Fig 2: ADP-520 TRuC T cells demonstrate superior persistence, expansion, and resistance to exhaustion during chronic antigen stimulation in vitro. Flow cytometric characterization of response of NT, C10 TRuC and ADP-520 TRuC T cells to multiple rechallenges with ACHN tumor cells: fold expansion (A), percentage of tumor cell lysis (B), percentage of live CD45+ T cells (C), and the percentage of TRuC+ T cells (D) are shown over 16 days of coculture. Corresponding expression of T-cell exhaustion markers are shown as a percentage of TRuC+ cells, either PD-1 alone (E, F) or the combination of PD-1, LAG3 and TIGIT (G, H), for CD4+ and CD8+ populations, respectively. Arrows indicate timepoints of ACHN cell challenge. Data are mean ± SEM for n=3 donors, measured in triplicate. Treatment groups were compared via two-way ANOVA, followed by Dunnett’s multiple comparison test; *p < 0.05, **p < 0.01, ****p < 0.0001. ANOVA, analysis of the variance; NT, non-transduced; SEM, standard error of the mean; TRuC, T-cell receptor fusion construct.

Fig 3: ADP-520 TRuC T cells demonstrate superior mbIL-15-dependent persistence in the presence or absence of exogenous cytokine or antigen stimulation. Flow cytometric analysis of the autonomic persistence of NT, C10 TRuC and ADP-520 TRuC T cells, showing growth curves in the absence of exogenous cytokines or antigen stimulation (A) and corresponding quantification of the percentage of TRuC+ cells at each timepoint (B). Antigen-dependent persistence of NT, C10 and ADP-520 TRuC T cells, showing growth curves in the presence of 786-O tumor cells at a 5:1 E:T ratio (C) and corresponding quantification of the percentage of live CD45+ T cells at each timepoint (D). Effect of TCR (E) or IL-15 (F) signaling inhibition on antigen-dependent expansion of ADP-520 TRuC T cells co-cultured with 786-O tumor cells at a 5:1 E:T ratio. The TCR signaling inhibitor dasatinib (Das), JAK1/2 inhibitor ruxolitinib (Ruxo) or JAK3 inhibitor tofacitinib (Tofac), or corresponding vehicle (Veh) or media (Med), were added to cocultures at the timepoints indicated and cell numbers determined by cytometric analysis. All data are mean ± SEM for n=3 donors, measured in triplicate. Treatment groups were compared via two-way ANOVA, followed by Dunnett’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ANOVA, analysis of the variance; Das, dasatinib; E:T, effector:target; mbIL-15, membrane-bound interleukin-15; n.s., not significant; NT, non-transduced; SEM, standard error of the mean; TRuC, T-cell receptor fusion construct.

Fig 4: ADP-520 TRuC T cells exhibit enhanced intratumoral infiltration and anti-tumor efficacy in vivo with evidence of pseudoprogression. Temporal profile of tumor burden following treatment of mouse models of subcutaneous 786-O (A) and ACHN (B) tumors with intravenous NT, C10 TRuC or ADP-520 TRuC T cells. Bioluminescence images are depicted for the ACHN model (C). Tumor burden is measured by means of caliper measurement of volume or bioluminescence intensity of luciferase-expressing tumor cells. In vivo data are mean ± SEM, n=1 donor per tumor and five mice per group. Treatment groups were compared using a two-way repeated-measures ANOVA, followed by the Bonferroni post hoc test. (D, E) TRuC T cell infiltration was analyzed by flow cytometric quantification of human vs mouse CD45 expression in the blood and tumor of the mice treated in (A, B), respectively. Data presented as percentage of CD45+ live cells and number of cells per tumor or microliter of blood at 14 days post-treatment. Ex vivo data are mean ± SEM, n=3 mice per group. Treatment groups were compared using one-way ANOVA followed by Dunnett’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ANOVA, analysis of the variance; BUV, Brilliant Ultra Violet; Cy, cyanine; NT, non-transduced; PerCP, peridinin-chlorophyll-protein; s.c., subcutaneous; SEM, standard error of the mean; TRuC, T-cell receptor fusion construct.