cd19 recombinant human r d systems 9269 cd  (R&D Systems)

Journal: Science translational medicine

Article Title: IFN-γ-resistant CD28 CAR-T cells demonstrate increased survival, efficacy, and durability in multiple murine tumor models

doi: 10.1126/scitranslmed.adp8166

Figure Lengend Snippet: (A) Schematic diagram of genetic constructs used to generate human chimeric antigen receptor (CAR)-T cells with a CD19 single-chain fragment variable (scFv), CD28 costimulatory domain, and CRISPR/Cas9 editing of T cell receptor A constant region locus ( TRAC ; CAR CD19 ), TRAC and IFNG (IFNγKO CAR CD19 ), or TRAC and IFNGR (IFNγRKO CAR CD19 ). (B) Expression of CD3 (Allophycocyanin, APC; left ), IFNγR (Alexa Fluor 647, AF647; middle ) and phosphoSTAT1 (pSTAT1) signaling (AF647; right ) following exposure to recombinant human IFNγ on resting CAR-T cells and donor-matched untransduced (UTD) T cells) with representative histograms from 3 biological replicates. (C) IFNγ (Brilliant Violet 510, BV510) expression on CAR-T cells was assessed by flow cytometry following four-hour activation with phorbol myristate acetate (PMA) and ionomycin; (n=3 biological replicates). Y-axis is an empty channel. (D) CD4 and CD8 expression on CAR-T cells and UTD T cells were determined using flow cytometry; n=6 biological replicates. Data are shown as mean ± SEM with P values determined by two-way ANOVA with Tukey’s multiple comparisons test. (E) CAR-T cells were activated overnight with CD19 antigen and secreted cytokines were measured by Luminex; bubble plot represents average cytokine expression (ng/ml) from 3 biological replicates. (F) Expansion of CD28 CAR-T cells in response to Nalm6 cells was monitored using real-time Incucyte imaging. Average expansion (±SEM) was measured over 72 hours. Data represents 4 biological replicates which were analyzed by one-way ANOVA with Tukey’s multiple comparisons analysis at 24, 48, and 72 hours. Significance of IFNγKO CAR CD19 vs. CAR CD19 ( pink * ) or IFNγRKO CARCD19 vs. CAR CD19 (turquoise *,**) is shown. (G) Schematic diagram of constructs used to generate control, IFNγKO, and IFNγRKO CAR-T cells with a CD19 scFv and 4–1BB costimulatory domain. (H) Representative histograms of CD3 (APC; left ) and IFNγR (AF647; middle ) expression on resting CAR-T cells and donor-matched UTD T cells measured by flow cytometry alongside IFNγ following a four-hour activation with PMA and Ionomycin (BV510; right ). Data was analyzed from 3 biological replicates. (I) Expansion of 4–1BB CAR-T cells in response to Nalm6 cells was monitored using real-time Incucyte imaging. Average expansion (±SEM) was measured over 72 hours. Data represents 3 biological replicates which were analyzed by one-way ANOVA. * P <0.05, ** P <0.01, **** P <0.0001; ns, not significant.

Article Snippet: For functional assessment, CAR-T cells were activated for four hours using Cell Activation Cocktail with Brefeldin A (BioLegend; Cat. 423302) or 18 hours with plate-bound CD19 (Recombinant Human CD19 Fc Chimera Protein, CF; R&D Systems Cat. 9269-CD-050), mesothelin (Recombinant Human Mesothelin; PeproTech Cat. 100–63), or EGFR (Recombinant Human EGF Receptor (EGFR); PeproTech Cat. 100–15R) prior to staining.

Techniques: Knock-Out, In Vitro, Construct, CRISPR, Expressing, Recombinant, Flow Cytometry, Activation Assay, Luminex, Imaging, Control

Journal: Science translational medicine

Article Title: IFN-γ-resistant CD28 CAR-T cells demonstrate increased survival, efficacy, and durability in multiple murine tumor models

