Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) Schematic representation of the lentiviral two-in-one vector carrying a CD19-CAR and shRNA expressing module. (b) PD-1 expression levels of CAR T cells with different shPD-1 candidates as determined by flow cytometry on day two after stimulation with γ-irradiated Nalm-6-GL cells. Gray denotes the isotype control. Data are the pooled mean ± SD from three independent experiments, each performed in triplicates. (c) Cell counts from the homeostatic expansion of LNGFR + CAR T cells with PD-1 downregulation candidates on days 3 and 6 after cell seeding. Data are the pooled mean ± SD from three independent experiments performed in triplicates. (d) The effects of hH1-, hU6-, and mU6-shPD1 on PD-1 expression was analyzed by flow cytometry two days after stimulation with γ-irradiated K562-CD19 cells. Data are the pooled mean ± SD from two independent experiments performed in triplicates. (e) Cell counts from the homeostatic expansion of CAR T cells with each Pol III promoter after LNGFR + isolation on days 3 and 6 after cell seeding. Data are the pooled mean ± SD from two independent experiments performed in triplicates. (f) CAR T cells were incubated with GFP-expressing Nalm-6-GL or Nalm-6-GL-PD-L1 cells at a 1:1, 0.3:1, and 0.1:1 effector: target (E:T) ratio. GFP intensity was measured every two hours using the IncuCyte S3 live-cell imaging system. The relative percentage of total integrated GFP intensity was calculated as [GFP intensity at each time point / GFP intensity at 0 h]*100. Representative mean ± SD from two independent experiments performed in triplicates. (g) CAR T cells were incubated with γ-irradiated K562-CD19 or K562-CD19-PD-L1 cells at a 1:3 E:T ratio and counted on day 7. Data are the pooled mean ± SD from two independent experiments performed in triplicates. (h) NSG mice were injected intravenously with 1×10 6 Nalm-6-GL-PD-L1 leukemia cells. 5 days later, 1×10 6 CAR T cells were injected intravenously. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Data are from n = 3 mock and n = 5 19BBz, 19GBBz, and 19PBBz mice, respectively, (i) PD-1 expression levels of CAR T cells from Nalm-6-GL-PD-L1-bearing mice at day 43. Gray denotes isotype control. Data are the mean ± SD from three mice per group. Statistical analysis was done by One-Way ANOVA for (b-f) and unpaired two-tailed t-test for (g and i). *p < 0.05, ** p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Plasmid Preparation, shRNA, Expressing, Flow Cytometry, Irradiation, Isolation, Incubation, Live Cell Imaging, Injection, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: Surface CD19, PD-L1, and CD80 expression levels of Nalm-6-GL-PD-L1-CD80, Nalm-6-GL-PD-L1, Nalm-6, K562-CD19-PD-L1, K562-CD19, and K562 cells was determined by flow cytometry.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) PD-1 expression level of CAR T cells with CD28 or 41BB costimulatory domains two days after stimulation with γ-irradiated K562-CD19 cells. Numbers denote the gMFI of PD-1. (b) Unstimulated and (c) stimulated CAR T cells were incubated with Nalm-6-GL-PD-L1 cells at a 1:1,0.3:1,0.1:1 E:T ratio and analyzed using the IncuCyte S3 system. Stimulated CAR T cells were generated by coincubation with Nalm-6-PD-L1-CD80 cells for 6 days prior to cytotoxicity assay. Data are the representative mean ± SD from two independent experiments performed in triplicates. (d) CAR T cells were incubated with γ-irradiated Nalm-6-GL-PD-L1-CD80 or K562-CD19-PD-L1 cells at 1:3 effector: target (E: T) ratio and counted on day 6 after each stimulation. Data are the mean ± SD from two independent experiments performed in triplicates. (e) NSG mice were injected intravenously with 1×10 6 Nalm-6-GL-PD-L1 leukemia cells. 5 days later, 1×10 6 CAR T cells were injected intravenously. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Data are from n = 3 mock, n = 5 19G28z and 19GBBz, and n = 4 19P28z and 19PBBz. (f) The number of CAR T cells in mouse blood was determined on day 20 and 43 after CAR T cell injection. Data are mean ± SD from three mice per group. Statistical analysis for (a-d and f) was done by One-Way ANOVA. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Expressing, Irradiation, Incubation, Generated, Cytotoxicity Assay, Injection, Imaging

