pd l1 protein  (Sino Biological)


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    Name:
    PD L1 Protein Human Recombinant
    Description:
    A DNA sequence encoding the N terminal segment Met 1 Thr 239 of the extracellular domain of human B7 H1 NP 054862 1 was expressed with C terminal fused Fc region of human IgG1
    Catalog Number:
    10084-H02H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    B7-H Protein Human, B7-H1 Protein Human, B7H1 Protein Human, PD-L1 Protein Human, PDCD1L1 Protein Human, PDCD1LG1 Protein Human, PDL1 Protein Human
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological pd l1 protein
    A DNA sequence encoding the N terminal segment Met 1 Thr 239 of the extracellular domain of human B7 H1 NP 054862 1 was expressed with C terminal fused Fc region of human IgG1
    https://www.bioz.com/result/pd l1 protein/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pd l1 protein - by Bioz Stars, 2021-09
    94/100 stars

    Images

    Related Articles

    Binding Assay:

    Article Title: Soluble PD-L1 is associated with local and systemic inflammation markers in primary and secondary brain tumours
    Article Snippet: .. Following anti-PD-L1 antibody binding and two subsequent washes with PBS, samples and standard PD-L1 human recombinant protein (Sino Biologicals 10084-H02H, Wayne, Pennsylvania, USA) were incubated for 12 hours at 4°C. ..

    Article Title: Novel anti-4-1BB×PD-L1 bispecific antibody augments anti-tumor immunity through tumor-directed T-cell activation and checkpoint blockade
    Article Snippet: .. For the dual-antigen capture ELISA, human PD-L1 (10084-H02H; Sino Biological) was coated and ABL503 binding detected using human 4-1BB-His (10041-H08H; Sino Biological) and HRP-conjugated anti-His antibody (Roche). ..

    Recombinant:

    Article Title: Soluble PD-L1 is associated with local and systemic inflammation markers in primary and secondary brain tumours
    Article Snippet: .. Following anti-PD-L1 antibody binding and two subsequent washes with PBS, samples and standard PD-L1 human recombinant protein (Sino Biologicals 10084-H02H, Wayne, Pennsylvania, USA) were incubated for 12 hours at 4°C. ..

    Article Title: Soluble PD-L1 is associated with local and systemic inflammation markers in primary and secondary brain tumours
    Article Snippet: .. The lower limit of sPD-L1 detection was 0.05 ng/mL as determined by serial dilutions of recombinant human PD-L1. ..

    Incubation:

    Article Title: Soluble PD-L1 is associated with local and systemic inflammation markers in primary and secondary brain tumours
    Article Snippet: .. Following anti-PD-L1 antibody binding and two subsequent washes with PBS, samples and standard PD-L1 human recombinant protein (Sino Biologicals 10084-H02H, Wayne, Pennsylvania, USA) were incubated for 12 hours at 4°C. ..

    Labeling:

    Article Title: Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors
    Article Snippet: .. In Fig A, human peripheral blood mononuclear cells from healthy donors were labeled with CFSE and seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 and with culture media supplemented with 10 μg/ml hPD‐L1 protein (Fc tag, Sino Biological, 10084‐H02H‐100) in the presence of 100 nM of MB or 25 μg/ml of pembrolizumab or 20 μg/ml nivolumab (kindly gifted by Dr. Li Zhang, SYSUCC). ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: Nanobody against PDL1.
    Article Snippet: .. Programmed death ligand 1 (PDL1, CD274, B7-H1) has been identified as the ligand for the immune inhibitory receptor programmed death 1 protein (PD1/PDCD1). ..

    Article Title: Novel anti-4-1BB×PD-L1 bispecific antibody augments anti-tumor immunity through tumor-directed T-cell activation and checkpoint blockade
    Article Snippet: .. For the dual-antigen capture ELISA, human PD-L1 (10084-H02H; Sino Biological) was coated and ABL503 binding detected using human 4-1BB-His (10041-H08H; Sino Biological) and HRP-conjugated anti-His antibody (Roche). ..

    other:

    Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model
    Article Snippet: Docking Simulation and Interaction Analysis The SPE component cosmosiin was docked onto the interaction space between PD-1 and PD-L1 retrieved from the Protein Data Bank ( www.rcsb.org, PDB code: 4ZQK) and a previous report , using AutoDock Vina integrated with UCSF Chimera-alpha v1.13 ( ).

    Isolation:

    Article Title: Nanobody against PDL1.
    Article Snippet: .. Programmed death ligand 1 (PDL1, CD274, B7-H1) has been identified as the ligand for the immune inhibitory receptor programmed death 1 protein (PD1/PDCD1). ..

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  • 86
    Sino Biological hpd l1 fc protein
    The <t>hPD-L1</t> binding ability of hPD-1 mutants. The binding of hPD-1 mutants with <t>hPD-L1-Fc</t> were measured by FACS. a , c Representative flow cytometry analyses of hPD-L1 binding to the HEK-293 T cells expressing WT hPD-1 or the mutants. b , d The binding affinity between hPD-1 mutants and hPD-L1 at different protein concentrations. Each point represents the mean ± S.E. of four independent measurements. e1 , e2 Relative PD-L1 binding potency (RP) values of the hPD-1 mutants. (mean ± S.E., n = 4). *, p
    Hpd L1 Fc Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hpd l1 fc protein/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    93
    Sino Biological mpd l1 protein
    MB is effective to activate human CD8 +  T cells 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. 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. 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
    Mpd L1 Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mpd l1 protein/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    93
    Sino Biological mouse pd l1
    PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or CD86 SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled <t>SNAP-PD-L1-His</t> was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p
    Mouse Pd L1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse pd l1/product/Sino Biological
    Average 93 stars, based on 1 article reviews
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    93
    Sino Biological human pd l1
    89 Zr-C4 can specifically detect mouse <t>PD-L1</t> in tumors established in an immunocompetent background. (A) Representative coronal and transaxial PET/CT images of male C57BL/6 mice bearing subcutaneous B16 F10 tumors, a mouse model of melanoma, show that peak tumor uptake of 89 Zr-C4 occurs 48 h after injection. (B) Biodistribution data also show peak tumor uptake of the radiotracer 48 h after injection. High uptake is also observed in PD-L1 positive tissues like the liver a spleen. (C) Representative data from a blocking study acquired 48 h after injection show the tumor specific uptake of 89 Zr-C4. Blocking was performed with 10-fold excess C4. Radiotracer uptake exceeded that observed in the blood pool and muscle: (∗) P
    Human Pd L1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human pd l1/product/Sino Biological
    Average 93 stars, based on 1 article reviews
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    Image Search Results


    The hPD-L1 binding ability of hPD-1 mutants. The binding of hPD-1 mutants with hPD-L1-Fc were measured by FACS. a , c Representative flow cytometry analyses of hPD-L1 binding to the HEK-293 T cells expressing WT hPD-1 or the mutants. b , d The binding affinity between hPD-1 mutants and hPD-L1 at different protein concentrations. Each point represents the mean ± S.E. of four independent measurements. e1 , e2 Relative PD-L1 binding potency (RP) values of the hPD-1 mutants. (mean ± S.E., n = 4). *, p

    Journal: Cell Communication and Signaling : CCS

    Article Title: The design of high affinity human PD-1 mutants by using molecular dynamics simulations (MD)

    doi: 10.1186/s12964-018-0239-9

    Figure Lengend Snippet: The hPD-L1 binding ability of hPD-1 mutants. The binding of hPD-1 mutants with hPD-L1-Fc were measured by FACS. a , c Representative flow cytometry analyses of hPD-L1 binding to the HEK-293 T cells expressing WT hPD-1 or the mutants. b , d The binding affinity between hPD-1 mutants and hPD-L1 at different protein concentrations. Each point represents the mean ± S.E. of four independent measurements. e1 , e2 Relative PD-L1 binding potency (RP) values of the hPD-1 mutants. (mean ± S.E., n = 4). *, p

    Article Snippet: The cells were washed and incubated with hPD-L1-Fc protein (Sino Biological Inc., China), then stained with APC conjugated anti-human IgG (Biolegend, US) on ice for 30 min. Next, the cells were acquired on a FACS Caliber flow cytometry (BD Biosciences, US) and analyzed by CELLQuest™ software.

    Techniques: Binding Assay, FACS, Flow Cytometry, Cytometry, Expressing

    MB is effective to activate human CD8 +  T cells 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. 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. 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

    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: MB is effective to activate human CD8 + T cells 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. 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. 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

    Article Snippet: These cells were then seeded in 6‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media supplemented with 20 μg/ml of mPD‐L1 protein and 100 nM of MB for 2 min.

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

    MB suppress PD‐1 signaling through blocking SHP2 recruitment by PD‐1 Confocal cell images of Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry. Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry were incubated with 10 μg/ml of human PD‐L1 protein in the presence of 100 nM and 1 μM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 μm). Fluorescence complementation analysis of the effect of MB on the interaction between PD‐1 and SHP2. Jurkat cells co‐expressing PD1‐C‐Luc or PD1 Y248F ‐C‐Luc and SHP2‐N‐luc were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. Co‐IP analysis of impact of MB on interaction between PD‐1 and SHP2. 293T cells co‐expressing PD‐1 and SHP2‐FLAG were treated with MB at indicated concentration for 6 h. Ability of MB to block interaction between PD‐1 with SHP2 revealed through ELISA. Y248‐phosphorylated human PD‐1 ITSM peptide was coated at 4 μg/ml in 96‐well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2. Representative Western blot showing the levels of total and phosphorylated ZAP70, PKCθ, and PLCγ1 in the lysates of stimulated CTL, inhibited by PD‐L1 in the presence of 1 μM of MB. Representative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7‐L1, and EG7‐L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti‐pY from the lysates of the indicated CTL‐EG7 co‐culture was subjected to SDS–PAGE separation and blotted with aCD28. β‐Actin served as loading control. EG7‐L1: EG7 overexpressing PD‐L1. Phos‐tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7‐L1 in the presence of 1 μM MB. Cell lysate was then separated by SDS–PAGE containing 50 μM of Phos‐tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow‐moving) species were visualized through exposure. Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat‐PD‐1 cells were co‐transfected with CD28‐C‐Luc or CD28 Y191F ‐C‐Luc and P85‐N‐Luc. Cells were seeded in a 96‐well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD‐L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28‐P85 interaction. Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. * P

    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: MB suppress PD‐1 signaling through blocking SHP2 recruitment by PD‐1 Confocal cell images of Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry. Jurkat cells co‐expressing PD‐1‐EGFP and SHP2‐mCherry were incubated with 10 μg/ml of human PD‐L1 protein in the presence of 100 nM and 1 μM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 μm). Fluorescence complementation analysis of the effect of MB on the interaction between PD‐1 and SHP2. Jurkat cells co‐expressing PD1‐C‐Luc or PD1 Y248F ‐C‐Luc and SHP2‐N‐luc were seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD‐L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity. Co‐IP analysis of impact of MB on interaction between PD‐1 and SHP2. 293T cells co‐expressing PD‐1 and SHP2‐FLAG were treated with MB at indicated concentration for 6 h. Ability of MB to block interaction between PD‐1 with SHP2 revealed through ELISA. Y248‐phosphorylated human PD‐1 ITSM peptide was coated at 4 μg/ml in 96‐well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2. Representative Western blot showing the levels of total and phosphorylated ZAP70, PKCθ, and PLCγ1 in the lysates of stimulated CTL, inhibited by PD‐L1 in the presence of 1 μM of MB. Representative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7‐L1, and EG7‐L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti‐pY from the lysates of the indicated CTL‐EG7 co‐culture was subjected to SDS–PAGE separation and blotted with aCD28. β‐Actin served as loading control. EG7‐L1: EG7 overexpressing PD‐L1. Phos‐tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7‐L1 in the presence of 1 μM MB. Cell lysate was then separated by SDS–PAGE containing 50 μM of Phos‐tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow‐moving) species were visualized through exposure. Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat‐PD‐1 cells were co‐transfected with CD28‐C‐Luc or CD28 Y191F ‐C‐Luc and P85‐N‐Luc. Cells were seeded in a 96‐well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD‐L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28‐P85 interaction. Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P

