Additional file 1 of Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC

Additional file 1: Fig. S1. Assessment of anti-PD-1 scFv binding. (A) Transgene schematic of bicistronic vector for constitutive secretion of anti-PD-1 scFv; (B) Supernatant from transfected HEK-293T cells producing anti-PD-1scFv was tested to confirm binding to PD-1+SupT1 cell line via flow cytometry; (C) Binding affinity measured via SPR using an anti-PD-1 scFv-Fc coated CM5 Chip: representative sensogram (of 3 independent repeats) of response units (RU) to increasing concentrations of PD-1 protein (0µg/ml–2.5µg/ml). Fig. S2. Transduction efficiency and additional in vitro characterization of F CAR and F i-CAR-T cells effector functions. (A) Representative flow cytometry plots of F CAR and F i-CAR T cells demonstrating comparable transduction levels between constructs as assessed by mCherry expression; (B) Representative flow cytometry of ROR1 and PD-L1 expression on positive MDA-MB-231, H1975 and negative control SUPT1 cell lines; (C) Anti-PD1 scFv was Luciferase-tagged and co-culture conditions were tested for scFv production via addition of substrate (coelenterazine) and luminescence quantified. Results are mean from 3 donors in triplicate; (D) Cytotoxicity assay following 72h of co-culture with CAR-T cells, viable ROR1- target cell lines were assessed via flow cytometry, results are mean+SD of 3 donors in triplicates normalized to control CD19 CAR-T cells; (E) Immune checkpoint expression was assessed via flow cytometry and shown as % of CAR-T cells. 2way ANOVA used: *p