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) Schematic representation of the generation of CD19-specific CAR T cells with selected shRNA sequences targeting TIM-3, TIGIT, LAG-3, and CTLA-4. ΔLNGFR + T cells were stimulated with γ-irradiated K562-CD19 cells on day 10 and knockdown efficiency was measured on day 12. (b) The expression level of inhibitory receptors in CAR T cells expressing the indicated shRNA cassetttes was evaluated by flow cytometry on day 12. (c) CAR expression levels in CAR T cells expressing the indicated shRNA cassettes were determined on day 10 by flow cytometry. Data are the pooled mean ± SD from two indepdendent experiments performed in duplicates. Statistical analysis was done by One-Way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: shRNA, Irradiation, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) Schematic representation of the engineered two-in-one vector system carrying dual shRNA cassettes for two ICRs. (b) Dual downregulation efficiency of each ICR in CAR T cells stimulated for 48 hours with γ-irradiated K562-CD19 cells. FACS plots are representative data from two independent experiments performed in duplicates and the bar graphs are the pooled mean ± SD. (c) NSG mice were injected intravenously with 1×10 6 Nalm-6-GL-PD-L1 leukemia cells. 5 days later, 1×10 6 CAR T cells with each dual downregulation (shGFP, shPD-1/shGFP, shPD-1/shTIM-3, shPD-1/shLAG-3, shPD-1/shTIGIT, shPD-1/shCTLA-4) were injected intravenously. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Data are from n = 3 mock, shPD-1/shTIM-3 and shPD-1/shLAG-3, n = 4 shGFP, shPD-1/shGFP, and shPD-1/shCTLA-4, n = 5 shPD-1/shTIGIT. (d) Kaplan-Meier survival analysis with the Log-rank (Mantel-Cox) test comparing each CAR T treated mice from (c). (e) NSG mice were injected intravenously with 1×10 6 Nalm-6-GL-PD-L1 leukemia cells. 5 days later, 0.5×10 6 or 0.25×10 6 CAR T cells with PD-1 (shPD-1/shGFP) or PD-1/TIGIT (shPD-1/shTIGIT) downregulation were injected intravenously. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Data are from n = 7 mice for the 0.5×10 6 dose groups and n = 6 mice for the 0.25×10 6 dose groups. (f) Kaplan-Meier survival analysis with Log-rank (Mantel-Cox) test comparing CAR T treated mice from (e). Statistical analysis for (b) was done by One-Way ANOVA. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 ns, not significant.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Plasmid Preparation, shRNA, Irradiation, Injection, Imaging

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) The transduction efficiency of dual shRNA constructs was determined by measuring CD19 CAR ΔLNGFR expression by FACS on day 4 after transduction. (b) The gMFI of CD19-CAR in ΔLNGFR + T cells on day 10 after transduction. Data are from the mean ± SD from three donors. (c) CD112 or CD155 expression level in Raji, Nalm-6-GL-PD-L1, K562-CD19-PD-L1, and IM-9 cells with or without IFN-γ treatment for 24 h. CD112 or CD115 expression (d) HLA-DR expression level in Nalm-6-GL-PD-L1 cells with or without IFN-γ treatment at the indicated doses for 24 hours. (e) The intracellular expression level of galectin-9 in Nalm-6-GL-PD-L1 cells. Statistical analysis was done by One-Way ANOVA. ns, not significant.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Transduction, shRNA, Construct, Expressing

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) Schematic illustration of the vector constructs used to generate 19GBBz, 19PBBz, 19TBBz, and 19PTBBz CAR T cells. (b) Surface expression level of CD19 CAR was evaluated by flow cytometry on day 6 after isolation of transduced cells. Data are the pooled mean ± SD from two independent experiments performed in duplicates. (c) PD-1 and (d) TIGIT expression levels of 19GBBz, 19PBBz, 19TBBz, and 19PTBBz cells stimulated with γ-irradiated K562-CD19 cells for 2 days. Data are the pooled mean ± SD from two indepdendent experiments performed in duplicates. Statistical analysis was done by One-Way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Plasmid Preparation, Construct, Expressing, Flow Cytometry, Isolation, Irradiation