doi: 10.1126/scitranslmed.adp8166

Figure Lengend Snippet: (A) CAR-T cells were cultured with Nalm6 cells (1:1) and tumor cytolysis was measured by Incucyte. At 3 and 6 days, cells were collected, counted, and CAR-T cells were re-plated with fresh Nalm6 cells (1:1) before being returned to the Incucyte. Data represents mean (±SEM) for 2 biological replicates. (B to F) NSG mice bearing intravenously administered CBGGFP + JeKo-1 tumor cells (1e 6 IV) were left untreated (tumor only; TO) or treated with 1e 6 CAR CD19 , IFNγKO CAR CD19 , or IFNγRKO CAR CD19 (1e 6 IV). Data was obtained from 4–5 mice/group per donor (2 donors total). (B) Schematic timeline of in vivo CAR-T cell treatment. Tumor burden was assessed by bioluminescent imaging for each CAR-T cell group and tumor only control over 75 days post-CAR-T cell infusion (C) and graphed by flux (D). (E) Overall survival was measured for each CAR-T cell treatment cohort and represented by Kaplan-Meier survival curve. (F) Mice were bled 14 days post-CAR-T cell infusion and T cell persistence was determined using flow cytometry. Data are shown as mean ± SEM with P values determined by one-way ANOVA with Tukey’s multiple comparisons analysis (D, F) or Mantel-Cox test (E). ** P <0.01, **** P <0.0001; ns, not significant.

Article Snippet: For functional assessment, CAR-T cells were activated for four hours using Cell Activation Cocktail with Brefeldin A (BioLegend; Cat. 423302) or 18 hours with plate-bound CD19 (Recombinant Human CD19 Fc Chimera Protein, CF; R&D Systems Cat. 9269-CD-050), mesothelin (Recombinant Human Mesothelin; PeproTech Cat. 100–63), or EGFR (Recombinant Human EGF Receptor (EGFR); PeproTech Cat. 100–15R) prior to staining.

Techniques: Activity Assay, In Vivo, Cell Culture, Imaging, Control, Flow Cytometry

Journal: Science translational medicine

Article Title: IFN-γ-resistant CD28 CAR-T cells demonstrate increased survival, efficacy, and durability in multiple murine tumor models

doi: 10.1126/scitranslmed.adp8166

Figure Lengend Snippet: (A) CAR-T cells were activated overnight with plate-bound CD19 antigen prior to RNA sequencing using NanoString and graphed as volcano plots. Proliferation (blue dots) and cell death (pink dots) genes are highlighted in CAR CD19 (purple shading) compared to either IFNγKO CAR CD19 (pink shading) or IFNγRKO CAR CD19 (turquoise shading). Three adjusted P value cutoffs are shown as a dotted bottom line ( P <0.05), dashed middle line ( P <0.01), and a dot-dash top line ( P <0.001). Data was derived from 3 biological replicates. (B) Proliferation in response to CD19 + Nalm6 cells was assessed by Cell Trace Violet at 6 days post-activation. Data are represented as histograms and pie charts showing the percentage of cells (white numbers) for each number of cell divisions (0=gray, 1=red, 2=turquoise, 3=purple, 4=orange) derived from 3 biological replicates. (C and D) Annexin V (C) and cleaved Caspase 3 (D) expression were determined by flow cytometry for CAR-T cell groups at rest (no activation; NA) and 48 hours post-activation (CD19); n=3 biological replicates . (E) Annexin V expression on CAR-T cells activated with plate-bound CD19 antigen was assessed using Incucyte real-time analysis over a 72-hour period in 3–6 biological replicates]. Significance at 72 hours was noted for IFNγRKO CAR CD19 vs. CAR CD19 (turquoise *). (F and G) CAR-T cells were activated overnight with CD19 antigen in the absence or presence of exogenous recombinant human IFNγ prior to RNA sequencing using NanoString. Data is presented as bar graphs (F) and volcano plots with adjusted P value cutoffs shown as a dotted bottom line ( P <0.05) and dashed top line ( P <0.01) (G); n=3 biological replicates. Interferon signaling (purple dots) and cell death (magenta dots)-related genes are highlighted. (H) Wildtype CD19-specific CAR-T cells with no CRISPR/Cas9 editing were activated with CD19 in the absence or presence of blocking antibodies to IFNγ (αIFNγ) or IFNγR (αIFNγR) and Annexin V expression was assessed by Incucyte; n=2 biological replicates. Data are shown as mean ± SEM with P values determined by one-way ANOVA with Tukey’s multiple comparisons analysis (C, D, E, F, H). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001; ns, not significant.

Article Snippet: For functional assessment, CAR-T cells were activated for four hours using Cell Activation Cocktail with Brefeldin A (BioLegend; Cat. 423302) or 18 hours with plate-bound CD19 (Recombinant Human CD19 Fc Chimera Protein, CF; R&D Systems Cat. 9269-CD-050), mesothelin (Recombinant Human Mesothelin; PeproTech Cat. 100–63), or EGFR (Recombinant Human EGF Receptor (EGFR); PeproTech Cat. 100–15R) prior to staining.

Techniques: RNA Sequencing, Derivative Assay, Activation Assay, Expressing, Flow Cytometry, Recombinant, CRISPR, Blocking Assay