Journal: Molecular therapy. Methods & clinical development

Article Title: Autologous antigen-presenting cells efficiently expand piggyBac transposon CAR-T cells with predominant memory phenotype.

doi: 10.1016/j.omtm.2021.03.011

Figure Lengend Snippet: Figure 4. PB-HER2-CAR-T cells have robust and sustained antitumor activity against HER2-positive tumors in vitro (A) An impedance-based tumor-cell-killing assay was used to evaluate PB-HER2-CAR-T cells. Tumor cells (U- 2OS) were plated, and 24 h later, CAR-T cells were added at different effector-to-target (E:T) ratios. Real-time impedance traces of tumor cells without treatment (green) and with co-culture of CAR-T cells (magenta, blue, and red) were shown. Each experiment was duplicated, and mean ± SD of the normalized cell index at each time point is shown in the graph. (B) Antitumor efficacy of PB-HER2- CAR-T cells against HER2-positive tumor cell lines. Real- time impedance traces of tumor cells without treatment (green) and tumor cells co-cultured with PB-CD19-CAR-T cells (blue) or PB-HER2-CAR-T cells (red) at an E:T ratio of 1:1 were acquired for 72 h. (C) Sequential killing assay of PB-HER2-CAR-T cells. Real-time impedance traces of tumor cells without treatment (green) and tumor cells co- cultured with PB-HER2-CAR-T cells (red) were acquired for 72 h in each round. Mean ± SD of normalized cell index at 72 h in each round were also evaluated (n = 3). (D) The expression of HER2-CAR molecules on T cells during sequential co-culture. Representative dot plots are shown. (E) Mean fluorescent Index of HER2-CAR expression on T cells during sequential co-culture. (F) The expression of PD-1, TIM-3, and LAG3 on HER2-CAR-T cells during sequential co-culture.

Article Snippet: Cell-surface expression of the HER2-CAR molecules on PB-HER2CAR-T cells or CD19-CAR molecules on PB-CD19-CAR-T cells was determined using the flow cytometry using recombinant human ErbB2/HER2-Fc chimera protein or recombinant human CD19 Fc chimera protein, respectively (R&D Systems, Minneapolis, MN, USA), followed by a goat anti-human IgG Fc fragment-specific antibody conjugated to fluorescein isothiocyanate (FITC; Merck Millipore, Burlington, MA, USA).

Techniques: Activity Assay, In Vitro, Co-Culture Assay, Cell Culture, Expressing

Journal: Molecular therapy. Methods & clinical development

Article Title: Autologous antigen-presenting cells efficiently expand piggyBac transposon CAR-T cells with predominant memory phenotype.

doi: 10.1016/j.omtm.2021.03.011

Figure Lengend Snippet: Figure 5. PB-HER2-CAR-T cells efficiently control HER2-positive tumor in vivo (A) Schematics of in vivo study and tumor re-challenge experiments in the HER2-positive tumor xenograft murine model. SJCRH30-FFluc was subcutaneously injected at the dorsal wall of the mice to form a tumor. One week after tumor engraftment, mice intravenously received either vehicle, approximately 6 106 of PB-CD19-CAR-T cells, or PB-HER2-CAR-T cells. (B) Tumor growth was measured as bioluminescence signal intensity (BLI) and expressed as total flux (p/s). The BLI images of each mouse and the tumor volumes measured as total flux (p/s) are shown. The PB-HER2-CAR-T cells group demon- strated a statistically significant tumor reduction measured as the mean total flux at day 28 (*), compared to the PB- CD19-CAR-T cells group (Mann-Whitney U test, p < 0.01). The SJCRH30-FFLuc tumor cells were re-inocu- lated into the corresponding contralateral thoracic wall of the mice on day 28 after the primary tumor inoculation. BLI images on days 7 and 14 after tumor re-challenges are indicated by arrowheads. (C) Blood samples were collected from each mouse on day 42 after primary tumor inoculation, and the presence of PB-HER2-CAR-T cells was assessed using flow cytometry. HER2CAR+/human CD3 population could be explained as caused by the non-specific binding of anti-human IgG-Fc antibody to mouse PBMCs, as shown in Figure S11.

Article Snippet: Cell-surface expression of the HER2-CAR molecules on PB-HER2CAR-T cells or CD19-CAR molecules on PB-CD19-CAR-T cells was determined using the flow cytometry using recombinant human ErbB2/HER2-Fc chimera protein or recombinant human CD19 Fc chimera protein, respectively (R&D Systems, Minneapolis, MN, USA), followed by a goat anti-human IgG Fc fragment-specific antibody conjugated to fluorescein isothiocyanate (FITC; Merck Millipore, Burlington, MA, USA).

