Extended Data Fig. 7 |. Disrupting GADS interactions eliminates LINK CAR leakiness.

a, Schematic illustrating LINK CAR bearing both the Cysteine-to-Alanine (2CA) mutations and GADS-binding site deletions/truncations (ΔGADS). b, Flow cytometric expression of LINK CARs (±2CA, ±ΔGADS) on T cells utilized in Fig. 5b–d. c, Quantification of CD107a⁺ and CD69⁺ on indicated LINK CAR T cells following co-culture with indicated cell lines as shown in Fig. 5d. Representative of five independent experiments with four different blood donors. Shown are mean values ± s.d. of three experimental replicates. Comparisons were performed with one-way ANOVA with correction for multiple comparisons. d, Flow cytometric expression of LINK CAR T cells bearing either Y171F/Y191F point mutations (2YF) or deletion/truncation of the GADS-binding regions (ΔGADS). e, IL-2 secretion (as measured by ELISA) by indicated LINK CAR T cells following co-culture with indicated cell lines shown in Fig. 4c. Representative of two independent experiments with different blood donors. Shown are mean values ± s.d. of three experimental replicates. Note that data for CD19-28TM-LAT + HER2-8TM-SLP-76 and CD19-28TM-LAT^ΔGADS + HER2-8TM-SLP-76^ΔGADS conditions are identical to Fig. 5b. Comparisons were performed via one-way ANOVA with correction for multiple comparisons. f, Tumour cell killing of Nalm6 by CD19-CD28ζ, CD19–4-1BBζ, LINK (CD19-28TM-LAT + CD19-8TM-SLP-76), or LINK^2CA+ΔGADS (CD19-28TM^2CA-LAT^ΔGADS + CD19-8TM-SLP-76^ΔGADS) CAR T cells at a 1:1 ratio of T cells to tumour cells. Shown are mean values ± s.d. of three experimental replicates. Representative of three independent experiments with different blood donors.