Extended Data Figure 26:

Low affinity designed Phox2B:HLA-C*07:02 scFvs do not show cytotoxicity

Cell index (CI) was monitored over time for neuroblastoma cell lines ((A) SK-N-AS, (B) SK-N-SH, (C) NBSD, (D) NB-1691, (E) SK-N-FI) and a colorectal adenocarcinoma cell line ((F) LS123) following the addition of CAR-T cells at total T cell effector-to-target ratio 15:1. CI values, representing cell viability and growth, were measured by the xCELLigence RTCA DP system at hourly intervals and normalized to baseline values to assess cell-mediated cytotoxicity. Mean and SEM are shown with each sample performed in triplicate, and rare outlier CI values were filtered. 10LH (positive control) demonstrated cytotoxicity against most Phox2b+ neuroblastoma lines expressing HLA-A*24:02 or HLA-A*23:01 and no cytotoxicity against Phox2b- LS123, as expected. In contrast, designed scFvs show no cytotoxicity against Phox2b+ neuroblastoma lines expressing HLA-C*07:02 or HLA-C*07:01. RA-7 and RA-8, as well as RA-11 and RA-12 comprise the same scFv with alternate heavy and light chain orderings. G) Transduced CAR T cells were stained with anti-(G₄S)₃ linker antibody, anti-CD3, anti-CD4 and anti-CD8 to confirm CAR expression. Staining for one example of positive control 10LH is shown.