Techniques: Control, In Vivo, Injection, MANN-WHITNEY, Cytometry, Binding Assay

Journal: Cytotherapy

Article Title: Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors.

doi: 10.1016/j.jcyt.2021.04.010

Figure Lengend Snippet: Figure 2. CD19-CAR NK cells recognize and kill CD19+ target cells. (A) Scheme of CD19-CARs (green = antigen recognition domain, gray = hinge and transmembrane domain, rose = co-stimulatory domain, blue = signaling domain). (B) Representative flow cytometry histogram plot depicting CD19-CAR expression with indicated MOIs (untreated = NK cells transduced in the absence of MRT67307 at MOI of 30). (C) Flow cytometry-based cytotoxicity assay of CD19-CAR NK cells targeting leukemia cell lines BV173 WT and BV173 CD19KO at indicated E:T ratios (N = 4 donors, average technical triplicates per donor, mean § SEM). Unpaired two-tailed Student’s t-test was used to determine significance. **P < 0.01. scFv, single-chain variable fragment; SEM, standard error of the mean.

Article Snippet: Surface HER2-CAR detection was determined via immunolabeling with anti-F(ab')2-AF647 at 1:100 (109-606-006; The Jackson Laboratory, Bar Harbor, ME, USA), CD19CAR was detected with phycoerythrin-conjugated recombinant human CD19 protein at 1:100 (3309HP; Creative BioMart, Shirley, NY, USA) and YFP was utilized for detection on a BD FACSLyric machine and analyzed with FlowJo v10 (BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Cytometry, Expressing, Flow Cytometry, Cytotoxicity Assay, Two Tailed Test

Journal: Immuno-Oncology and Technology

Article Title: Overcoming antigen loss in CAR T therapy with Vγ9Vδ2 CAR T-cells

doi: 10.1016/j.iotech.2025.101053

Figure Lengend Snippet: Antigen-specific expansion of Vγ9Vδ2 and αβ T-cells transduced with a CD19-specific CAR. (A) Contour plots of a representative donor showing the percentages of CAR-positive cells in Vγ9Vδ2 T-cells expanded with either IL-2 (black) or IL-2/IL-15 (red) and αβ T-cell (blue) cultures before the Ag-specific expansion started, and after 7 and 14 days in a CD19-coated plate. (B) Percentages of CAR-positive T-cells in all cultures through the 14-day Ag-specific expansion. Bars represent the mean value, and error bars show the standard deviation ( n = 6, three independent experiments). Ag, antigen; CAR, chimeric antigen receptor; IL, interleukin.

Article Snippet: After 1 week, the cells were transferred to a plate coated with a recombinant human CD19 protein (1 μg/ml, R&D Systems, Minneapolis, MN) for 2 weeks to specifically expand the engineered CAR T-cells.

Techniques: Transduction, Standard Deviation

Journal: Immuno-Oncology and Technology

Article Title: Overcoming antigen loss in CAR T therapy with Vγ9Vδ2 CAR T-cells

doi: 10.1016/j.iotech.2025.101053

Figure Lengend Snippet: Antitumor effect of Vγ9Vδ2 and αβ CAR T-cells against CD19-positive tumor cell lines. (A) Intracellular staining analysis was used to determine the levels of the degranulation marker CD107a and the cytokines TNF-α and IFN-γ in Vγ9Vδ2 T-cells expanded with either IL-2 (black) or IL-2/IL-15 (red) and αβ T-cell (blue) cultures after a 5-h co-culture with the CD19-positive tumor cell lines K562-CD19, Daudi and Nalm-6 ( n = 4, three independent experiments). (B) Representative plot for long-term cytotoxicity of Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures against the CD19-positive cell lines K562-CD16 (left plot), Daudi (middle plot) and Nalm-6 (right plot). Data are presented as the average of triplicates. One-way analysis of variance (ANOVA) with Tukey multiple comparisons test was used to compare the different curves. (C) Time to kill 50% of the tumor cells (KT50) was compared between Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures. Each donor is represented with a different symbol, and lines represent the average of all ( n = 4, three independent experiments). Two-way ANOVA with Tukey multiple comparisons test was used to compare the different cultures. (D) Proliferation of Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T cell upon encountering the cell lines Daudi or Nalm-6. Stacked bar graphs represent the percentage of cells in each generation after a 5-day co-culture with the tumor cells. Bars represent the mean value, and error bars show the standard deviation ( n = 4, three independent experiments). CAR, chimeric antigen receptor; IFN, interferon; IL, interleukin; TNF-α, tumor necrosis factor-α. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: After 1 week, the cells were transferred to a plate coated with a recombinant human CD19 protein (1 μg/ml, R&D Systems, Minneapolis, MN) for 2 weeks to specifically expand the engineered CAR T-cells.

