Extended Data Fig. 2. Transgenic expression of TGFβRDN significantly increases the proliferative capacity, but not the effector function of CART-PSMA cells compared to knockout of the endogenous TGFβRII.

(a) Efficiency of CRISPR/Cas9-mediated knockout (KO) of the endogenous TGFβRII (TGFβRKO) in CART-PSMA cells derived from n = 4 different subjects, as determined by Sanger sequencing and TIDE analysis. Editing efficiency is presented relative to AAVS1 knockout in donor-matched CART-PSMA cells. Error bars depict the SEM. (b) Representative flow cytometry showing levels of pSMAD2/3 in CART-PSMA cells with knockout of AAVS1, TGFβRII or co-expression of TGFβRDN that were unstimulated or stimulated with recombinant human TGFβ (representative of 3 independent experiments). (c) Expansion capacity of CART-PSMA-TGFβRDN versus CART-PSMA-TGFβRKO cells following serial re-stimulation (indicated by black arrows) with TGFβ-expressing irradiated PC3 prostate tumor cells. Proliferation is presented as a change in fold expansion over the longitudinal growth of stimulated CART-PSMA-AAVS1KO cells. Cells were manufactured from 4 different subjects, with pooled data from 3 independent experiments. Error bars depict the s.e.m. P values were calculated using a two-tailed t-test. (d) Killing kinetics of CART-PSMA-TGFβRDN, CART-PSMA-TGFβRKO and CART-PSMA-AAVS1KO cells co-cultured with PC3 tumor targets. CAR T cells directed against CD19 (irrelevant CAR) served as a negative control. Data are representative of 3 individual experiments performed with engineered T cells from 3 independent subjects. Error bars indicate the s.e.m. (e) Cytokine production from CART-PSMA-TGFβRKO and CART-PSMA-TGFβRDN compared to control CART-PSMA cells following stimulation with PC3 cells. Each data point represents a CAR T cell sample derived from an independent donor. Error bars depict the s.e.m. P values were calculated with a two-tailed t-test.