    Article Snippet: These cells were then seeded in 6‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media supplemented with 20 μg/ml of mPD‐L1 protein and 100 nM of MB for 2 min.

    Techniques: Blocking Assay, Expressing, Incubation, Staining, Microscopy, Fluorescence, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot, Immunoprecipitation, Co-Culture Assay, SDS Page, Transfection

    MB enhanced activation and effector function of CTL 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. Bar graph of (Fig   EV2 A). 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. Bar graph of IFNγ production of OT‐1 CTLs in (Fig   EV2 C). Bar graph of GZMB production of OT‐1 CTLs in (Fig   EV2 C). Data information: Data are representative of three independent experiments and were analyzed by unpaired  t ‐test. Error bars denote SEM. * P

    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: MB enhanced activation and effector function of CTL 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. Bar graph of (Fig  EV2 A). 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. Bar graph of IFNγ production of OT‐1 CTLs in (Fig  EV2 C). Bar graph of GZMB production of OT‐1 CTLs in (Fig  EV2 C). Data information: Data are representative of three independent experiments and were analyzed by unpaired t ‐test. Error bars denote SEM. * P

    Article Snippet: These cells were then seeded in 6‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media supplemented with 20 μg/ml of mPD‐L1 protein and 100 nM of MB for 2 min.

    Techniques: Activation Assay, Mouse Assay, Labeling, FACS, Incubation, Expressing, Flow Cytometry, Positive Control

    MB enhanced activation and effector function of CTL 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. Statistical results of (A). 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. Statistical results of (C). 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. 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. 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. MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). 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. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired  t ‐test; error bars denote SEM. * P

    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: MB enhanced activation and effector function of CTL 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. Statistical results of (A). 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. Statistical results of (C). 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. 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. 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. MB enhancing IL‐2 expression by JP‐luc stimulated with Raji‐L1 for 24 h quantified through ELISA analysis (aPD1 served as positive control). 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. Statistical results of (I). Data information: Data are representative of three independent experiments. Unpaired t ‐test; error bars denote SEM. * P

    Article Snippet: These cells were then seeded in 6‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media supplemented with 20 μg/ml of mPD‐L1 protein and 100 nM of MB for 2 min.

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

    PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or CD86 SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled SNAP-PD-L1-His was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p

    Journal: Immunity

    Article Title: PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

    doi: 10.1016/j.immuni.2019.11.003

    Figure Lengend Snippet: PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or CD86 SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled SNAP-PD-L1-His was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p

    Article Snippet: For , SLB was functionalized by a mixture of 5 nM pMHC-I-His, 2 nM mouse ICAM–His (Sino Biological, 50440-M08H) and either 3 nM mouse PD-L1–His (Sino Biological, 50010-M08H), 3 nM mouse PD-L1–His plus 9 nM mouse CD80–His (Sino Biological, 50446-M08H), or 3 nM mouse PD-L1–His plus 9 nM mouse CD86–His (Sino Biological, 50068-M08H).

    Techniques: Fluorescence, Expressing, Labeling, Two Tailed Test

    Cis -PD-L1 Inhibits CD80:CTLA-4 Interaction through Disrupting CD80 Homodimers (A) Representative flow-cytometry histograms of CTLA-4-huFc staining of the indicated types of Raji cells. Bound CTLA-4-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CTLA-4-huFc). Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n ≥ 3. (B) Representative flow-cytometry histograms of CTLA-4-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CTLA-4-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n ≥ 3. (C) Representative flow-cytometry histograms of CTLA-4-GCN4*SC647 staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab and of Raji (CD80 − CD86 − ) cells with atezolizumab. MFI of SC647 was plotted against the input concentration (means ± SEM, n ≥ 3). (D) At the top are flow-cytometry histograms showing both PD-L1 and CD80 amounts on a population of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with tight PD-L1 expression and a wide range of CD80 expression. The cells were stained with either phycoerythrin (PE) anti-CD80, PE anti-PD-L1, or PE isotype, and the 3 histograms overlaid. On the bottom is a flow-cytometry dot plot showing CTLA-4-GCN4*SC647 staining of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with or without atezolizumab. Gray dots correspond to control signals of unstained cells. CD80 + cells were gated by the vertical dash line, determined by the mGFP signal of parental Raji (CD80 − CD86 − ) cells. (E) A FRET assay probing CD80:CD80 homodimerization on cell membranes. In the first row, the leftmost cartoon depicts a HEK293T cell expressing SNAP-CD80, with a subpopulation labeled with SS549 (donor) and the rest labeled with SSAF647 (acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell. Further on the right is the calculated pseudo-color FRET efficiency image (yellow to purple spectrum denotes strong to weak FRET) and the DIC image. The second and third rows are the same as the first row except replacing SNAP-CD80 with SNAP-CD80 (I92R) or with SNAP-CD86. The fourth row is the same as the first row except with co-expressed unlabeled PD-L1. The fifth row is the same as fourth row except in the presence of atezolizumab. The bar graph summarizes the FRET efficiencies as mean ± SEM, n > 22 cells from 3 independent experiments. Scale bars, 10 μm. (F) An LUV FRET assay for probing CD80:CD80 homodimerization and PD-L1 effects. Shown is a representative time course of normalized FI of LUV-bound SC505*CD80-His, challenged by TMR*CD80-His and then by indicated concentrations of unlabeled PD-L1-His, with or without atezolizumab (Atezo) (20 μg/mL). (G) An LUV FRET assay showing that a single point mutation in CD80 disrupts both CD80:CD80 homodimerization and PD-L1:CD80 heterodimerization. Each indicated SC505 (energy donor)-labeled protein was pre-coupled to DGS-NTA-Ni containing LUVs through its His-tag, and challenged with TMR (energy acceptor)-labeled proteins as indicated. Shown are representative time courses of 3 independent replicates. (H) Representative TIRF images of Raji (CD80-mGFP + CD86 − PD-L1-SNAP + . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Journal: Immunity