Techniques: Staining, Marker, Co-Culture Assay, Standard Deviation

Journal: Immuno-Oncology and Technology

Article Title: Overcoming antigen loss in CAR T therapy with Vγ9Vδ2 CAR T-cells

doi: 10.1016/j.iotech.2025.101053

Figure Lengend Snippet: Antitumor effect of Vγ9Vδ2 and αβ CAR T-cells against CD19-negative tumor cell lines. (A) Intracellular staining analysis was used to determine the levels of the degranulation marker CD107a and the cytokines TNF-α and IFN-γ in Vγ9Vδ2 T-cells expanded with either IL-2 (black) or IL-2/IL-15 (red) and αβ T-cell (blue) cultures after a 5-h co-culture with the CD19-negative tumor cell lines K562 and Molm-14 ( n = 4, three independent experiments). (B) Representative plot for long-term cytotoxicity of Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures against the CD19-negative cell lines K562 (left plot) and Molm-14 (right plot). Data are presented as the average of triplicates. One-way analysis of variance (ANOVA) with Tukey multiple comparisons test was used to compare the different curves. (C) Time to kill 20% of the tumor cells (KT20) was compared between Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures. Each donor is represented with a different symbol, and lines represent the average of all ( n = 4, three independent experiments). One-way ANOVA with Tukey multiple comparisons test was used to compare the different curves. (D) Proliferation of Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell upon encountering the cell lines K562 and Molm-14. Stacked bar graphs represent the percentage of cells in each generation after a 5-day co-culture with the tumor cells. Bars represent the mean value, and error bars show the standard deviation ( n = 4, three independent experiments). CAR, chimeric antigen receptor; IFN, interferon; IL, interleukin; TNF-α, tumor necrosis factor-α. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: After 1 week, the cells were transferred to a plate coated with a recombinant human CD19 protein (1 μg/ml, R&D Systems, Minneapolis, MN) for 2 weeks to specifically expand the engineered CAR T-cells.

Techniques: Staining, Marker, Co-Culture Assay, Standard Deviation

Journal: Immuno-Oncology and Technology

Article Title: Overcoming antigen loss in CAR T therapy with Vγ9Vδ2 CAR T-cells

doi: 10.1016/j.iotech.2025.101053

Figure Lengend Snippet: Antitumor effect of Vγ9Vδ2 and αβ CAR T-cells during antigen loss. (A) Long-term cytotoxicity of Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures against mixed tumor population of the CD19-negative cell line K562 and the CD19-positive cell line K562-CD19. Data are presented as the average of duplicates. One-way analysis of variance with Tukey multiple comparisons test was used to compare the different curves. (B) Time to kill 20% of the tumor cells (KT20) was compared between Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures. Dots represent average, and error bars represent the range ( n = 3, two independent experiments). CAR, chimeric antigen receptor; IL, interleukin. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: After 1 week, the cells were transferred to a plate coated with a recombinant human CD19 protein (1 μg/ml, R&D Systems, Minneapolis, MN) for 2 weeks to specifically expand the engineered CAR T-cells.

Techniques:

Journal: Molecular Cancer

Article Title: Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study

doi: 10.1186/s12943-023-01886-9

Figure Lengend Snippet: The h1218 antibody is specific for CD19 and recognizes a non-FMC63 membrane proximal epitope. A Schematic of FMC63 and h1218 antibodies binding sites on CD19. B Binding of h1218 to FMC63 bound-human CD19 complex. Sensor chips were coated with FMC63 Fc and CD19-ECD-Ck from 1700 to 2300 s. Additional FMC63 or h1218 antibody was added to the FMC63-bound sensor chip at 2300 s and monitored for further binding activities. C h1218 antibody binding test on wild-type HEK293T (CD19 negative), HEK293T cell expressing human CD19 (huCD19, HHH), and HEK293T cells expressing one of the three chimeric CD19 forms that had cynomolgus residues replacement at different region of CD19, respectively (CHH, HCH, and HHC). D Mutagenesis study to identify key residues corresponding to the h1218 CD19 epitope. E Quantification of IFNγ release on HEK293T cells expressing WT or mutant CD19 to identify additional key residues corresponding to the h1218 CD19 epitope. F Binding affinity of FMC63 and h1218 scFv to recombinant human CD19-ECD-Ck (M1-P278). Each line represents affinity measured at a different scFv concentration. All the experiments were repeated at least twice

