subcloned into identically digested pCAGiN, respectively. pcDNA3.1‐hPD‐1‐EGFP and pcDNA3.1‐hSHP2‐mCherry (human SHP2) for colocalization assay: Fragments hPD‐1‐EGFP and hSHP2‐mCherry were synthesized. pcDNA3.1 was digested with Knp1 and Asc1. hPD‐1‐EGFP and hSHP2‐mCherry fragments were identically digested for cloning into pcDNA3.1. pWPI‐mPDL1 (mouse‐PDL1) for overexpression of mPD‐L1 and IRES‐EGFP element in the vector for FACS sorting virus‐infected cells: pWPI (Addgene, #12254) and mPD‐L1 fragment was digested with SwaI and AscI and joined by T4 ligase. pCAGiN‐hPD‐1‐C‐luciferase for overexpressing fusion gene containing hPD‐1 followed by C‐terminal half of luciferase.

Techniques: Luciferase, Activity Assay, Positive Control, Incubation, Labeling, Flow Cytometry, Staining

Journal: EMBO Molecular Medicine

Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

doi: 10.15252/emmm.201911571

Figure Lengend Snippet: A Effect of PD‐1 antibody on the proliferation of OT‐I CTLs. Splenocytes from OT‐I mice were labeled with CFSE and seeded in a 96‐well plate. Media were supplemented with 10 nM of SINFEEKL peptide, 10 ng/ml of human IL‐2 and 10 μg/ml of mouse PD‐L1 protein in the presence of 10 μg/ml of PD‐1 antibody. Cell proliferation was measured by FACS. B Bar graph of (Fig A). C MB enhancing production of cytokine and cytolytic granule by OT‐1 CTLs. CTLs were co‐incubated with CFSE‐labeled EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD‐1 antibody served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. D Bar graph of IFNγ production of OT‐1 CTLs in (Fig C). E Bar graph of GZMB production of OT‐1 CTLs in (Fig C). Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Digested hPD‐1 and hPD‐L1 were subcloned into identically digested pCAGiN, respectively. pcDNA3.1‐hPD‐1‐EGFP and pcDNA3.1‐hSHP2‐mCherry (human SHP2) for colocalization assay: Fragments hPD‐1‐EGFP and hSHP2‐mCherry were synthesized. pcDNA3.1 was digested with Knp1 and Asc1. hPD‐1‐EGFP and hSHP2‐mCherry fragments were identically digested for cloning into pcDNA3.1. pWPI‐mPDL1 (mouse‐PDL1) for overexpression of mPD‐L1 and IRES‐EGFP element in the vector for FACS sorting virus‐infected cells: pWPI (Addgene, #12254) and mPD‐L1 fragment was digested with SwaI and AscI and joined by T4 ligase. pCAGiN‐hPD‐1‐C‐luciferase for overexpressing fusion gene containing hPD‐1 followed by C‐terminal half of luciferase.

Techniques: Labeling, Incubation, Expressing, Flow Cytometry, Positive Control

Journal: EMBO Molecular Medicine

Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

doi: 10.15252/emmm.201911571

Figure Lengend Snippet: A Effect of MB on the proliferation of WT CTLs and PD‐1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28‐coated 96‐well plates. 10 μg/ml of PD‐L1 was administered in media. Cell proliferation was checked by monitoring dilution of CFSE 48 h after CD3/CD28 stimulation by gating on CD8‐positive population through FACS analysis. WT: splenic cell from wild‐type C57BL/J mice. PD‐1KO: splenic cell from PD‐1 knockout mice. B Statistical results of (A). C FACS analysis of the effect of MB on the activation of OT‐I CTLs. OT‐I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD‐L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 h, surface expression of CD25 and CD69 on OT‐1 CTLs was determined through FACS analysis. D Statistical results of (C). E Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP‐luc by co‐culture with Raji‐L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE‐loaded Raji (PD‐L1 negative) served as positive control). IC 50 is calculated to be 117 nM. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. F Impact of MB on luciferase activity of various engineered Jurkat T cells. J‐luc: Jurkat cell harboring NFAT‐luciferase transgene; J‐luc‐sgPD‐1: J‐luc cells treated with lentivirus expressing sgPD‐1/CAS9 simultaneously. G qRT–PCR analysis of IL‐2 mRNA level in JP‐luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD‐L1 and MB at indicated concentrations. H MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). I MB enhancing production of cytokine and cytolytic granule by OT‐I CTLs. CTLs were incubated with EG7‐L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule was determined by flow cytometry. aPD1 served as positive control. EG7‐L1: EG7 overexpressing PD‐L1. J Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired t ‐test; error bars denote SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Digested hPD‐1 and hPD‐L1 were subcloned into identically digested pCAGiN, respectively. pcDNA3.1‐hPD‐1‐EGFP and pcDNA3.1‐hSHP2‐mCherry (human SHP2) for colocalization assay: Fragments hPD‐1‐EGFP and hSHP2‐mCherry were synthesized. pcDNA3.1 was digested with Knp1 and Asc1. hPD‐1‐EGFP and hSHP2‐mCherry fragments were identically digested for cloning into pcDNA3.1. pWPI‐mPDL1 (mouse‐PDL1) for overexpression of mPD‐L1 and IRES‐EGFP element in the vector for FACS sorting virus‐infected cells: pWPI (Addgene, #12254) and mPD‐L1 fragment was digested with SwaI and AscI and joined by T4 ligase. pCAGiN‐hPD‐1‐C‐luciferase for overexpressing fusion gene containing hPD‐1 followed by C‐terminal half of luciferase.

