Staining:Article Title: Enhanced anti-tumor activity by zinc finger repressor-driven epigenetic silencing of immune checkpoints and TGFBR2 in CAR-T cells and TILs
Article Snippet: .. Human primary cells immunophenotyping For cellular immunophenotyping, T cells were stained with conjugated monoclonal antibodies (mAb) targeting HA (RRID:AB_2784359), PD-1 (RRID:AB_2752070), TIGIT (RRID:AB_2751336), TIM-3 (RRID:AB_RRID:AB_2657763), CD271/NGFR (RRID:AB_2725863) (Miltenyi Biotec), LAG-3 (RRID:AB_2632656), and TGFBR2 (RRID: AB_2876726, BioLegend, Amsterdam, the Netherlands). .. For TIL characterization, expanded cells were stained with fluorophore-labeled antibodies against CD3 (RRID:AB_493741), CD4 (RRID:AB_2563314), CD8 (RRID:AB_1967149), CD271/NGFR (RRID:AB_2282828), PD-1 (RRID:AB_2563658), TIGIT (RRID:AB_2632929), TIM-3 (RRID: AB_11218598), LAG-3 (RRID:AB_2632656), TGFBR2 (RRID: AB_2876726), CD45RA (RRID:AB_2563816), CCR7 (RRID:AB_ 2563640), CD183/CXCR3 (RRID:AB_11219383), CD25 (RRID:AB_ 314280), CD28 (RRID:AB_2800752), KLRG1 (RRID:AB_2566594), CD62L (RRID:AB_2562130), CD127 (RRID:AB_10720185), FoxP3 (RRID:AB_430887), CD40L (RRID:AB_2562721), CD107a (RRID: AB_2563851, BioLegend), TNF-α (RRID:AB_2751384, Miltenyi Biotec), and IFN-γ (RRID:AB_396760, BD Biosciences, Heidelberg, Germany).
Article Title: Enhanced anti-tumor activity by zinc finger repressor-driven epigenetic silencing of immune checkpoints and TGFBR2 in CAR-T cells and TILs
Article Snippet: Human primary cells immunophenotyping For cellular immunophenotyping, T cells were stained with conjugated monoclonal antibodies (mAb) targeting HA (RRID:AB_2784359), PD-1 (RRID:AB_2752070), TIGIT (RRID:AB_2751336), TIM-3 (RRID:AB_RRID:AB_2657763), CD271/NGFR (RRID:AB_2725863) (Miltenyi Biotec), LAG-3 (RRID:AB_2632656), and TGFBR2 (RRID: AB_2876726, BioLegend, Amsterdam, the Netherlands). .. For TIL characterization, expanded cells were stained with fluorophore-labeled antibodies against CD3 (RRID:AB_493741), CD4 (RRID:AB_2563314), CD8 (RRID:AB_1967149), CD271/NGFR (RRID:AB_2282828), PD-1 (RRID:AB_2563658), TIGIT (RRID:AB_2632929), TIM-3 (RRID: AB_11218598), LAG-3 (RRID:AB_2632656), TGFBR2 (RRID: AB_2876726), CD45RA (RRID:AB_2563816), CCR7 (RRID:AB_ 2563640), CD183/CXCR3 (RRID:AB_11219383), CD25 (RRID:AB_ 314280), CD28 (RRID:AB_2800752), KLRG1 (RRID:AB_2566594), CD62L (RRID:AB_2562130), CD127 (RRID:AB_10720185), FoxP3 (RRID:AB_430887), CD40L (RRID:AB_2562721), CD107a (RRID: AB_2563851, BioLegend), TNF-α (RRID:AB_2751384, Miltenyi Biotec), and IFN-γ (RRID:AB_396760, BD Biosciences, Heidelberg, Germany). .. After surface staining, FoxP3 was assessed after fixation and permeabilization using the BD Pharmingen Human FoxP3 Buffer Set (BD Biosciences).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells
Article Snippet: .. For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark. .. Stained cells were analyzed by applying MACSQuant ® Analyzer 10 and MACSQuantify ® software (both from Miltenyi Biotec GmbH).