    Article Title: PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

    doi: 10.1016/j.immuni.2019.11.003

    Figure Lengend Snippet: Cis -PD-L1 Inhibits CD80:CTLA-4 Interaction through Disrupting CD80 Homodimers (A) Representative flow-cytometry histograms of CTLA-4-huFc staining of the indicated types of Raji cells. Bound CTLA-4-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CTLA-4-huFc). Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n ≥ 3. (B) Representative flow-cytometry histograms of CTLA-4-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CTLA-4-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n ≥ 3. (C) Representative flow-cytometry histograms of CTLA-4-GCN4*SC647 staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab and of Raji (CD80 − CD86 − ) cells with atezolizumab. MFI of SC647 was plotted against the input concentration (means ± SEM, n ≥ 3). (D) At the top are flow-cytometry histograms showing both PD-L1 and CD80 amounts on a population of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with tight PD-L1 expression and a wide range of CD80 expression. The cells were stained with either phycoerythrin (PE) anti-CD80, PE anti-PD-L1, or PE isotype, and the 3 histograms overlaid. On the bottom is a flow-cytometry dot plot showing CTLA-4-GCN4*SC647 staining of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with or without atezolizumab. Gray dots correspond to control signals of unstained cells. CD80 + cells were gated by the vertical dash line, determined by the mGFP signal of parental Raji (CD80 − CD86 − ) cells. (E) A FRET assay probing CD80:CD80 homodimerization on cell membranes. In the first row, the leftmost cartoon depicts a HEK293T cell expressing SNAP-CD80, with a subpopulation labeled with SS549 (donor) and the rest labeled with SSAF647 (acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell. Further on the right is the calculated pseudo-color FRET efficiency image (yellow to purple spectrum denotes strong to weak FRET) and the DIC image. The second and third rows are the same as the first row except replacing SNAP-CD80 with SNAP-CD80 (I92R) or with SNAP-CD86. The fourth row is the same as the first row except with co-expressed unlabeled PD-L1. The fifth row is the same as fourth row except in the presence of atezolizumab. The bar graph summarizes the FRET efficiencies as mean ± SEM, n > 22 cells from 3 independent experiments. Scale bars, 10 μm. (F) An LUV FRET assay for probing CD80:CD80 homodimerization and PD-L1 effects. Shown is a representative time course of normalized FI of LUV-bound SC505*CD80-His, challenged by TMR*CD80-His and then by indicated concentrations of unlabeled PD-L1-His, with or without atezolizumab (Atezo) (20 μg/mL). (G) An LUV FRET assay showing that a single point mutation in CD80 disrupts both CD80:CD80 homodimerization and PD-L1:CD80 heterodimerization. Each indicated SC505 (energy donor)-labeled protein was pre-coupled to DGS-NTA-Ni containing LUVs through its His-tag, and challenged with TMR (energy acceptor)-labeled proteins as indicated. Shown are representative time courses of 3 independent replicates. (H) Representative TIRF images of Raji (CD80-mGFP + CD86 − PD-L1-SNAP + . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Article Snippet: For , SLB was functionalized by a mixture of 5 nM pMHC-I-His, 2 nM mouse ICAM–His (Sino Biological, 50440-M08H) and either 3 nM mouse PD-L1–His (Sino Biological, 50010-M08H), 3 nM mouse PD-L1–His plus 9 nM mouse CD80–His (Sino Biological, 50446-M08H), or 3 nM mouse PD-L1–His plus 9 nM mouse CD86–His (Sino Biological, 50068-M08H).

    Techniques: Flow Cytometry, Cytometry, Staining, Labeling, Isolation, Concentration Assay, Expressing, Mutagenesis, Two Tailed Test

    89 Zr-C4 can specifically detect mouse PD-L1 in tumors established in an immunocompetent background. (A) Representative coronal and transaxial PET/CT images of male C57BL/6 mice bearing subcutaneous B16 F10 tumors, a mouse model of melanoma, show that peak tumor uptake of 89 Zr-C4 occurs 48 h after injection. (B) Biodistribution data also show peak tumor uptake of the radiotracer 48 h after injection. High uptake is also observed in PD-L1 positive tissues like the liver a spleen. (C) Representative data from a blocking study acquired 48 h after injection show the tumor specific uptake of 89 Zr-C4. Blocking was performed with 10-fold excess C4. Radiotracer uptake exceeded that observed in the blood pool and muscle: (∗) P

    Journal: Bioconjugate Chemistry

    Article Title: Imaging PD-L1 Expression with ImmunoPET

    doi: 10.1021/acs.bioconjchem.7b00631

    Figure Lengend Snippet: 89 Zr-C4 can specifically detect mouse PD-L1 in tumors established in an immunocompetent background. (A) Representative coronal and transaxial PET/CT images of male C57BL/6 mice bearing subcutaneous B16 F10 tumors, a mouse model of melanoma, show that peak tumor uptake of 89 Zr-C4 occurs 48 h after injection. (B) Biodistribution data also show peak tumor uptake of the radiotracer 48 h after injection. High uptake is also observed in PD-L1 positive tissues like the liver a spleen. (C) Representative data from a blocking study acquired 48 h after injection show the tumor specific uptake of 89 Zr-C4. Blocking was performed with 10-fold excess C4. Radiotracer uptake exceeded that observed in the blood pool and muscle: (∗) P

