Journal: Journal of Translational Medicine

Article Title: Multi-omic profiling and preclinical efficacy of fratricide-driven, unedited CD7 CAR-T cells in T-cell leukemia

doi: 10.1186/s12967-026-07701-5

Figure Lengend Snippet: Culture conditions for optimized natural-selection-based manufacturing of UMCG-001. ( A ) Third-generation K12 CAR construct and lentiviral expression vector diagram. ( B ) Timeline of UMCG-001 manufacturing. By day 4, cells were cultured under four conditions (IL-7/IL-15, IL-2 with/without human platelet lysate (HPL) supplementation). ( C ) Flow cytometry-based detection of K12 CAR surface expression via biotinylated rhCD7 protein binding followed by Streptavidin-AF647. ( D ) Flow cytometry-based quantification of transduction efficiency as %GFP + cells. Mean ± SD, n = 4. ( E ) Flow cytometry-based quantification (MFI) of CD7 surface expression. IL-7/IL-15/HPL culture condition. Mean ± SD, n = 4. Paired-t Test performed at each time point. ( F ) Flow cytometry diagram illustrating final cell compositions of UMCG-001 and CD19 CAR DPs regarding CD7 and CAR (GFP + ) expression on day 12 post-transduction. ( G ) Cumulative expansion over 12-day culture across different conditions. Mean ± SD, n ≥ 4. ( H ) Overall expansion on day 12 post-transduction across different conditions. Kruskal-Wallis test ( p < 0.001), post-hoc Dunn test significance is shown. ( I ) Cumulative expansion of UMCG-001 over 12 days culture across different conditions. Two-way ANOVA, with culture media factor significance ( p = 0.0011), post-hoc Tukey-Kramer test significance shown. ( J ) Cumulative expansion over 12 days culture on IL-7/IL-15/HPL. Mean ± SD, n ≥ 3. Paired-t Test performed at each time point with p < 0.05 significant. ( K ) Cumulative expansion of UMCG-001 over 12 days culture on TexMACS IL-7/IL-15 supplemented with different HPL concentrations. Paired-t Test performed at each time point. ( L ) Schematic representation of flow cytometry-based viability assessment. Data on day 10 and IL-7/IL-15 with and without HPL. ( M ) Percentage of viable cells evaluated as in ( L ) across different conditions. Mean ± SD, n ≥ 4. ( N ) Percentage of viable cells evaluated as in ( L ) for UMCG-001 across different conditions. Two-way ANOVA, with culture media factor significance ( p < 0.0011) and post-hoc Tukey-Kramer test significance shown. Significance levels indicated as follows: ns = Not significant, * = p < 0.05; ** = p < 0.01; *** = p < 0.001, ****= p < 0.0001

Article Snippet: Samples were acquired on an Aurora spectral flow cytometer (Cytek Biosciences, Fremont, CA, USA) and data were analyzed using OMIQ flow cytometry software (Dotmatics, Boston, MA, USA).

Techniques: Selection, Construct, Expressing, Plasmid Preparation, Cell Culture, Flow Cytometry, Protein Binding, Transduction

Journal: Journal of Translational Medicine

Article Title: Multi-omic profiling and preclinical efficacy of fratricide-driven, unedited CD7 CAR-T cells in T-cell leukemia

