murine cd70 oe construct  (Addgene inc)

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Validation of CD70 as a GB target for CAR-T cell therapy (A) Expression of CD70 and selected markers from an in-house glioma patient single-cell RNA seq dataset. (B) CD70 expression in GB samples from Heidelberg University Hospital, analyzed by bulk RNAseq. Each dot represents a patient. A Mann-Whitney test was performed to assess statistical significance. (C) CD70 expression in 20 matched pairs from (B). A two-tailed paired t test was used to assess significance. (D) IHC staining of three Heidelberg University Hospital GB patients from (B) with high CD70 expression based on RNAseq. Scale bars, 200 μm. Patient-1 and Patient-3: primary GB; Patient-2: recurrent GB. (E) Measurement of CD70 protein levels on GB cells by flow cytometry. (F) Measurement of CD70 gene expression levels in glioma cell lines by RT-qPCR. N = 3 technical replicates per cell line. (G) Correlation between CD70 gene expression and protein levels from (E) and (F). (H) Measurement of CD70 on the surface of generated primary GB models by flow cytometry (blue histograms). An isotype control antibody (red histograms) was used. For (E) and (H), data were gated on single live cells. Data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: Biomarker Discovery, Expressing, Single Cell, RNA Sequencing, RNA sequencing, MANN-WHITNEY, Two Tailed Test, Immunohistochemistry, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Generated, Control

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Generation and phenotyping of anti-CD70 CAR-Tcells (A) CAR construct design. (B) Transduction efficiency of primary human T cells by flow cytometry. A non-transduced (NT) sample from each donor was used to determine gating. Data gated on single live CD3 + cells. (C) CD8a/CD4 expression on transduced T cells, determined by flow cytometry. A one-way ANOVA followed by a Dunnett’s multiple comparisons test was performed to assess significance. (D) Expression of exhaustion receptors on transduced T cells, determined by flow cytometry. (E) Quantification of PD1 + /LAG3 + /TIM3 + T cells from (D). (F) Expression of immune memory-associated markers on transduced T cells by flow cytometry. For (C), (D), (E), and (F), N = 3 biological replicates per group. Isotype controls were used to determine gating and displayed data are gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Data presented as mean (SD). A one-way ANOVA followed by a post-hoc Šídák’s multiple comparisons test was performed. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: Construct, Transduction, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vitro assessment of the CD70-directed CAR-T cell anti-GB cytotoxicity (A) Measurement of secreted TNF-α and IFN-γ in the SN of GB/CAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. For comparison between MCS and CD70 (upper bar plots), an unpaired two-tailed t test was used. For comparisons among constructs (bottom), a one-way ANOVA followed by a Holm-Šídák multiple comparisons test was used. (B) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. A two-tailed Student’s t test was performed using the values of the last measured time point to determine statistical significance. For (A) and (B), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test, Construct, Co-Culture Assay

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Assessment of anti-CD70 CAR-T cell killing in naturally CD70 expressing cell lines (A) CD70 expression levels (blue histogram) on primary GB cell line MMKI by flow cytometry. Data gated on single live cells. An isotype control (red histogram) was used. (B) Quantification of activation marker CD137 after O/N co-culture of MMKI cells with CD70-targeting CAR-T cells by flow cytometry. N = 4 biological replicates per group. Data gated on single live CD3 + cells (NT) or single live CD3 + /tdTomato + cells (SFG, CD27z, LF28z, and LFBBz). Isotype control antibodies were used for gating. A one-way ANOVA followed by a Tukey’s multiple comparisons test was applied for significance. (C) Measurement of CD70 (blue histograms) on the surface of generated genetic knockout models by flow cytometry. An isotype control (red histogram) was used. Data gated on single live EGFP + cells. (D) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. Statistical significance was assessed as in B). For (B) and (D), data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: Expressing, Flow Cytometry, Control, Activation Assay, Marker, Co-Culture Assay, Generated, Knock-Out

