Additional file 1 of Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC
Creators: name: Harrasser, Micaela
name: Gohil, Satyen Harish
name: Lau, Hiu
name: Della Peruta, Marco
name: Muczynski, Vincent
name: Patel, Dominic
name: Miranda, Elena
name: Grigoriadis, Kristiana
name: Grigoriadis, Anita
name: Granger, David
name: Evans, Rachel
name: Nathwani, Amit Chunilal
Publisher: name: figshare
Year: 2022
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: 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&lt;0.05; **p&lt;0.005. Fig. S3. In vivo monitoring of CAR-T cell treated mice. (A) BLI to assess tumors before and 4 weeks after treatment, 3-4 representative mice/group; (B) Tumor measurements (via caliper) up to day 37 post treatment, the arrow pointing at time point showed in Figure 2A, mean+SD of 6 mice/group; (C) Mice were monitored before and up to two weeks post CAR-T cell treatment for signs of CAR-T cell therapy-related side effects including weight loss (n=6/group shown); (D) MDA-MD-231 tumors were harvested at the end of the experiment and processed from tumor-bearing mice: immunohistochemistry analysis of tumors: a total of 5 tumors/group were stained for human CD8 and 5 sections/tumor were analyzed and quantified. One representative section for human CD8 from each treatment group. Positive control from human tonsil, negative control from untreated tumor. Fig. S4. Ex-vivo analysis shows that F i-CAR-T cells have a distinct transcription signature. (A) Heatmap showing raw numbers of T cell population frequencies (Log2 scale) from Nanostring analysis, including characteristic genes that were used to measure these populations' abundance (B) Pathway score comparisons between F CAR and F i-CAR T cells isolated from MDA-MB-231 tumors shown as box and whisker plots. Pathway scores are fit using the first principal component of each gene set’s data and oriented so that an increasing score corresponds to increasing expression (specifically, each pathway score has positive weights for at least half its genes). Data was analyzed through NanostringTM software.