Article Title: Enhanced anti-tumor activity by zinc finger repressor-driven epigenetic silencing of immune checkpoints and TGFBR2 in CAR-T cells and TILs
Article Snippet: .. For cellular immunophenotyping, T cells were stained with conjugated monoclonal antibodies (mAb) targeting HA (RRID: AB_2784359 ), PD-1 (RRID: AB_2752070 ), TIGIT (RRID: AB_2751336 ), TIM-3 (RRID:AB_RRID:AB_2657763), CD271/NGFR (RRID: AB_2725863 ) (Miltenyi Biotec), LAG-3 (RRID: AB_2632656 ), and TGFBR2 (RRID: AB_2876726 , BioLegend, Amsterdam, the Netherlands). .. For TIL characterization, expanded cells were stained with fluorophore-labeled antibodies against CD3 (RRID: AB_493741 ), CD4 (RRID: AB_2563314 ), CD8 (RRID: AB_1967149 ), CD271/NGFR (RRID: AB_2282828 ), PD-1 (RRID: AB_2563658 ), TIGIT (RRID: AB_2632929 ), TIM-3 (RRID: AB_11218598 ), LAG-3 (RRID: AB_2632656 ), TGFBR2 (RRID: AB_2876726 ), CD45RA (RRID: AB_2563816 ), CCR7 (RRID: AB_2563640 ), CD183/CXCR3 (RRID: AB_11219383 ), CD25 (RRID: AB_314280 ), CD28 (RRID: AB_2800752 ), KLRG1 (RRID: AB_2566594 ), CD62L (RRID: AB_2562130 ), CD127 (RRID: AB_10720185 ), FoxP3 (RRID: AB_430887 ), CD40L (RRID: AB_2562721 ), CD107a (RRID: AB_2563851 , BioLegend), TNF-α (RRID: AB_2751384 , Miltenyi Biotec), and IFN-γ (RRID: AB_396760 , BD Biosciences, Heidelberg, Germany).
Article Title: CNS infiltration by zamtocabtagene autoleucel tandem CD20/CD19 CAR T cells leading to complete remission in a patient with primary CNS lymphoma.
Article Snippet: Figure 2: Factors potentially promoting persistence of the zamto-cel CAR-T in our PCNSL patient. (A) Surface expression levels of CD27 (Miltenyi, Cat#130-120-036) on PB CAR T cells (left column) from our patient compared to their own non-CAR-T (right column) on day+7 and day+14 post CAR-T treatment, respectively. .. To control for unspecific background staining an FMO control was used (gray histogram). (B) Surface expression levels of exhaustion markers PD-1 (Miltenyi, Cat#130-120-385), LAG-3 (Miltenyi, Cat#130-118-549), and TIM-3 (Miltenyi, Cat130-119-781) on PB CD4+ (upper row) and CD8+ (lower row) CAR T cells from our patient on day+7 and day+14 post CAR-T treatment, respectively. .. To control for unspecific background staining an FMO control was used (gray histogram). (C) Surface expression levels of CD127 (IL-7R ; Miltenyi, Cat#130-113-413) on PB CD4+ or CD8+ CAR T cells on day+7 post CAR-T infusion from our patient compared to their own non-CAR-T. To control for unspecific background staining an FMO control was used (gray histogram).
Article Title: Autocrine signaling in hormonally active cancer induces antigen expression for immunotherapy
Article Snippet: .. The following antibodies were used for cell staining: ROR1 (AF647, clone 2A2, BioLegend), CD45 (VioBlue, clone REA747, Miltenyi), CD4 (PeVio770, clone REA, Miltenyi), CD8 (APC-Cy7, clone SK1, BioLegend), tEGFR (APC, clone AY13, Bio-Legend), PD-1 (PE, clone PD1.3.1., Miltenyi), Lag3 (PerCP-Cy5.5, clone 11C3C65, Bio-Legend), CTLA-4 (PeCy7, clone L3D10, BioLegend), Tim-3 (CD366), APC-Cy7, clone F382E2, BioLegend), CD45RO (FITC, clone UCHL1, BioLegend), CD45RA (PE, clone T6D11, Miltenyi), CD62L (PerCP-Cy5.5, clone DREG-56, BioLegend), CD25, PeCy7, clone BC96, BioLegend), CD69 (APC-Cy7, clone FN50, BioLegend). .. Quantitative flow cytometry was performed using PE-labeled antibodies and the BD-Quantibrite PE-Bead Assay (BD Biosciences).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells.
Article Snippet: .. For phenotypic analysis, 0.2 × 106 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 106 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [45] or the anti-La mAb (7B6) [46] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 ◦C in the dark. .. Stained cells were analyzed by applying MACSQuant® Analyzer 10 and MACSQuantify® software (both from Miltenyi Biotec GmbH).