    Article Snippet: Following 3 rounds of biopanning against the biotinylated ectodomain of the human PD-L1, 27 antigen-positive Fabs of unique sequences were identified after screening 249 clones.

    Techniques: Positron Emission Tomography, Mouse Assay, Injection, Blocking Assay

    89 Zr-C4 can detect pharmacologically induced PD-L1 expression changes on the tumor cell. (A) Representative coronal and transverse PET images showing the distribution of 89 Zr-C4 48 h after injection in a cohort of male nu/nu mice bearing subcutaneous H1975 xenografts and treated with vehicle, paclitaxel (Taxol), or doxorubicine. The mice were treated with 20 mg/kg paclitaxel or 2 mg/kg doxorubicine for 2 days prior to radiotracer injection. (B) Representative biodistribution data in the tumor and selected normal tissues showing that paclitaxel increases tumor PD-L1 expression levels, while doxorubicin suppresses it compared to vehicle. No impact was observed on PD-L1 expressing normal tissues like liver and spleen. (∗) P

    Journal: Bioconjugate Chemistry

    Article Title: Imaging PD-L1 Expression with ImmunoPET

    doi: 10.1021/acs.bioconjchem.7b00631

    Figure Lengend Snippet: 89 Zr-C4 can detect pharmacologically induced PD-L1 expression changes on the tumor cell. (A) Representative coronal and transverse PET images showing the distribution of 89 Zr-C4 48 h after injection in a cohort of male nu/nu mice bearing subcutaneous H1975 xenografts and treated with vehicle, paclitaxel (Taxol), or doxorubicine. The mice were treated with 20 mg/kg paclitaxel or 2 mg/kg doxorubicine for 2 days prior to radiotracer injection. (B) Representative biodistribution data in the tumor and selected normal tissues showing that paclitaxel increases tumor PD-L1 expression levels, while doxorubicin suppresses it compared to vehicle. No impact was observed on PD-L1 expressing normal tissues like liver and spleen. (∗) P

    Article Snippet: Following 3 rounds of biopanning against the biotinylated ectodomain of the human PD-L1, 27 antigen-positive Fabs of unique sequences were identified after screening 249 clones.

    Techniques: Expressing, Positron Emission Tomography, Injection, Mouse Assay

    Defining the optimal time after injection to study PD-L1 expression levels in a xenograft model. (A) Representative coronal and transaxial PET/CT images of male nu/nu mice bearing subcutaneous H1975 tumors, a human model of NSCLC , show that peak tumor uptake of 89 Zr-C4 occurs 48 h after injection. (B) Biodistribution data also show peak tumor uptake of the radiotracer 48 h after injection. High uptake is also observed in PD-L1 positive tissues like the liver a spleen. (C) Representative data from a blocking study acquired 48 h after injection show the tumor specific uptake of 89 Zr-C4. Blocking was performed with 30-fold excess C4. Radiotracer uptake exceeded that observed in the blood pool and muscle: (∗) P

    Journal: Bioconjugate Chemistry

    Article Title: Imaging PD-L1 Expression with ImmunoPET

    doi: 10.1021/acs.bioconjchem.7b00631

    Figure Lengend Snippet: Defining the optimal time after injection to study PD-L1 expression levels in a xenograft model. (A) Representative coronal and transaxial PET/CT images of male nu/nu mice bearing subcutaneous H1975 tumors, a human model of NSCLC , show that peak tumor uptake of 89 Zr-C4 occurs 48 h after injection. (B) Biodistribution data also show peak tumor uptake of the radiotracer 48 h after injection. High uptake is also observed in PD-L1 positive tissues like the liver a spleen. (C) Representative data from a blocking study acquired 48 h after injection show the tumor specific uptake of 89 Zr-C4. Blocking was performed with 30-fold excess C4. Radiotracer uptake exceeded that observed in the blood pool and muscle: (∗) P

