Journal: Blood Advances

Article Title: FLT3-directed BiTE molecules vs CAR T cells in AML: costimulatory signals mitigate T-cell exhaustion

doi: 10.1182/bloodadvances.2025018168

Figure Lengend Snippet: Superior antileukemia activity of CAR T cells in an AML xenograft model. (A) Schematic overview of the experimental setup. NXG mice were inoculated IV with 1 × 10 6 OCI-AML3-LUC-GFP tumor cells. Mice were treated with a single IV injection of T cells. BiTE molecule was given by daily IP injections (200 μg/kg per injection) as indicated by the arrows in the figure. A total of 5 × 10 6 HD T cells or 1 × 10 7 HD CAR T cells (50% transduction efficiency) were given IV as indicated. Treatment groups were as follows: PBS (n = 5), cBiTE (n = 5), BiTE (n = 5), UT (n = 5), and CAR (n = 5). (B) In vivo images with luminescence intensity counts for all experimental groups from treatment day onward (days 0, 3, 10, 14, 18, 21, 24, 31, and 38). (C) Kaplan-Meier plot of survival probability. (D) Analysis of blood samples on day 14 after the start of treatment. Frequencies of hCD3 + and hCD33 + cells were determined by flow cytometry. (E) Analysis of tumor burden in the bone marrow. Frequencies of hCD33 + cells were determined by flow cytometry. (F) Analysis of T-cell homing to the spleens of mice. Frequencies of hCD3 + cells were determined by flow cytometry. Bar and dot graphs represent the mean ± SEM values. Statistical analysis: log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon testing (panel C); Kruskal-Wallis and Dunn multiple comparison testing (panels D-E); ns_ P > .05; ∗P < .05; ∗∗P < .01. †Mice were euthanized because of non–disease-related toxicity. cBiTE, control BiTE; PBS, phosphate-buffered saline.

Article Snippet: Four-week-old NXG mice (NOD.Cg-Prkdcscid Il2rgtm1WjI/Rj) were purchased from Janvier (St Berthevin, France).

Techniques: Activity Assay, IV Injection, Injection, Transduction, In Vivo, Flow Cytometry, Comparison, Control, Saline

Journal: Blood Advances

Article Title: FLT3-directed BiTE molecules vs CAR T cells in AML: costimulatory signals mitigate T-cell exhaustion

doi: 10.1182/bloodadvances.2025018168

Figure Lengend Snippet: BiTE molecule–redirected T cells exhibit a more exhausted transcriptional profile. (A) Schematic overview of the experimental setup. NXG mice were inoculated IV with 1 × 10 6 OCI-AML3-LUC-GFP tumor cells. Mice were treated with a single IV injection of T cells. BiTE molecule was given by daily IP injections (200 μg/kg per injection) as indicated by the arrows in the figure. A total of 5 × 10 6 HD T cells or 1 × 10 7 HD CAR T cells (50% transduction efficiency) were given IV as indicated. Treatment groups were as follows: PBS (n = 5), cBiTE (n = 5), BiTE (n = 5), UT (n = 5), and CAR (n = 5). The experiment was terminated 18 days after the start of the treatment. T cells were isolated from the bone marrow of mice and sorted for bulk RNA sequencing. (B) Heat map depicting the top 100 differentially expressed genes in day-18 CAR vs BiTE molecule–redirected T cells; adjusted P value ( P adj ) < .05. Selected genes are highlighted. (C) Volcano plot of day-18 CAR vs BiTE molecule–redirected T cells; P adj < .05. Selected genes are highlighted as significantly downregulated (blue) and upregulated (red) in day 18 CAR vs BiTE-redirected T cells. (D) Pathways enriched in day 18 CAR vs BiTE molecule–redirected T cells; P adj < .05. (E) Gene set enrichment analysis of day 18 CAR vs BiTE-redirected T cells using the Molecular Signatures Database (MSigDB) and the gene sets GSE9650_EFFECTOR_VS_MEMORY_CD8_TCELL_UP. (F) Hallmark gene sets upregulated in CAR T cells compared with BiTE molecule–redirected T cells. cBiTE, control BiTE; CTLA4, Cytotoxic T-Lymphocyte-Associated Protein 4; FOS, FBJ Murine Osteosarcoma Viral Oncogene Homolog; GPCR, G-protein coupled receptor; PDCD1, Programmed Cell Death 1; TIGIT, T-cell immunoreceptor with Ig and ITIM domains; TOX, Thymocyte selection-associated high mobility group box.

Article Snippet: Four-week-old NXG mice (NOD.Cg-Prkdcscid Il2rgtm1WjI/Rj) were purchased from Janvier (St Berthevin, France).

Techniques: IV Injection, Injection, Transduction, Isolation, RNA Sequencing, Control, Selection

Journal: Blood Advances

Article Title: FLT3-directed BiTE molecules vs CAR T cells in AML: costimulatory signals mitigate T-cell exhaustion

doi: 10.1182/bloodadvances.2025018168

Figure Lengend Snippet: Positive costimulatory signals enhance BiTE molecule–mediated T-cell efficacy in vivo. (A) In vitro cell line model demonstrating the effect of positive costimulation by CD86 on BiTE molecule– and CAR-mediated cytotoxicity. (B) Schematic overview of the experimental setup. NXG mice were inoculated IV with 1 × 10 6 OCI-AML3-LUC-GFP-CD86 high tumor cells. Mice were treated with a single IV injection of T cells. The half-life extended (HLE) BiTE molecule was given by IP injections (392 μg/kg per injection) every 5 days. 5 × 10 6 HD T cells or 1 × 10 7 HD CAR T cells (50% transduction efficiency) were given IV as indicated. Treatment groups were as follows: PBS (n = 5), cBiTE (n = 5), BiTE (n = 5), UT (n = 5), and CAR (n = 5). (C) In vivo images with luminescence intensity counts for all experimental groups from treatment day onward (days 5, 12, 20, 26, 33, 40, 47, 54, 62). (D) Kaplan-Meier plot of survival probability. (E-F) Analysis of murine bone marrow and spleen. Frequencies of hCD3 + cells were determined by flow cytometry. (G) Comparison of median survival of HLE BiTE molecule–treated and CAR-treated mice between in vivo 1 ( ; without additional costimulation) and in vivo 3 ( ; with additional costimulation). Statistical analysis: log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon testing (panel D); Kruskal-Wallis and Dunn’s multiple comparison testing (panels A,E-F); ns_ P > .05; ∗P < .05; ∗∗P < .01. †Mice were euthanized because of non–disease-related toxicity. max, maximum; min, minimum; ns, not significant; PBS, phosphate-buffered saline; wt, wild type.

Article Snippet: Four-week-old NXG mice (NOD.Cg-Prkdcscid Il2rgtm1WjI/Rj) were purchased from Janvier (St Berthevin, France).

Techniques: In Vivo, In Vitro, IV Injection, Injection, Transduction, Flow Cytometry, Comparison, Saline