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.

Fig 3: Cystatin overexpression enhances long-term CAR T-cell persistence. a Schema of the in vitro serial cytotoxicity experiment to determine long-term CAR T-cell persistence and antitumor activity. b Survival of CD19-GFP-expressing NALM6-Fluc cells at a 0.5:1 effector-target ratio using a luminescence-based cytotoxicity assay. Data indicate the mean ± S.D. from three technical replicates. Tumor survival was normalized to untreated tumor cells. The data are representative of two independent experiments. Statistical significance comparing FMC63 CAR and CARCSTA T cells was determined by multiple two-tailed Student’s t tests. c FMC63 CAR and CARCSTA T-cell counts on days 1–3 of the in vitro serial coculture described in Fig. 6A. Data represent the mean ± S.D. of 3 technical replicates and are representative of two independent experiments. Statistical significance comparing FMC63 CAR and CARCSTA T cells was determined by multiple two-tailed Student’s t tests. d Schema of the in vivo experiment to measure NALM6 tumor control and CAR T-cell persistence. e Tumor burden in mice bearing systemic NALM6-Fluc tumors and treated with ∆scFv or FMC63 CAR or CARCSTA T cells as determined by an in vivo imaging system (IVIS). Data represent the mean ± S.D. of 5 mice per group. f Quantification of CAR T cells in murine spleen, peripheral blood, and bone marrow as determined by flow cytometry. Data represent the mean ± S.D. of 10 mice pooled from two independent experiments, with experiments denoted by symbols. Data were normalized to the average of the respective experiment’s CSTA– condition and are expressed as fold change. Statistical significance was determined by a two-tailed Student’s t-test. g Phenotype of FMC63 CAR/CARCSTA T cells from murine blood as determined by flow cytometry. Statistical significance was determined by two-way ANOVA. h PD-1, TIM-3, and LAG-3 expression on FMC63 CAR or CARCSTA T cells isolated from murine blood as determined by flow cytometry. Statistical significance was determined by a two-tailed Student’s t-test. g, h Data represent the mean ± S.D. of 5 mice per group. i Differential mRNA expression in sorted FMC63 CAR and CARCSTA T cells from two independent productions at the end of manufacturing as determined by bulk RNA sequencing. j Schema of the in vitro serial cytotoxicity experiment to determine long-term CAR T-cell persistence and antitumor activity. k Survival of NALM6-Fluc cells at a 2:1 effector target ratio using a luminescence-based serial cytotoxicity assay. On Days 2, 4, 6, 8, 10, 12, and 14, CAR T cells were normalized, and fresh tumor cells were added. l Total CAR T cells on day 10 of the in vitro serial coculture described in Fig. 6J. m PD-1 expression on CAR T cells on day 5 of in vitro serial coculture, as described in Fig. 6J. n TIM-3 expression on CAR T cells on day 5 of in vitro serial coculture, as described in Fig. 6J. k–n Data represent the mean ± S.D. of three technical replicates and are representative of two independent experiments. o Quantification of CAR T cells in bone marrow 28 days after intravenous treatment with the indicated CAR T-cell conditions as determined by flow cytometry. Data represent the mean ± S.D. of 2–5 mice per group. Statistical significance was determined by one-way ANOVA. p Schema of the in vivo experiment to measure solid tumor control and CAR T-cell infiltration into solid tumors. q Tumor burden in mice bearing intratibial CD19-expressing A673-Fluc tumors and treated intravenously with ∆scFv or FMC63 CAR CISHWT or CARCSTA CISHKO T cells as determined by an IVIS. Data represent the mean ± S.D. of 3–4 mice per group. r Quantification of CAR T cells per 1000 acquired live cells in peripheral blood four days after treatment with the indicated CAR T cell conditions as determined by flow cytometry (n = 2–3 per group). Statistical significance was determined by one-way ANOVA. s Quantification of CAR T cells per 1000 acquired live cells in intratibial A673 tumors four days after treatment with the indicated CAR T-cell conditions as determined by flow cytometry (n = 2–3 per group). Statistical significance was determined by one-way ANOVA. t PD-1 and u TIM-3 levels on intratumoral CAR T cells four days after CAR T-cell injection as determined by flow cytometry. Statistical significance was determined by one-way ANOVA. r–u Data represent the mean ± S.D. of 2–3 mice per group

