Fig 1: Expression of human cystatins increases CAR T cell expansion by reducing CTSB-mediated CMT.(A) Crystal structures of bovine cathepsin B in complex with Ca-074 (left, PDB: 1QDQ) and human cathepsin B in complex with cystatin A (right, PDB: 3K9M). The active site cysteine of cathepsin B is shown in pink; Ca-074 and cystatin A are shown in teal. (B) Schema of construct used for production of CARCSTA T cells. (C) Levels of CSTA in CAR or CARCSTA T cells measured by ELISA. Data represent mean ± S.D. of two technical replicates. (D) Cathepsin B activity in CAR or CARCSTA T cells measured by fluorescence. Data represent mean ± S.D. of two technical replicates. (E) Expansion of CAR or CARCSTA CAR T cells during manufacturing as determined by cell counting. (F) CAR T cells were analyzed via flow cytometry after production for (I) CAR expression, (II) T cell subsets, and (III-IV) T cell phenotype. (G) Survival of Raji-Fluc, NALM6-Fluc, Daudi-Fluc, or Toledo-Fluc cells after 16-hour coculture with FMC63 or CARCSTA T cells at the indicated effector-target ratios. Tumor survival was measured using a luciferase-based cytotoxicity assay. Data represent mean ± S.D. of three technical replicates. (H) CMT and (I) area under curve quantification as determined by CompLuc using K562 cells expressing CD19-cLuc after 3 hours. Data represent mean ± S.D. of three technical replicates. (J) CD19 loss on tumor cells, (K) CD19 transfer to CAR T cells, and (L) total CAR T cells following a 30-minute coculture of FMC63 or CARCSTA T cells and K562 cells expressing CD19-cLuc at a 0.5:1 effector-target ratio. CAR T cell numbers are normalized to wells containing only CAR T cells using counting beads. Data represent mean ± S.D. of three technical replicates.
Fig 2: CSTA overexpression reduces CMT and increases expansion in BCMA CAR T cells.(A) Schema of various CARBCMA T cells. (B) BCMA transfer to CAR T cells and BCMA loss on BCMA-GFP-expressing K562 cells. (C) BCMA loss on MM.1S, RPMI8226, and U266B1 cells when cocultured with CARBCMA, Abecma, and Carvykti CAR T cells. (D) Expansion of BCMA CAR or CARCSTA CAR T cells during manufacturing as determined by cell counting. (E) BCMA CAR T cells were analyzed via flow cytometry after production for (I) CAR expression, (II) T cell subsets, and (III-IV) T cell phenotype. (F) Survival of MM.1S-, RPMI8226-, and U266B1-Fluc cells after 16-hour coculture with BCMA CAR or CARCSTA T cells at the indicated effector-target ratios. Tumor survival was measured using a luciferase-based cytotoxicity assay. Data represent mean ± S.D. of three technical replicates. (G) BCMA CAR T cell expansion after 16-hour coculture with the indicated tumor cell lines at an 8:1 effector-target ratio. (H) CMT and (I) area under curve quantification as determined by CompLuc using K562 cells expressing BCMA-GFP-cLuc after 3 hours. Data represent mean ± S.D. of three technical replicates. (J) BCMA loss on U266B1 tumor cells after 2-hour coculture with BCMA CAR or CARCSTA T cells. (K) BCMA-GFP transfer to CAR T cells, and (L) total CAR T cells following a 30-minute coculture of BCMA CAR or CARCSTA T cells and K562 cells expressing CD19-cLuc at a 1:1 effector-target ratio. CAR T cell numbers are normalized to wells containing only CAR T cells using counting beads. (H-L) Data represent mean ± S.D. of three technical replicates. (G, I-L) Statistical significance was determined by two-tailed Student’s t-test.
Supplier Page from Thermo Fisher Scientific for Human Cystatin A ELISA Kit