Journal: bioRxiv

Article Title: Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT enhances the effector function of CD19-targeting CAR T cells and promotes an early-memory phenotype

doi: 10.1101/2020.11.07.372334

Figure Lengend Snippet: (a) Schematic representation of CD226 knockout by CRISPR/Cas9 and the flow cytometric evaluation of the expression level of CD226 following knockout by 4 different sgRNA candidates. sgRNA targeting CAG (CMV-IE, chicken actin, rabbit beta globin) was used as a negative control. (b) The knockout efficiency of gRNA #4 targeting CD226 was estimated by T7 endonuclease I assay. (c) Schematic representation of the generation of CD226 KO CD19 specific CAR T cells and the measurement of intracellular IL-2 by flow cytometry, (c) Intracellular IL-2 expression levels in CD226 + and CD226 - populations measured by flow cytometry. Data are the mean ± SD from one experiment performed in triplicates.

Article Snippet: Nalm-6-GL, or K562-CD19 cells were transduced to express human PD-L1 (Sino Biological; HG10084-UT cDNA subcloned into a lentiviral vector) to generate Nalm-6-GL-PD-L1 and K562-CD19-PD-L1 cells.

Techniques: Knock-Out, CRISPR, Expressing, Negative Control, T7EI Assay, Flow Cytometry

Journal: bioRxiv

Article Title: A Digital Microfluidic Platform for the Microscale Production of Functional Immune Cell Therapies

doi: 10.1101/2024.09.03.611092

Figure Lengend Snippet: a) Overview of immunotherapeutic testing assay detailing an anti-CD19 CAR T cell interacting with a CD19 negative or positive tumor cell line. Created with BioRender.com. b) Fluorescence intensity histograms showing FITC expressions for cells stained with a FITC-tagged CD19 protein from a control (grey), triDrop (green), Nucleofection (blue), and Neon (red). c) Line graphs depicting expression of an anti-CD19 CAR molecule at 6, 24, and 72 hours post EP when using the triDrop (green, 100ng of mRNA), Neon (red, 500ng of mRNA), and Nucleofection (blue, 1000ng of mRNA). Bar graphs depicting measured levels of d) TNF-α and e) IFN-γ after 24 hours (light purple and light blue) and 48 hours (dark purple and dark blue) of culture for cells electroporated using all three systems using either manufacturer recommended conditions or 0.5 x 10 cells per reaction. Engineered cells are either cultured by themselves (T), or at a 1:1 ratio with MCF-7 cells (T/M), or with Raji cells (T/R). (n = 6) f) Line graphs depicting relative killing of Raji cells cocultured with activated Pan T cells at 1:1 ratio and 4:1 ratio after 24 hours (light grey, solid line) and 48 hours (dark grey, dashed line) or with triDrop engineered CAR T cells at a 1:1 and 4:1 ratio after 24 hours (light green, solid line), and 48 hours (dark green, dashed line). Statistical significance markers indicate difference between engineered cells and activated pan T cells for each timepoint. (n = 4). All error bars represent mean +/- SD. n.s indicated no significant difference, *, **,***, and **** represent p-values below 0.05, 0.01, 0.001 and 0.0001 respectively. Statistical analysis was performed using a student’s t-test.

Article Snippet: CD19 expression was detected using a FITC Mouse anti-Human CD19 antibody (BD Bioscience, catalog #560994).

Techniques: Fluorescence, Staining, Control, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Patient-tailored design of AML cell subpopulation-selective drug combinations

doi: 10.1101/2020.07.28.222034

Figure Lengend Snippet: Gating strategy for the high-throughput flow cytometry (HTFC) assay. Live cells were gated as cells negative to 7-AAD and Annexin V markers. Leukemic stem cells were gated as CD34+ CD38-population. Lymphocytes were gated based on CD56 and CD3/CD19 to distinguish NK as well as T and B cell populations, respectively.

Article Snippet: The cells were washed with a cell staining buffer (PBS with 2% fetal bovine serum), centrifuged at 600× g for 5 min. To profile cell subpopulation responses, the cells were stained with BV605 Mouse Anti-Human CD56, BV421 Mouse Anti-Human CD19, BV421 Mouse Anti-Human CD3, FITC Mouse Anti-Human CD45, APC Mouse Anti-Human CD34, BV786 Mouse Anti-Human CD38 antibodies (all antibodies from BD Biosciences) for 30 min at room temperature in the dark.

Techniques: High Throughput Screening Assay, Flow Cytometry

Journal: Cell Transplantation

Article Title: Persistent Cytopenia After CD19 CAR T Therapy in Relapsed/Refractory DLBCL Patients Could Be a Predictor of Efficacy and Side Effects

doi: 10.1177/09636897241247951

Figure Lengend Snippet: Hematological toxicity of anti-CD19 CAR T cell therapy. (A) Grades 0–4 neutropenia, anemia, and thrombocytopenia were diagnosed after anti-CD19 CAR T cell therapy. (B) The different types and persistent times of cytopenia in the 14 patients who had Grades 3–4 persistent cytopenia. (C–E) Blood cell changes in patients with Grades 3–4 neutropenia, anemia, and thrombocytopenia. CAR: chimeric antigen receptor.

Article Snippet: Humanized CD19 CAR antibodies were provided by GenBase Biotechnology (patent owner of CD19 CAR).

Techniques:

Journal: Cell Transplantation

Article Title: Persistent Cytopenia After CD19 CAR T Therapy in Relapsed/Refractory DLBCL Patients Could Be a Predictor of Efficacy and Side Effects

doi: 10.1177/09636897241247951

Figure Lengend Snippet: (A) All 14 patients with Grades 3–4 persistent cytopenia obtained CR and PR, whereas no patient who obtained SD and PD had cytopenia during CAR T cell therapy. (B) The clinical response to anti-CD19 CAR T cell therapy, PD, and survival time of all 38 patients. (C) The incidence of Grades 3–4 persistent cytopenia was higher in 26 patients who obtained ORR with high tumor load than in patients with lower tumor loads. CAR: chimeric antigen receptor; CR: complete response; PR: partial remission; SD: stable disease; PD: disease progression; ORR: objective response rate.

Article Snippet: Humanized CD19 CAR antibodies were provided by GenBase Biotechnology (patent owner of CD19 CAR).

Techniques:

Journal: Cell Transplantation

Article Title: Persistent Cytopenia After CD19 CAR T Therapy in Relapsed/Refractory DLBCL Patients Could Be a Predictor of Efficacy and Side Effects

doi: 10.1177/09636897241247951

Figure Lengend Snippet: Adverse Events During Anti-CD19 CAR T Cell Therapy.

Article Snippet: Humanized CD19 CAR antibodies were provided by GenBase Biotechnology (patent owner of CD19 CAR).

Techniques:

Journal: Cell Transplantation

Article Title: Persistent Cytopenia After CD19 CAR T Therapy in Relapsed/Refractory DLBCL Patients Could Be a Predictor of Efficacy and Side Effects

doi: 10.1177/09636897241247951

Figure Lengend Snippet: Adverse events of anti-CD19 CAR T cell therapy. (A) The mean peak of IL-6 levels in patients with Grades 3–4 persistent cytopenia was higher than in patients without Grades 3–4 persistent cytopenia. (B) CRS was higher in patients with Grades 3–4 persistent cytopenia than in patients without persistent cytopenia. (C) No difference was observed in Grades 3–4 persistent cytopenia between different ICANS grades. (D) The median amplification peak of CAR T cells was higher in patients with Grades 3–4 persistent cytopenia than in patients without persistent cytopenia. (E, F) Most patients whose CAR T cells were observed at 21 and 28 days after CAR T cell infusion had Grades 3–4 persistent cytopenia. CAR: chimeric antigen receptor; IL: interleukin; CRS: cytokine release syndrome; ICANS: immune effector cell-associated neurotoxic syndrome.

Article Snippet: Humanized CD19 CAR antibodies were provided by GenBase Biotechnology (patent owner of CD19 CAR).

Techniques: Amplification

Journal: Cell Transplantation

Article Title: Persistent Cytopenia After CD19 CAR T Therapy in Relapsed/Refractory DLBCL Patients Could Be a Predictor of Efficacy and Side Effects

doi: 10.1177/09636897241247951

Figure Lengend Snippet: Follow-up after anti-CD19 CAR T cell therapy. (A, B) The progression-free and overall survival were higher in patients with Grades 3–4 persistent cytopenia than in those who did not have persistent cytopenia. CAR: chimeric antigen receptor.

Article Snippet: Humanized CD19 CAR antibodies were provided by GenBase Biotechnology (patent owner of CD19 CAR).

Techniques: