Extended Data Fig. 2: Screening of CAR30 scFv binders.
From: The BTLA–HVEM axis restricts CAR T cell efficacy in cancer
a, Eleven anti-CD30 CAR constructs were developed and screened for transduction efficiency in primary human T cells from three healthy human donors. CAR30 surface expression was detected using an anti-G4S-linker antibody. HRS3 is a control gold-standard clone. Low transduction efficiency candidates (T1-8, T1-9, T2-2) were not assessed further. b,c, Short-term tumor killing of CAR30 candidates from two donors against (b) CD30-HEK293T (n = 2 technical replicates) and (c) SU-DHL-1 (n = 2 technical replicates). d, IFNγ concentration (ELISA) of supernatant following 24 h coculture with SU-DHL-1 (effector: target = 0.3, n = 2 technical replicates, representative of three independent biological donors). e, Expansion of UTD, HRS3, T1-36, and T1-159, revealing fratricide of T1-36 during manufacturing. f, Assessment of off-target toxicity of lead candidates using CD30- cell lines (OCI-Ly18, Nalm6, and Raji-B). Tumor killing is shown after 48 h against OCI-Ly18 (left) and Nalm6 (middle, effector: target = 2, n = 2 technical replicates), and IFNγ concentration (ELISA) is shown after 24 h against Raji-B (right, effector: target = 1, n = 2 technical replicates). g, Average tumor volumes (± s.d.) of in vivo study comparing T1-159 and HRS3 CAR30 T cells. Briefly, 15 × 106 HDLM-2 cells were inoculated into the right flank of NSG mice, and mice were infused at week 8 with 3 × 105 T1-159 CART30 (n = 4) or HRS3 CART30 (n = 4). Caliper measurements were taken once per week following infusion. h, Schema depicting overall selection process which led to the lead CAR30 construct, T1-159. For all bar plots, data are presented as mean ± s.d. Statistical significance was determined by two sided, multiple unpaired t-test (g). Except for g, ‘n’ defines the number of technical replicates.