Bioprocessing:Article Title: Enhanced anti-tumor activity by zinc finger repressor-driven epigenetic silencing of immune checkpoints and TGFBR2 in CAR-T cells and TILs
Article Snippet: .. Human primary cells immunophenotyping For cellular immunophenotyping, T cells were stained with conjugated monoclonal antibodies (mAb) targeting HA (RRID:AB_2784359), PD-1 (RRID:AB_2752070), TIGIT (RRID:AB_2751336), TIM-3 (RRID:AB_RRID:AB_2657763), CD271/NGFR (RRID:AB_2725863) (Miltenyi Biotec), LAG-3 (RRID:AB_2632656), and TGFBR2 (RRID: AB_2876726, BioLegend, Amsterdam, the Netherlands). .. For TIL characterization, expanded cells were stained with fluorophore-labeled antibodies against CD3 (RRID:AB_493741), CD4 (RRID:AB_2563314), CD8 (RRID:AB_1967149), CD271/NGFR (RRID:AB_2282828), PD-1 (RRID:AB_2563658), TIGIT (RRID:AB_2632929), TIM-3 (RRID: AB_11218598), LAG-3 (RRID:AB_2632656), TGFBR2 (RRID: AB_2876726), CD45RA (RRID:AB_2563816), CCR7 (RRID:AB_ 2563640), CD183/CXCR3 (RRID:AB_11219383), CD25 (RRID:AB_ 314280), CD28 (RRID:AB_2800752), KLRG1 (RRID:AB_2566594), CD62L (RRID:AB_2562130), CD127 (RRID:AB_10720185), FoxP3 (RRID:AB_430887), CD40L (RRID:AB_2562721), CD107a (RRID: AB_2563851, BioLegend), TNF-α (RRID:AB_2751384, Miltenyi Biotec), and IFN-γ (RRID:AB_396760, BD Biosciences, Heidelberg, Germany).
Article Title: Enhanced anti-tumor activity by zinc finger repressor-driven epigenetic silencing of immune checkpoints and TGFBR2 in CAR-T cells and TILs
Article Snippet: .. For cellular immunophenotyping, T cells were stained with conjugated monoclonal antibodies (mAb) targeting HA (RRID: AB_2784359 ), PD-1 (RRID: AB_2752070 ), TIGIT (RRID: AB_2751336 ), TIM-3 (RRID:AB_RRID:AB_2657763), CD271/NGFR (RRID: AB_2725863 ) (Miltenyi Biotec), LAG-3 (RRID: AB_2632656 ), and TGFBR2 (RRID: AB_2876726 , BioLegend, Amsterdam, the Netherlands). .. For TIL characterization, expanded cells were stained with fluorophore-labeled antibodies against CD3 (RRID: AB_493741 ), CD4 (RRID: AB_2563314 ), CD8 (RRID: AB_1967149 ), CD271/NGFR (RRID: AB_2282828 ), PD-1 (RRID: AB_2563658 ), TIGIT (RRID: AB_2632929 ), TIM-3 (RRID: AB_11218598 ), LAG-3 (RRID: AB_2632656 ), TGFBR2 (RRID: AB_2876726 ), CD45RA (RRID: AB_2563816 ), CCR7 (RRID: AB_2563640 ), CD183/CXCR3 (RRID: AB_11219383 ), CD25 (RRID: AB_314280 ), CD28 (RRID: AB_2800752 ), KLRG1 (RRID: AB_2566594 ), CD62L (RRID: AB_2562130 ), CD127 (RRID: AB_10720185 ), FoxP3 (RRID: AB_430887 ), CD40L (RRID: AB_2562721 ), CD107a (RRID: AB_2563851 , BioLegend), TNF-α (RRID: AB_2751384 , Miltenyi Biotec), and IFN-γ (RRID: AB_396760 , BD Biosciences, Heidelberg, Germany).
Article Title: Enhanced solid tumor cell targeting by a neoepitope-encoding oncolytic measles virus combined with CAR therapy
Article Snippet: For evaluation of CAR-T cell activation, staining with CD137 (CD137 Antibody, PE-Vio770, anti-human, REAfinity) (Miltenyi Biotech, Bergisch Gladbach, Germany) was carried out after 24 h of co-incubation. .. For evaluation of CAR-T cell exhaustion, the monoclonal antibodies LAG-3 (CD223 Antibody, PE-Vio770, anti-human, REAfinity), PD-1 (CD279 Antibody, APC, anti-human, REAfinity), and TIM-3 (CD366 Antibody, PE-Vio615, anti-human, REAfinity) (all Miltenyi Biotec, Bergisch Gladbach, Germany) were used. ..
Expressing:Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells
Article Snippet: .. For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark. .. Stained cells were analyzed by applying MACSQuant ® Analyzer 10 and MACSQuantify ® software (both from Miltenyi Biotec GmbH).
Article Title: CNS infiltration by zamtocabtagene autoleucel tandem CD20/CD19 CAR T cells leading to complete remission in a patient with primary CNS lymphoma.
