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, Single cell RNA sequencing data derived from[ 29 ] showing expression of KDR (VEGFR2), FLT1 (VEGFR1), VEGFA , NRP1, and NRP2 in CAR T cells from patients that received Tisa-cel and Axi-cel at baseline (prior to CAR T cell production), pre-infusion (post CAR T-cell production), and day 7 after CAR-T cell infusion into the patient (separated into CAR + and CAR − populations). Only cell populations with at least 200 cells in a given patient were included, yielding n=133 measurements taken from N=32 patients, including N=20 baseline, N=31 infusion product, N=29 Day +7 CAR-negative, and N=22 Day +7 CAR-positive sorted PBMC samples. B, Comparison of VEGF signaling family members among Tisa-cel (4–1BB) and Axi-cel (CD28z) patient pre-infusion products. C, Expression of VEGFR1, VEGFR2, NRP1, and NRP2 at baseline or in the infusion product (IP) or monocytes from responding and non-responding patients that received Tisa-cel or Axi-cel. P-values represent a wilcoxon ranksum test, the q-values are FDR-corrected by Benjamini-Hochberg. D, Mean fluorescence intensity (MFI) of VEGFR1 expression in meso-targeting CD8 + CAR + T cells (or untransduced T cells, UTD) with (+) and without (−) stimulation with MESO + K562s for 96 hours and representative histograms. Symbols represent technical triplicates from 2 normal donors (NDs), bars represent mean±SEM, p values by unpaired t-test). E, MFI of VEGFR1 expression in CD70 targeting CD8 + CAR + T cells after activation with CD70 + K562s for 96 hours and representative histograms. Symbols represent technical triplicates from 3 NDs, bars represent mean±SEM, p-value by unpaired t-test) F, VEGF concentration in culture supernatant following mesothelin CAR T-cell production measured by ELISA. (Symbols represent technical triplicates from 2 NDs, bars represent mean±SEM, p-value by one-way ANOVA). IP-infusion product

Article Snippet: The following antibodies were used: anti-mouse IgG (Cell Signaling Technology 4410S) VEGFR1 (R&D systems, FAB321V-100ug), Mouse IgG1k Isotype (R&D systems-IC002V), CD69 (Biolegend, 310918), CD8 (BD-560179, BD-647458), CD4 (BD-651850), CD3 (641406), CD71 (Biolegend-334104), PD1(BD Horizon-563789), TIM3 (BD Horizon-565566), LAG3 (BD Pharmingen-565716), CCR7 (BD Pharmingen-561271), CD45RA (Biolegend-304126), CD45 (Biolegend-368562), TOX (Miltenyi Biotec, 130-118-335), Human IgG1 isotype (Miltenyi Biotec, 130-120-709) .

Techniques: RNA Sequencing, Derivative Assay, Expressing, Comparison, Fluorescence, Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay