Journal: Cancer immunology research

Article Title: Secretion of a VEGF-blocking scFv enhances CAR T-cell potency

doi: 10.1158/2326-6066.CIR-24-0876

Figure Lengend Snippet: A and B , Bioluminescence of HEK-293T VEGFR reporter cells treated with supernatant containing either anti-VEGF or anti-CD19 and recombinant VEGF (rVEGF) (Data from technical triplicates, points represent the mean, error bars represent SEM, p-value by two-way ANOVA) ( A ) or supernatant from confluent VEGF-producing 786O tumor cells (data from technical triplicates, lines represent the median, p-value by one-way ANOVA) ( B ). C, Schematic of in vitro angiogenesis assay to visualize blood vessel formation upon blocking VEGF. GFP + human umbilical vein endothelial cells (HUVECs) were cocultured with normal human dermal fibroblasts (NHDFs), suramin, rVEGF, and culture supernatant from CAR αCD19 or CAR αVEGF constructs and primitive blood vessel formation was imaged over time on the incucyte. D, Representative images of GFP + HUVECs with vessels labeled by the automated incucyte angiogenesis software package. E, HUVEC blood vessel network length (length of blood vessel network (mm) per mm 2 in image). (data representative of 2 technical replicates, mean±SEM, p-value by two-way ANOVA).

Article Snippet: The assay was imaged and analyzed using the Sartorius Incucyte Angiogenesis Software package every 6 hours for 14 days.

Techniques: In Vitro, Recombinant, Angiogenesis Assay, Blocking Assay, Construct, Labeling, Software

Journal: Cancer immunology research

Article Title: Secretion of a VEGF-blocking scFv enhances CAR T-cell potency

doi: 10.1158/2326-6066.CIR-24-0876

Figure Lengend Snippet: A, CD69 surface expression of meso-targeting CAR T-cell constructs cultured with SKOV3 for 18 hours. B, CD69 surface expression of CD70-targeting CAR T-cell constructs cultured with plate bound CD70 antigen for 24 hours. C, 70 αVEGF and 70 αCD19 CAR T cells (n=4 donor T cells each) were incubated for 48 hours on plates coated with recombinant CD70 protein, then lysed and evaluated via targeted RNA expression. Heatmap results highlight individual pathway scores for each donor and construct. See supplemental figure 5 for additional details. D, Pathway score for the most differentially scored pathway from 3C. Symbols represent individual T-cell donors. Dashed line represents the median and the dotted line represents the top and bottom quartiles. All pathways were subjected to paired t tests and corrected for multiple comparisons with the two-stage step-up method of Bejanmini, Kreiger, and Yekutieli with a desired FDR (q) of 1%. E, Fold expansion of Meso αCD19 and meso αVEGF CAR-T cells from restimulation assays with meso + K562 ( 2 NDs and 2 technical replicates, mean±SEM, p-values by 2 way ANOVA). F and G, Cytotoxicity and proliferation of meso-targeting CAR T cells cultured with SKOV3 at 3:1 E:T ratio (2 NDs with 2+ technical replicates) ( F ) and OVCAR3 at 3:1 E:T (2 NDs) ( G ). H and I, Concentration of VEGF, IFNγ, TNFα, and Granzyme B in supernatant taken from endpoint of associated cytotoxicity assay, SKOV3 ( H ) and OVCAR3 ( I ) measured using Ella automated immunoassay system ( 2 ND with 2+ technical replicates). J and K, Cytotoxicity and proliferation of meso-targeting CAR T cells cultured with Nomo-1 at 3:1 E:T (1 ND, representative of 2 NDs) ( J ) and A549 at 3:1 E:T (2 NDs) ( K ), images depict GFP-expressing tumor and mcherry-expressing CAR T cells from the same respective wells at time 0 and after 72 hours of co-culture. L , M , and N, Cytotoxicity assays of mcherry+ CD70 αCD19 and CD70 αVEGF CAR T cells cultured with GFP + 786O at 2:1 E:T (3 NDs) ( L ), SKOV3 at 3:1 E:T ( 2 NDs) ( M ), and Nomo-1 at 3:1 E:T (1 ND) ( N ). O, Concentration of VEGF from supernatant of cytotoxicity assay in N (2 NDs with 2+ technical replicates). Cytotoxicity assays were performed using incucyte imaging system and wells were imaged every 1–2 hours. Data represents values normalized to the starting condition of red or green fluorescence. All cytotoxicity assays show mean±SEM with p-values by two-way ANOVA. All cytokine assays show mean±SEM with p-values by one-way ANOVA. ND-normal donor.

Article Snippet: The assay was imaged and analyzed using the Sartorius Incucyte Angiogenesis Software package every 6 hours for 14 days.

Techniques: In Vitro, Expressing, Construct, Cell Culture, Incubation, Recombinant, RNA Expression, Concentration Assay, Cytotoxicity Assay, Co-Culture Assay, Imaging, Fluorescence