doi: 10.1186/s12967-026-07701-5

Figure Lengend Snippet: Tech transfer of UMCG-001 manufacturing to GMP-facility. ( A ) Overview for three UMCG-001 batches produced in a CliniMACS Prodigy ® bioreactor. Batch 1 and 3 were manufactured for 6 days, with cell harvest on day 6. Culture of batch 2 was expanded to day 12. For all, cell selection and activation were performed on day 0, LV transduction on day 1, HPL supplementation on day 4 and quality and characterization tests on harvesting day. Parallel lab batches were generated with matched-donor material for batch 2 and 3. ( B ) Flow cytometry-based quantification of transduction efficiency as %GFP + cells. ( C ) UMCG-001 expansion in million cells over time. Dotted line: DP release specification. ( D ) Fold-change expansion exhibited by UMCG-001 batch 3 generated at the GMP facility or Lab. ( E ) Fold-change expansion exhibited by UMCG-001 batch 2 generated at the GMP facility or Lab. ( F ) Flow cytometry scatterplots illustrating cell viability on harvesting day assessed by LIVE/DEAD TM staining within T-cell gate. ( G ) Histogram representation of CD7 expression on manufacturing day 6 among the different batches and CD19 CAR-T cells as reference. ( H ) Flow cytometry scatterplots representing CD7 vs. K12 CAR (GFP + ) expression across the batches on day 6. ( I ) Killing capacity across UMCG-001 batches of CD7 + and CD7 Neg cell lines at different E: T ratios for 24 h as AnnexinV/PI flow cytometry-based quantification. ( J ) Specific OV-CAR-3.Luc.CD7 killing calculated as indicated in Formula 1 by GMP batch 1 up to 5 rounds of 24 h-tumor re-challenges at different E: T ratios or ( K ) Percentage of viable cells at selected 2:1, 1:1 and 1:4 E: T. ( L ) IFN-γ secretion by the GMP batches when co-cultured with non-loaded (NL), CD7-loaded (CD7L), CD19-loaded (CD19L) or no beads for 24 h on day 6. Delta IFN-γ secretion in respect to NL beads. ( M ) IFN-γ secretion by UMCG-001 batches vs. CD19 CAR-T cells batches or ( N ) matched GMP and lab batch 3 as in ( L )

Article Snippet: Samples were acquired on an Aurora spectral flow cytometer (Cytek Biosciences, Fremont, CA, USA) and data were analyzed using OMIQ flow cytometry software (Dotmatics, Boston, MA, USA).

Techniques: Produced, Selection, Activation Assay, Transduction, Generated, Flow Cytometry, Staining, Expressing, Cell Culture

Journal: Journal of Translational Medicine

Article Title: Multi-omic profiling and preclinical efficacy of fratricide-driven, unedited CD7 CAR-T cells in T-cell leukemia

doi: 10.1186/s12967-026-07701-5

Figure Lengend Snippet: Impact of HPL-supplementation on cytotoxicity and phenotype of UMCG-001. ( A ) UMCG-001 killing of MOLT-4 and K-562 cell lines at different E: T ratios for 24 h as quantified by flow cytometry with AnnexinV/PI. UMCG-001 expanded in TexMACS Il-7/IL-15 without (solid line) or with HPL (dashed line). Mean ± SD, n = 3 ( B ) Percentage of UMCG-001 and CD19 CAR + cells expressing CD4, PD-1 and CD62L/CD45RO (TCM) from paired donors at day 10 of culture. Paired T-test, with p < 0.05 significant. ( C ) Flow cytometry scatterplot illustrating the difference in CD62L/CD45RO expression between UMCG-001 and CD19 CAR-T cells on day 10 from paired donors. ( D ) Percentage of CAR-T cells with a naïve (N) (CD45RO-/CD62L + ), central memory (CM) (CD45RO + /CD62L + ), effector memory (EM) (CD45RO + /CD62L − ) and terminally effector (EMRA) (CD45RO-/CD62L−) phenotype across samples cultured in TexMACS/IL-7/-15 with (+) or without (-) HPL. Mean ± SD, n > 3. ( E ) Percentage of TCM (CD45RO + /CD62L + ) cells within UMCG-001 on day 5 vs. day 10 of culture across different expansion protocols. ( F ) Concentration of soluble CD62L measured in culture media supernatants of CD19 and UMCG-001 products. Paired T-test, with p < 0.05 significant ( G ) CD62L mRNA expression quantified by qPCR in UMCG-001 and normalized to CD19 CAR T cells. Mean ± SD, n = 3. Significance levels indicated as follows: ns = Not significant, * = p < 0.05; ** = p < 0.01; *** = p < 0.001, ****= p < 0.0001

Article Snippet: Samples were acquired on an Aurora spectral flow cytometer (Cytek Biosciences, Fremont, CA, USA) and data were analyzed using OMIQ flow cytometry software (Dotmatics, Boston, MA, USA).