Techniques: Staining, Knock-Out, Activation Assay, Positive Control, Expressing, Luciferase, Activity Assay, Co-Culture Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

doi: 10.15252/emmm.201911571

Figure Lengend Snippet: A Effect of different concentration of MB on EG7‐L1 xenograft in C57BL/6J mice ( n = 5). EG7‐L1: EG7 overexpressing PD‐L1. B EG7‐L1 xenograft growth curve in RAG1 −/− mice ( n = 2). C Effect of different concentration of MB on xenograft in RAG1 −/− mice ( n = 2). D Tumor growth curves of subcutaneous tumor allograft. EG7‐L1 cell (2 × 10 6 cells) was subcutaneously injected into the right flank of C57BL/6J mice ( n = 4 per group). The mice were then injected with WT or PD‐1KO CTLs (i.v.) on days 3 and 6, respectively, and treated with vehicle, aPD1 (i. p . 10 mg/kg, every other day), or MB (i.g. 20 mg/ kg/day). Tumor volume is shown as mean ± SEM. E Expression of IL‐2, granzyme B, and perforin by CD8 + T Cells was analyzed in tumors of EC mice after treatment with MB by FACS. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Digested hPD‐1 and hPD‐L1 were subcloned into identically digested pCAGiN, respectively. pcDNA3.1‐hPD‐1‐EGFP and pcDNA3.1‐hSHP2‐mCherry (human SHP2) for colocalization assay: Fragments hPD‐1‐EGFP and hSHP2‐mCherry were synthesized. pcDNA3.1 was digested with Knp1 and Asc1. hPD‐1‐EGFP and hSHP2‐mCherry fragments were identically digested for cloning into pcDNA3.1. pWPI‐mPDL1 (mouse‐PDL1) for overexpression of mPD‐L1 and IRES‐EGFP element in the vector for FACS sorting virus‐infected cells: pWPI (Addgene, #12254) and mPD‐L1 fragment was digested with SwaI and AscI and joined by T4 ligase. pCAGiN‐hPD‐1‐C‐luciferase for overexpressing fusion gene containing hPD‐1 followed by C‐terminal half of luciferase.

Techniques: Concentration Assay, Injection, Expressing

Journal: EMBO Molecular Medicine

Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

doi: 10.15252/emmm.201911571

Figure Lengend Snippet: A Effect of MB on proliferation of human CD8 + T cells. CFSE‐labeled human peripheral blood mononuclear cells (PBMC) were preincubated with DMSO, MB, 25 μg/ml of pembrolizumab, or 20 μg/ml of nivolumab for 1 h and then seeded in the 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD‐L1 protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. B Cytokine and cytolytic granule production in CD8 + T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem), or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD‐L1 protein. Cytokine and cytolytic granule production was determined by intracellular flow cytometry by gating on CD8 + populations. C Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP‐luc cells were stimulated with Raji‐L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 6 h. Luciferase activity was measured by luminometer. Data are representative of three independent experiments and were analyzed by unpaired t ‐test. JP‐luc: Jurkat cell harboring NFAT‐luciferase transgene and overexpressing PD‐1; Raji‐L1: Raji overexpressing PD‐L1. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. ** P < 0.01; **** P < 0.0001.

Article Snippet: Digested hPD‐1 and hPD‐L1 were subcloned into identically digested pCAGiN, respectively. pcDNA3.1‐hPD‐1‐EGFP and pcDNA3.1‐hSHP2‐mCherry (human SHP2) for colocalization assay: Fragments hPD‐1‐EGFP and hSHP2‐mCherry were synthesized. pcDNA3.1 was digested with Knp1 and Asc1. hPD‐1‐EGFP and hSHP2‐mCherry fragments were identically digested for cloning into pcDNA3.1. pWPI‐mPDL1 (mouse‐PDL1) for overexpression of mPD‐L1 and IRES‐EGFP element in the vector for FACS sorting virus‐infected cells: pWPI (Addgene, #12254) and mPD‐L1 fragment was digested with SwaI and AscI and joined by T4 ligase. pCAGiN‐hPD‐1‐C‐luciferase for overexpressing fusion gene containing hPD‐1 followed by C‐terminal half of luciferase.

Techniques: Labeling, Flow Cytometry, Comparison, Concentration Assay, Luciferase, Activity Assay