Fig 4: Cystatin abundance regulates CTSB activity and CMT. a Crystal structures of bovine cathepsin B (bCTSB) in complex with Ca-074 (left, PDB: 1QDQ) and human cathepsin B (hCTSB) in complex with cystatin A (right, PDB: 3K9M). The active site cysteine of CTSB is shown in pink; Ca-074 and cystatin A are shown in teal. b Schematic of the construct used for the production of FMC63-based CARCSTA T cells. c Amounts of CSTA in CAR or CARCSTA T-cell lysates, as measured by ELISA, are expressed as fold change of WT. Data represent mean ± S.D. of four technical replicates from two independent experiments. Statistical significance was determined by a two-tailed Student’s t-test. d CTSB activity in two different CD19-targeting (○ FMC63; □ CAT) CAR or CARCSTA T-cell products measured by fluorescence-based CTSB activity assay expressed as a percentage of WT. Data represent the mean ± S.D. of four replicates from two independent experiments. Statistical significance was determined by a two-tailed Student’s t-test. e Survival of Raji-Fluc cells after a 16-h coculture with FMC63 CAR or CARCSTA T cells at the indicated effector-target ratios. Data represent the mean ± S.D. of three technical replicates. Data are representative of at least three independent experiments using cells produced from three healthy donors. f CMT of FMC63 CAR or CARCSTA T cells as determined by CompLuc using K562 cells expressing CD19-cLuc after 3 h. Data are representative of three independent experiments using cells produced from three independent donors. g CMT in BCMA CAR or CARCSTA T cells as determined by CompLuc using K562 cells expressing BCMA-GFP-cLuc after 3 h. Data are representative of three independent experiments. f, g The data represent the best fit of three technical replicates. h CD19 transfer to CAR T cells following a 30-min coculture of FMC63 CAR or CARCSTA T cells with K562 cells expressing CD19-cLuc at a 0.5:1 effector-target ratio as determined by flow cytometry. Data are representative of at least three independent experiments using cells produced from three healthy donors. i BCMA-GFP transfer to CAR T cells following a 2-h coculture of BCMA CAR or CARCSTA T cells and K562 cells expressing BCMA-GFP-cLuc at a 1:1 effector-target ratio as determined by flow cytometry. Data are representative of three independent experiments. j GFP transfer to CAR T cells following a 1-h coculture with A673 cells engineered to express a CD19-GFP fusion protein at a 1:1 effector target ratio. k Amounts of CD19 on tumor cells following a 30-min coculture of FMC63 CAR or CARCSTA T cells and K562 cells expressing CD19-cLuc at a 0.5:1 effector-target ratio as determined by flow cytometry. Data are representative of at least three independent experiments using CAR T cells produced from three healthy donors. l Amounts of BCMA on U266B1 tumor cells following a 2-h coculture with BCMA CAR or CARCSTA T cells at a 1:1 effector-target ratio as determined by flow cytometry. Data are representative of three independent experiments. m Amount of GFP in A673 tumor cells following a 1-h coculture of FMC63 CAR or CARCSTA T cells and A673 cells expressing CD19-GFP at a 1:1 effector-target ratio as determined by flow cytometry. j, m Data are representative of two independent experiments using cells produced from three healthy donors. n Total CAR T cells following a 30-min coculture of FMC63 CAR or CARCSTA T cells and K562 cells expressing CD19-cLuc at a 0.5:1 effector-target ratio as determined by flow cytometry. Data are representative of at least three independent experiments using cells produced from three healthy donors. o Total CAR T cells following a 2-h coculture of BCMA CAR or CARCSTA T cells and K562 cells expressing BCMA-GFP-cLuc at a 1:1 effector-target ratio as determined by flow cytometry. Data are representative of three independent experiments. n, o CAR T-cell numbers are normalized to wells containing only CAR T cells using counting beads. p Membrane transfer to CAR T cells following a 1-h coculture of FMC63 CAR or CARCSTA T cells and primary B-cell acute lymphoblastic leukemia (B-ALL) cells at a 1:1 effector-target ratio as determined by flow cytometry. Circles indicate three technical replicates from a single patient B-ALL sample. q Amounts of CD19 on primary B-ALL cells following a 1-h coculture of FMC63 CAR or CARCSTA T cells and primary B-ALL cells at a 1:1 effector-target ratio as determined by flow cytometry. r Total CAR T cells following a 1-h coculture of FMC63 CAR or CARCSTA T cells and primary B-ALL cells at a 1:1 effector-target ratio as determined by flow cytometry. q, r Data are representative of three independent experiments using a single patient B-ALL sample. h–r Statistical significance was determined by one-way ANOVA