Techniques: Flow Cytometry, Expressing, Cell Culture, Concentration Assay

Journal: Journal of Translational Medicine

Article Title: Multi-omic profiling and preclinical efficacy of fratricide-driven, unedited CD7 CAR-T cells in T-cell leukemia

doi: 10.1186/s12967-026-07701-5

Figure Lengend Snippet: Multi-omic profiling of UMCG-001. ( A ) Flow cytometry-based quantification (MFI) of CD62L expression. Mean ± SD, n = 3 to 6. ( B ) UMAP plot of GMP UMCG-001 batches and CD19 CAR-T cells. Cluster representation per sample and ( C ) for CD4 + /CD8 + T cells. ( D ) Batch-to-batch comparison of CD4:CD8 T cell ratio ( E ) Batch-to-batch comparison of T cell maturity composition. (TN CD45RA + /CD62L + ), Central Memory (TCM CD45RA - / CD62L + ), Effector Memory (TEM) (CD45RA - /CD62L - ) and Terminally differentiated Effector Memory (TEMRA) (CD45RA + /CD62L - ). ( F ) Heatmap-overlay of expression levels for markers of interest from ( B ). Individual scaling for each marker is based on the fluorescence intensity of each fluorochrome. Batch-to-batch comparison of marker expressions associated with ( G ) Exhaustion and ( H ) Activation. Gating performed on live CD3 + CD45 + CAR + cells. ( I ) Schematic diagram for single-cell data acquisition and analysis. ( J ) UMAP colored by sample subsets present in the Seurat object. ( K ) Relative abundance of the different clonotype groups based on the frequency of clonotype. ( L ) Number of unique TCR clones across batches. ( M ) Scaled-Venn plot representation of overlapping genes from differential expression gene (DEG) analysis between CD19 CAR and UMCG-001 batches respectively (Table ). ( N ) Heatmap representation of the top 20 differentially expressed genes across samples. Yellow indicates high, purple indicates low expression of a particular gene (Table ). ( O ) Dot-plot of cycling score genes. ( P ) Heatmap representation showing the expression of several public available exhaustion, activation/proliferation, differentiation and cytotoxicity signatures across the UMCG-001 batches and CD19 CAR as reference group. The Ucell method was used to score each signature (Table ). ( Q ) Dot-plot of selected genes of relevance for CAR-T cell therapy manufacturing and function

Article Snippet: Samples were acquired on an Aurora spectral flow cytometer (Cytek Biosciences, Fremont, CA, USA) and data were analyzed using OMIQ flow cytometry software (Dotmatics, Boston, MA, USA).

Techniques: Flow Cytometry, Expressing, Comparison, Marker, Fluorescence, Activation Assay, Single Cell, Clone Assay, Quantitative Proteomics

Journal: Journal of Translational Medicine

Article Title: Multi-omic profiling and preclinical efficacy of fratricide-driven, unedited CD7 CAR-T cells in T-cell leukemia

