Journal: Toxicological Sciences

Article Title: Nonclinical safety assessment of SBT777101, a CAR-engineered regulatory T cell therapy for autoimmune diseases

doi: 10.1093/toxsci/kfag125

Figure Lengend Snippet: SBT777101 demonstrates minimal antigen-specific activation without impact on normal human cell survival. (A) Representative flow cytometry plots showing surface (top) and intracellular (bottom) binding of SBT777101 scFv-Fc (right) relative to isotype control (left) in SNU449 cells. (B) In vitro functional characterization using SNU449 cells ± CV-beads. Left: Mean ± SD CD69 expression (only SBT777101 data shown; no activation observed in UT controls). Data analyzed by two-way ANOVA with Sidak’s multiple comparisons test. Right: Cytotoxicity measured as the number of viable target cells remaining, normalized to no-Treg controls. Data analyzed by one-way ANOVA with Tukey’s multiple comparisons test. (C) Percentage of UT (top) or SBT777101 (bottom) Tregs expressing CD69 after 24-h co-culture with normal human primary cells or CV-beads (positive control). Data represent mean ± SD from three replicates. Independent experiments are separated by dashed vertical lines. Comparisons were made for media alone compared with in the presence of primary cells for each type and analyzed by two-tailed unpaired t -test. (D) Cytotoxicity (mean ± SD) of SBT777101 and UT Tregs against the same primary cell panel, normalized to no-Treg controls. Data analyzed by one-way ANOVA with Tukey’s multiple comparisons test. (E) Fold-change in CD71 expression (mean ± SD; relative to media control) across three donors after 72-h culture with media, CitP − SKNBE2 cells, CitP + SNU449 cells, human brain astrocytes (HBA), or CV-beads. CV beads were prepared in RPMI or AGM media as appropriate. Full lysis controls (B, D) represent target cells treated with Triton X-100. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. All experiments included 3 technical replicates. AGM, astrocyte growth medium; CitP, citrullinated protein; CV, citrullinated vimentin; HMEC, human mammary epithelial cells; NHBE, normal human bronchial epithelial cells; NHDF, normal human dermal fibroblasts; PrEC, prostate epithelial cells; RPTEC, renal proximal tubule epithelial cells; scFv, single-chain variable fragment; UT, untransduced.

Article Snippet: Primary human cells (mammary epithelial cells [HMEC; Lonza Cat#CC-2551], normal bronchial epithelial cells [NHBE; Lonza Cat#CC2451], renal proximal tubule epithelial cells [RPTEC; Lonza Cat#CC2553], prostate epithelial cells [PrEC; Lonza Cat#CC2555], hepatocytes [Lonza Cat#HUCPG], brain astrocytes [HBA; Neuromics Cat#HMP202], iPSC-derived myocytes [Tempo Bioscience Cat#1014], iPSC-derived cortical neurons [Tempo Bioscience Cat#201], and monocyte-derived macrophages [internally derived]) and the positive-control SNU449 cell line (ATCC Cat#CRL-2234) were assessed for binding of the SBT777101 scFv by flow cytometry.

Techniques: Activation Assay, Flow Cytometry, Binding Assay, Control, In Vitro, Functional Assay, Expressing, Co-culture Assay, Positive Control, Two Tailed Test, Lysis