    Article Snippet: Following 3 rounds of biopanning against the biotinylated ectodomain of the human PD-L1, 27 antigen-positive Fabs of unique sequences were identified after screening 249 clones.

    Techniques: Injection, Expressing, Positron Emission Tomography, Mouse Assay, Blocking Assay

    89 Zr-C4 detects PD-L1 expression levels in a PDX derived from a NSCLC patient that experienced a durable clinical response to anti-PD-1 and anti-CTLA4 therapies. (A) Transaxial CT slices showing a soft tissue lesion in the lung prior to the initiation of pembrolizumab and ipilimumab (left), and a smaller mass 3 months after the start of therapy (right). The position of the tumor is indicated with a white arrow. This patient experienced a partial response for 8 months. The PDX was derived 7 months prior to the first CT scan. (B) Small animal PET/CT data showing the biodistribution of 89 Zr-C4 in mice bearing bilateral PDX tumors in the flank. The tumors can be clearly resolved, and radiotracer uptake in abdominal tissues like the liver is observed, as expected for a large biomolecule. Mice treated with 89 Zr-C4 that was heat denatured (HD) for 10 min prior to injection show no evidence of radiotracer uptake in the tumor. (C) Biodistribution data showing the uptake of 89 Zr-C4 in the PDX tissue 48 h after injection. The uptake is higher in the tumor compared to heat denatured 89 Zr-C4 (HD) and standard reference tissues like the blood and muscle. (D) Biodistribution data acquired 48 h after injection in mice bearing subcutaneous H1975, PC3, A549, and the PDX tumors show the different degree of 89 Zr-C4 uptake in the tumors.

    Journal: Bioconjugate Chemistry

    Article Title: Imaging PD-L1 Expression with ImmunoPET

    doi: 10.1021/acs.bioconjchem.7b00631

    Figure Lengend Snippet: 89 Zr-C4 detects PD-L1 expression levels in a PDX derived from a NSCLC patient that experienced a durable clinical response to anti-PD-1 and anti-CTLA4 therapies. (A) Transaxial CT slices showing a soft tissue lesion in the lung prior to the initiation of pembrolizumab and ipilimumab (left), and a smaller mass 3 months after the start of therapy (right). The position of the tumor is indicated with a white arrow. This patient experienced a partial response for 8 months. The PDX was derived 7 months prior to the first CT scan. (B) Small animal PET/CT data showing the biodistribution of 89 Zr-C4 in mice bearing bilateral PDX tumors in the flank. The tumors can be clearly resolved, and radiotracer uptake in abdominal tissues like the liver is observed, as expected for a large biomolecule. Mice treated with 89 Zr-C4 that was heat denatured (HD) for 10 min prior to injection show no evidence of radiotracer uptake in the tumor. (C) Biodistribution data showing the uptake of 89 Zr-C4 in the PDX tissue 48 h after injection. The uptake is higher in the tumor compared to heat denatured 89 Zr-C4 (HD) and standard reference tissues like the blood and muscle. (D) Biodistribution data acquired 48 h after injection in mice bearing subcutaneous H1975, PC3, A549, and the PDX tumors show the different degree of 89 Zr-C4 uptake in the tumors.

    Article Snippet: Following 3 rounds of biopanning against the biotinylated ectodomain of the human PD-L1, 27 antigen-positive Fabs of unique sequences were identified after screening 249 clones.

    Techniques: Expressing, Derivative Assay, Computed Tomography, Positron Emission Tomography, Mouse Assay, Injection