doi: 10.1186/s12967-026-07701-5

Figure Lengend Snippet: In vivo efficacy of UMCG-001 in a T-ALL xenograft mouse model. ( A ) Experimental timeline detailing tumor engraftment, CAR T-cell infusion, and imaging/analysis time points. ( B ) Bioluminescence imaging (BLI) monitoring of tumor burden at different time points across the different groups. ( C ) Jurkat presence in bleeds samples on day 25 measured as %CD7 + cells within the hCD45 + gate. ( D ) Quantification of the total radiance (photons/s) from the images shown in ( B ). Mean ± SD Vehicle ( n = 4), CD19 CAR ( n = 4), UMCG-001 ( n = 11). ( E ) Kaplan-Meier survival curves for grouped mice. Log-rank (Mantel-Cox) test ( p < 0.0001). ( F ) Individual quantification of the total radiance (photons/s) from the images shown in ( B ). ( G ) Quantification of Jurkat engraftment, measured as %hCD45 + CD7 + within total lymphocyte (FSC/SSC) gate in bone marrow. One-way ANOVA ( p = 0.003) and post-hoc Tukey-Kramer Test. ( H ) Flow-cytometry based quantification of K12 CAR + (GFP + ) cells within the hCD45 + gate and further classified as ( I ) > 99% CAR + CD3 + CD7 Neg cells across different organs, exemplified for squared mice in ( H ). ( J ) Expansion of CAR + cells in two representative mice when Jurkat was injected (+ T-ALL) or not (-T-ALL) before UMCG-001 treatment. Significance levels were indicated as follows: ns = Not significant, * = p < 0.05; ** = p < 0.01; *** = p < 0.001, ****= p < 0.0001

Article Snippet: Samples were acquired on an Aurora spectral flow cytometer (Cytek Biosciences, Fremont, CA, USA) and data were analyzed using OMIQ flow cytometry software (Dotmatics, Boston, MA, USA).

Techniques: In Vivo, Imaging, Flow Cytometry, Injection

Journal: Journal of Translational Medicine

Article Title: Multi-omic profiling and preclinical efficacy of fratricide-driven, unedited CD7 CAR-T cells in T-cell leukemia

doi: 10.1186/s12967-026-07701-5

Figure Lengend Snippet: Autologous manufacturing and malignant contamination assessment in UMCG-001. ( A ) UMCG-001 manufactured from T-ALL patient 1 (T-ALL_1) derived material. ( B ) IFN-γ secretion upon 72 h co-culture of UMCG-001 batches with CD7 + (Jurkat, MOLT-4), autologous T-ALL and CD7 Neg K-562 cells. ( C ) Flow cytometry-based assessment of the percentage of killing of CD7 + (Jurkat, MOLT-4), autologous T-ALL and CD7 Neg K-562cells by UMCG-001. ( D ) Flow cytometry dot blot illustrating CD7 and surface CD3 (sCD3) expression in UMCG-001 and CD19 CAR-T cells upon fratricide. ( E ) Percentage of surface CD7 and CD3 over time in T.ALL_1 UMCG-001 final DP. Day − 1 represents the day before activation, day 0 after activation and transduction, and day 16 end of the fratricidal phase. ( F ) Schematic representation of sample preparation for subsequent single-cell RNA sequencing. T-ALL_1 original sample was composed of healthy (sCD3 + ) and blasts (sCD3 - ), which were sorted based on sCD3 expression. The whole sample was subjected to CAR - T manufacturing, after which CAR + cells were sorted based on GFP expression. All sorted samples were processed for V( D )J and gene expression (GEX) single-cell libraries. ( G ) Violin plot representation of genes related to malignant phenotype across TALL_1 sample. ( H ) Relative abundance of TCR clones across CD19 CAR-T cells UMCG-001 and sCD3 + samples, calculated as the number of unique TCRs per total number of cells. ( I ) Clone sizes across CD19 CAR-T cells and UMCG-001 batches generated from independent healthy donors (gray) and T-ALL_1 (black). The number of cells per clone group is shown

Article Snippet: Samples were acquired on an Aurora spectral flow cytometer (Cytek Biosciences, Fremont, CA, USA) and data were analyzed using OMIQ flow cytometry software (Dotmatics, Boston, MA, USA).

Techniques: Derivative Assay, Co-Culture Assay, Flow Cytometry, Dot Blot, Expressing, Activation Assay, Transduction, Sample Prep, Single Cell, RNA Sequencing, Gene Expression, Clone Assay, Generated