Article Snippet: Figure 2: Factors potentially promoting persistence of the zamto-cel CAR-T in our PCNSL patient. (A) Surface expression levels of CD27 (Miltenyi, Cat#130-120-036) on PB CAR T cells (left column) from our patient compared to their own non-CAR-T (right column) on day+7 and day+14 post CAR-T treatment, respectively. .. To control for unspecific background staining an FMO control was used (gray histogram). (B) Surface expression levels of exhaustion markers PD-1 (Miltenyi, Cat#130-120-385), LAG-3 (Miltenyi, Cat#130-118-549), and TIM-3 (Miltenyi, Cat130-119-781) on PB CD4+ (upper row) and CD8+ (lower row) CAR T cells from our patient on day+7 and day+14 post CAR-T treatment, respectively. .. To control for unspecific background staining an FMO control was used (gray histogram). (C) Surface expression levels of CD127 (IL-7R ; Miltenyi, Cat#130-113-413) on PB CD4+ or CD8+ CAR T cells on day+7 post CAR-T infusion from our patient compared to their own non-CAR-T. To control for unspecific background staining an FMO control was used (gray histogram).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells.
Article Snippet: .. For phenotypic analysis, 0.2 × 106 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 106 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [45] or the anti-La mAb (7B6) [46] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 ◦C in the dark. .. Stained cells were analyzed by applying MACSQuant® Analyzer 10 and MACSQuantify® software (both from Miltenyi Biotec GmbH).
Construct:Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells
Article Snippet: .. For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark. .. Stained cells were analyzed by applying MACSQuant ® Analyzer 10 and MACSQuantify ® software (both from Miltenyi Biotec GmbH).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells.
Article Snippet: .. For phenotypic analysis, 0.2 × 106 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 106 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [45] or the anti-La mAb (7B6) [46] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 ◦C in the dark. .. Stained cells were analyzed by applying MACSQuant® Analyzer 10 and MACSQuantify® software (both from Miltenyi Biotec GmbH).
Incubation:Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells
Article Snippet: .. For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark. .. Stained cells were analyzed by applying MACSQuant ® Analyzer 10 and MACSQuantify ® software (both from Miltenyi Biotec GmbH).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells.
Article Snippet: .. For phenotypic analysis, 0.2 × 106 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 106 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [45] or the anti-La mAb (7B6) [46] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 ◦C in the dark. .. Stained cells were analyzed by applying MACSQuant® Analyzer 10 and MACSQuantify® software (both from Miltenyi Biotec GmbH).
Labeling:Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells
Article Snippet: .. For phenotypic analysis, 0.2 × 10 6 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 10 6 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [ ] or the anti-La mAb (7B6) [ ] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 °C in the dark. .. Stained cells were analyzed by applying MACSQuant ® Analyzer 10 and MACSQuantify ® software (both from Miltenyi Biotec GmbH).
Article Title: CliniMACS Prodigy Manufacturing of Switchable, AND-Gate CAR T Cells.
Article Snippet: .. For phenotypic analysis, 0.2 × 106 cells were stained with monoclonal Abs (mAbs) against CD4, CD8, CD45RO, CD69, PD-1, Tim-3, Lag-3 (all purchased from Miltenyi Biotec GmbH), and CD62L (BioLegend, San Diego, CA, USA) for 15 min. To determine the percentage of cells expressing the respective RevCAR construct, 0.2 × 106 cells were incubated with 0.25 μg of the anti-La mAb (5B9) [45] or the anti-La mAb (7B6) [46] for 1 h, followed by staining with a fluorescently labeled secondary goat anti-mouse IgG Ab (BioLegend) for 30 min. All staining steps were performed at 4 ◦C in the dark. .. Stained cells were analyzed by applying MACSQuant® Analyzer 10 and MACSQuantify® software (both from Miltenyi Biotec GmbH).
Control:Article Title: CNS infiltration by zamtocabtagene autoleucel tandem CD20/CD19 CAR T cells leading to complete remission in a patient with primary CNS lymphoma.
Article Snippet: Figure 2: Factors potentially promoting persistence of the zamto-cel CAR-T in our PCNSL patient. (A) Surface expression levels of CD27 (Miltenyi, Cat#130-120-036) on PB CAR T cells (left column) from our patient compared to their own non-CAR-T (right column) on day+7 and day+14 post CAR-T treatment, respectively. .. To control for unspecific background staining an FMO control was used (gray histogram). (B) Surface expression levels of exhaustion markers PD-1 (Miltenyi, Cat#130-120-385), LAG-3 (Miltenyi, Cat#130-118-549), and TIM-3 (Miltenyi, Cat130-119-781) on PB CD4+ (upper row) and CD8+ (lower row) CAR T cells from our patient on day+7 and day+14 post CAR-T treatment, respectively. .. To control for unspecific background staining an FMO control was used (gray histogram). (C) Surface expression levels of CD127 (IL-7R ; Miltenyi, Cat#130-113-413) on PB CD4+ or CD8+ CAR T cells on day+7 post CAR-T infusion from our patient compared to their own non-CAR-T. To control for unspecific background staining an FMO control was used (gray histogram).
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