Article Snippet: Lipid bilayers were first blocked with 5% casein for 20 min and then incubated with 6.3 nM streptavidin (ThermoFisher; Cat# 434301) for 15 min. After washing with imaging buffer (HEPES-buffered saline), lipids were incubated with biotinylated CD19 protein (R&D Systems; Cat#AVI9269) conjugated with Alexa Fluor 647 at room temperature for 30 min. After washing with the imaging buffer, lipid bilayers were blocked with 2.5 μM D-biotin to saturate the streptavidin-binding sites.

Techniques: Membrane, Binding Assay, Expressing, Mutagenesis, Recombinant, Concentration Assay

Journal: Molecular Cancer

Article Title: Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study

doi: 10.1186/s12943-023-01886-9

Figure Lengend Snippet: h1218-CART19 recognize and kill malignant B cells carrying FMC63-resistant CD19 mutations. A Schematic representation of B cell leukemia cells with point mutations in the membrane-distal CD19 domain (CD19 R163L or CD19 L174V ) as clinically identified post FMC63-CART19 treatment. B CD19 expression levels in Nalm6-CD19 KO, Nalm6 (wild type), Nalm6-CD19 L174V (left) and Nalm6-CD19 R163L (right) cell lines as measured by flow cytometry. C CART19 cytotoxicity against Nalm6-CD19 L174V and Nalm6-CD19 R163L at various effector to target (E:T) ratios ( n = 3 independent donors) (luciferase assay). D IL-2 and TNF cytokine release measured by ELISA 24 h after CART19 and cancer cell co-culture at an E:T ratio of 5:1 ( n = 2 donors). All values determined to be negative by comparison with the standard curve are shown as zero. E (Left) Schematic of the xenograft NSG mouse model: 1 × 10 6 luciferase + Nalm6-CD19 L174V cells and 0.75 × 10 6 UTD, FMC63-CART19, or h1218-CART19 cells were intravenous injected with a 5-day interval. (Right) Tumor burden of engrafted mice treated with UTD ( n = 5), FMC63-CART19 ( n = 5), or h1218-CART19 ( n = 5) as measured by bioluminescence imaging. The bold lines represent the median luminescence of each group. F Overall survival in each treatment group ( p = 0.0027). G Absolute cell counts of huCD45 + huCD3 + T cells in 100μL mouse blood on day 9. All bar graphs and cytotoxicity curves are presented as the mean ± SEM. Survival curves were compared using the log-rank (Mantel-Cox) test, and one-way ANOVA was performed with Tukey’s correction for multiple comparisons; **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05. All bar graphs are presented as the mean ± SEM. All experiments were repeated at least twice

Article Snippet: Lipid bilayers were first blocked with 5% casein for 20 min and then incubated with 6.3 nM streptavidin (ThermoFisher; Cat# 434301) for 15 min. After washing with imaging buffer (HEPES-buffered saline), lipids were incubated with biotinylated CD19 protein (R&D Systems; Cat#AVI9269) conjugated with Alexa Fluor 647 at room temperature for 30 min. After washing with the imaging buffer, lipid bilayers were blocked with 2.5 μM D-biotin to saturate the streptavidin-binding sites.

Techniques: Membrane, Expressing, Flow Cytometry, Luciferase, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Comparison, Injection, Imaging