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Evaluation of CD70-directed CAR-T cell effector function in cerebral organoids (A) Confocal microscopy of cerebral organoids, infiltrated by generated GB models. (B) Quantification of CD70 signal in organoids from (A). Each dot represents an organoid. A Welch’s t test was used to assess significance. (C) Immunofluorescence analysis of endogenous CD70 expression in cerebral organoids. (D) Confocal microscopy of cerebral organoids previously invaded by GB cells and subsequently treated with CAR-T cells for 3 d. (E) Quantification of Granzyme-B signal from (D). A two-tailed t test was used to determine significance. (F) Measurement of secreted Granzyme-B and IFN-γ levels in the SN of co-cultures from (D) by ELISA. N = 3 biological replicates per group. (G) CAR construct direct comparisons from (F). A one-way ANOVA followed by a Tukey’s post hoc test for multiple comparisons was used. For (A), (C), and (D), scale bars, 200 μm. For (D) and (E), N ≥ 3 organoids per group. For (E) and (F), a two-tailed t test was used to assess significance. For (B), (E), and (F), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: Confocal Microscopy, Generated, Immunofluorescence, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Construct

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vivo evaluation of the anti-CD70 CAR-T cell killing efficacy (A) In vivo pipeline. (B) Measurement of tumor cell signal in mice orthotopically implanted with P3/CD70_NLuc/EGFP cells and subsequently treated ICT with anti-CD70 CAR-T cells by BLI. (C) Quantification of BLI signals from B). N = 8 animals per group. (D) Overall survival of treated mice from B). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0083. (E) Analysis of CAR-T cell persistence and CD70 expression in recurrent tumors of treated mice from (A) by IF. Representative images from N = 3 animals per group. Scale bars, 100 μm.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: In Vivo, Expressing

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based anti-murine CD70 CAR-T cells are potent against murine GB in vitro (A) Murine CD27-based construct design. (B) Transduction efficiency of primary murine T cells by flow cytometry. A non-transduced (NT) sample from each donor mouse was used to determine gating. Data gated on single live mCD3 + cells. (C) Measurement of mCD70 gene expression levels in generated OE models by RT-qPCR. N = 3 technical replicates per cell line. (D) Measurement of mCD70 on the surface of generated murine OE GB models by flow cytometry (blue histograms). Signal was compared to that of an isotype control (red histograms). (E) Quantification of secreted TNF-α in the SN of mGB/mCAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. (F) Pairwise comparisons of secreted TNF-α levels from (E) among targeting constructs. A one-way ANOVA with a post hoc Holm-Šídák test was used for significance. (G) Schematic representation of the live-cell imaging pipeline. (H) Quantification of tumor cell signal from (G) over time. A one-way ANOVA with a Dunnett’s multiple comparisons test was used with data from the t = 660 min mark. For (C) and (E), an unpaired two-tailed t test was used for significance. Data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: In Vitro, Construct, Transduction, Flow Cytometry, Gene Expression, Generated, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based CAR constructs lead to regression of syngeneic GB tumors in vivo (A) Schematic representation of the syngeneic model in vivo experiment. (B) Evaluation of mCAR expression on transduced murine T cells on treatment day by flow cytometry. A non-transduced sample was used to determine gating. Data gated on single live mCD3 + cells. (C) Weekly tumor volume evaluation in C57BL/6J mice orthotopically implanted with GL261/CD70 cells and subsequently treated ICT with anti-mCD70 CAR-T cells by MRI. A one-way ANOVA with a Dunnett’s multiple comparisons test was used to assess significance. (D) Indicative T2-weighed MRI sections (coronal plane) from N = 3 mice of each treatment group from (C), before and after treatment with anti-mCD70 CAR-T cells. (E) Comparison of tumor volumes among treatment groups from (C). For day-14, a one-way ANOVA followed by a Tukey’s multiple comparisons test was used. For day-21, a Welch’s ANOVA followed by a Dunnett’s T3 multiple comparisons test was used. For day-28, a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test was used. Data presented as mean (SD). (F) Overall survival of treated mice from (C). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0166. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: For cloning of the lentiviral human and murine CD70 OE construct, the pLEX306 was a gift from David Root (Addgene plasmid #41391; http://n2t.net/addgene:41391 ; RRID:Addgene_41391).

Techniques: Construct, In Vivo, Expressing, Flow Cytometry, Comparison