Apc conjugated anti cd3 antibody

To determine the killing effect of EPHB4-CAR-T cells on the target cells, RMS cell lines were co-cultured with EPHB4-CAR-T cells for 24 h at an E:T ratio of 1:1. The cell mixture was then collected and stained with PE-conjugated anti-insulin growth factor 1 receptor (IGF1R) antibody (BioLegend) and APC-conjugated anti-CD3 antibody (BioLegend) to identify RMS cells and T cells, respectively, and mixed with 50,000 CountBright Absolute Counting Beads (Thermo Fisher Scientific, Waltham, MA, USA). The number of live tumor cells (IGF1R-positive, CD3-negative cell fraction) was then determined via flow cytometry until the number of counting beads reached 5,000.

The expression of EPHB4 was detected via phycoerythrin (PE)‐conjugated EPHB4 antibody (R&D Systems, Inc., Minneapolis, MN, USA) staining. For the detection of EPHB4‐CAR expression, transduced T cells were stained using goat anti‐Ephrin B2 antibody (R&D Systems) and then stained using PE‐conjugated anti‐goat IgG antibody (R&D Systems). Allophycocyanin (APC)‐conjugated anti‐CD3 antibody, APC‐conjugated anti‐CD8a antibody, fluorescein isothiocyanate (FITC)‐conjugated CD4 antibody, FITC‐conjugated anti‐CD45RA antibody, and APC‐conjugated anti‐CCR7 antibody (all from BioLegend) were used for the characterisation of the CAR‐T cell phenotype. To determine the binding capacity of human Ephrin B2 to cynomolgus EPHB4, 293‐human EPHB4‐GFP and 293‐cynoEPHB4‐GFP cells were incubated with the recombinant human Ephrin B2‐Fc Chimera Protein (R&D Systems) for 20 min on ice and then stained using APC‐conjugated anti‐human IgG‐Fc antibody (BioLegend). Detailed antibody information is presented in Supplementary table 2. All flow cytometry data were acquired using BD FACS Accuri C6 Plus (BD Biosciences) and analysed using the FlowJo Software (Tree Star, Inc., Ashland, OR, USA).

PBMCs were activated with 5 µg mL⁻¹ prebound anti‐CD3/anti‐CD28 antibodies (BioLegend), followed by CFSE labeling (Invitrogen) according to the manufacturer's instructions. The activated PBMCs were cocultured with organoids and treated with a conditioned medium from organoids or lung cancer cell lines. After 96 h, the cells were stained with an APC‐conjugated anti‐CD3 antibody (BioLegend), and T cell proliferation was determined by assessing CFSE intensity via flow cytometry.