CAR-T cell immunophenotyping
CD45RA-BV510
Standard staining and flow cytometry techniques were used to perform immunophenotyping of surface markers on BNT211 CAR-T cell products at the manufacturing site. Cells were stained with CD3-FITC, CD4-APC and CD8-PE (BD Multitest catalog no. 342417; 1:11 dilution) in the bulk drug product, and CD3-PerCP, CD4-FITC, CD8-PE (BD, Tritest, catalog no. 342414; 1:6.25 dilution) and murine F(ab’)2-fragment-AF647 (anti-CAR antibody) (Jackson ImmunoResearch catalog no. 115-605-006; 1:100 dilution) in the drug substance. An AF647 conjugated murine isotype control (Jackson ImmunoResearch catalog no. 005-600-003; 1:143 dilution) was used as a control. The frequencies of naïve-like T cells, central memory T cells, effector memory T cells and effector memory re-expressing CD45RA T cells were defined by CD3-PerCP (BD catalog no. 345766; 1: 5 dilution), CD4-BV421(BD catalog no. 565997; 1:50 dilution), CD8-BB515 (BD catalog no. 564526; 1:20 dilution), CD197-PE (BD catalog no. 560765; 1:5 dilution) and CD45RA-BV510 (BD catalog no. 563031; 1:25 dilution) in the bulk drug product. All antibodies for analysis were purchased from BD Biosciences and Jackson ImmunoResearch. Samples were analyzed on a FACSVerse flow cytometer (BD Biosciences) using the gating strategies shown in Supplementary Fig. 