Journal: Molecular Cancer

Article Title: Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study

doi: 10.1186/s12943-023-01886-9

Figure Lengend Snippet: h1218-CART19 demonstrates enhanced efficacy than FMC63-CART19. A Schematic of h1218-CART19 targeting the membrane-proximal domain of CD19 in B cell lymphoma/leukemia as compared to standard FMC63-CART19. B Nalm6 tumor killing by UTD, h1218-CART19, or FMC63-CART19 cells at various E:T ratios ( n = 3 donors) by flow cytometry. C IFNγ release as measured by ELISA upon stimulation of CD19-expressing tumor cells (Raji, Pfeiffer, Toledo, and Nalm6) at an E:T ratio of 3:1 ( n = 2 donors). D (Left) Schematic of the in vivo experiment: either 1.5 × 10 6 UTD or CART19 cells were infused 7 days after intravenous injection of luciferase + Raji or lNalm6 cells. Tumor progression in mice bearing Raji (middle) or Nalm6 (right) cells is shown. E Quantification of Nalm6 fold change over 14 days in the presence of UTD, FMC63-CART19, or h1218-CART19 at low-E:T ratio model ( n = 2 donors) using flow cytometry. F (Left) Schematic of the in vivo xenograft model: 0.75 × 10 6 UTD or CAR T cells were infused intravenously 5 days after luciferase + Nalm6 engraftment. (Middle) Tumor progression over time in mice bearing Nalm6 cells treated with UTD ( n = 3), FMC63-CART19 ( n = 7), or h1218-CART19 ( n = 7) measured by luminescence. Bolded lines represent the median tumor burden in the corresponding group. (Right) Tumor burden on day 42 after CART19 injection. G Overall survival in mice bearing Nalm6. H (Left) CAR T cell expansion kinetics in the peripheral blood after CART19 injection. Bolded lines represent the median CAR T expansion. (Right) Quantification of CAR T cells in the blood 14 days after CART19 injection using flow cytometry. All bar graphs and cytotoxicity curves are presented as the mean ± SEM. Student's t-test was used to compare two groups; one-way ANOVA was performed with Tukey’s correction for multiple comparisons; survival curves were compared using the log-rank (Mantel-Cox) test; **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05. All bar graphs are presented as the mean ± SEM. All experiments were repeated at least twice

Article Snippet: Lipid bilayers were first blocked with 5% casein for 20 min and then incubated with 6.3 nM streptavidin (ThermoFisher; Cat# 434301) for 15 min. After washing with imaging buffer (HEPES-buffered saline), lipids were incubated with biotinylated CD19 protein (R&D Systems; Cat#AVI9269) conjugated with Alexa Fluor 647 at room temperature for 30 min. After washing with the imaging buffer, lipid bilayers were blocked with 2.5 μM D-biotin to saturate the streptavidin-binding sites.

Techniques: Membrane, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, In Vivo, Injection, Luciferase

Journal: Molecular Cancer

Article Title: Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study

doi: 10.1186/s12943-023-01886-9

Figure Lengend Snippet: h1218-CART19 demonstrates lower avidity and less activation-induced cell death than FMC63-CART19. A Quantification of UTD, FMC63-CART19, and h1218-CART19 binding avidity to Nalm6 after 15-min co-culture ( n = 2 donors) by Lumicks analysis. B Representative confocal microscopy images of F-actin, CD19, perforin, and phosphorylated-CD3ζ (pCD3ζ) (CAR) expressed in FMC63-CART19 or h1218-CART19 cells when engaged with biotinylated CD19 protein. C Quantification of F-actin, perforin polarization, and pCD3ζ in FMC63-CART19 or h1219-CART19 cells engaged with biotinylated CD19 protein ( n = 2 donors). In total, 200 events were recorded for each group. D (Left) Representative flow cytometric analysis of Caspase3/7 + FMC63-CART19 and h1218-CART19 cells with and without 4-h stimulation by Nalm6. Caspase3/7 + population of CART19 cells is boxed in red. (Middle) Baseline level of Caspase3/7 + population in FMC63- or h1218-CART19 cells at 0 h. (Right) Differential increase in the Caspase3/7 + population after 4-h stimulation. All graphs are represented as the mean ± SEM. Student's t-test was used to compare two groups; one-way ANOVA was performed with Tukey’s correction for multiple comparisons; **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05. All bar graphs are presented as the mean ± SEM. All experiments were repeated at least twice

Article Snippet: Lipid bilayers were first blocked with 5% casein for 20 min and then incubated with 6.3 nM streptavidin (ThermoFisher; Cat# 434301) for 15 min. After washing with imaging buffer (HEPES-buffered saline), lipids were incubated with biotinylated CD19 protein (R&D Systems; Cat#AVI9269) conjugated with Alexa Fluor 647 at room temperature for 30 min. After washing with the imaging buffer, lipid bilayers were blocked with 2.5 μM D-biotin to saturate the streptavidin-binding sites.

Techniques: Activation Assay, Binding Assay, Co-Culture Assay, Confocal Microscopy