Journal: Frontiers in Immunology

Article Title: Development and in vivo evaluation of novel humanized CD19 CAR-T cells for advanced B cell malignancies

doi: 10.3389/fimmu.2026.1798748

Figure Lengend Snippet: Design and characterization of humanized and harmonized anti-CD19 scFvs. (A) Sequence identity analysis comparing the FMC63 VH domain against the two most closely related human VH germlines (H1 and H2) utilized for humanization and harmonization. Schematic representation of the H1 (harmonized) and H2 (humanized) scFv constructs in a VL-linker-VH orientation. Both constructs utilize an identical humanized VL domain . (B) In silico prediction of aggregation propensity for FMC63, humanized and harmonized versions of H1 and humanized H2. (C) Prediction of HLA epitopes within scFvs sequences. Bar plots represent the number of predicted HLA-presented peptides for each unique or shared set among FMC63, H1, and H2, as indicated by the intersection matrix below. Filled dots denote the groups included in each intersection (D) Equilibrium dissociation constant (K D ) of soluble FvFc fusion proteins (scFvs fused to a human IgG1 Fc region) as measured by biolayer interferometry (BLI). (E) Flow cytometric analysis of CD19+ Raji cells stained with the soluble FvFc molecules comparing antigen-binding properties.

Article Snippet: Molecular dynamics simulations were performed using homology models of FMC63 scFv (scFv FMC63 ) and its humanized variants (H1 and H2) named scFv H1 and scFv H2 , constructed based on the FMC63–CD19 crystal structure (PDB ID: 7URV).

Techniques: Sequencing, Construct, In Silico, Staining, Binding Assay

Journal: Frontiers in Immunology

Article Title: Development and in vivo evaluation of novel humanized CD19 CAR-T cells for advanced B cell malignancies

doi: 10.3389/fimmu.2026.1798748

Figure Lengend Snippet: New anti-CD19 CAR-T cells maintain sustained cytotoxic activity during prolonged in vitro challenge. (A) Kinetic killing assays using GFP + Nalm-6 target cells expressing either wild-type CD19 [CD19 WT , (B) or reduced CD19 levels (CD19 Low , (C) ]. The count of residual GFP + cells was quantified every 24 hours throughout a 96-hour co-culture period. Effector-to-target (E:T) ratios were assessed at 1:1, 0.5:1, 0.25:1, and 0.1:1. CAR+ cells (FMC63- black, H1- red and H2- blue). killing activity was compared to that exhibited by Mock-transduced negative control (grey). Cumulative cytotoxicity was compared using Area Under the Curve (AUC) analysis derived from the 96-hour killing kinetics. Assays were performed in triplicate and data are presented as mean ± SD. Statistical significance was determined via Two-Way ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Molecular dynamics simulations were performed using homology models of FMC63 scFv (scFv FMC63 ) and its humanized variants (H1 and H2) named scFv H1 and scFv H2 , constructed based on the FMC63–CD19 crystal structure (PDB ID: 7URV).

Techniques: Activity Assay, In Vitro, Expressing, Co-Culture Assay, Negative Control, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Development and in vivo evaluation of novel humanized CD19 CAR-T cells for advanced B cell malignancies

doi: 10.3389/fimmu.2026.1798748

Figure Lengend Snippet: Comparative in vivo efficacy of CAR-T variants in a standard tumor burden xenograft model. (A) Schematic representation of the xenograft model used to evaluate CAR T-cell activity in vivo . NSG mice were inoculated with 1 × 10 5 Nalm-6 CD19 WT cells. Two days after tumor inoculation, mice received a single intravenous dose of CAR-T cells (7 × 10 5 ). Tumor burden was monitored by bioluminescence imaging using the IVIS Spectrum in vivo imaging system every 7 days. Mice were euthanized according to predefined clinical criteria, and survival was recorded. (B) Kaplan-Meier survival curves and statistical analysis of the advanced disease model. Experimental groups are indicated: Control Tumor Only (untreated mice, PBS-inoculated; brown) and CAR-T cells treated- FMC63 CAR-T (black), H1 CAR-T (red), and H2 CAR-T (blue). (C) Longitudinal assessment of tumor burden quantified by bioluminescence intensity (Total Flux, photons/second). (D) Kinetics of tumor progression and regression monitored by representative bioluminescence imaging across all experimental cohorts. End points were determined by ethical criteria for euthanasia.

Article Snippet: Molecular dynamics simulations were performed using homology models of FMC63 scFv (scFv FMC63 ) and its humanized variants (H1 and H2) named scFv H1 and scFv H2 , constructed based on the FMC63–CD19 crystal structure (PDB ID: 7URV).

Techniques: In Vivo, Activity Assay, Imaging, In Vivo Imaging, Control

Journal: Frontiers in Immunology

Article Title: Development and in vivo evaluation of novel humanized CD19 CAR-T cells for advanced B cell malignancies

doi: 10.3389/fimmu.2026.1798748

Figure Lengend Snippet: Antitumor activity of engineered CAR-T cells against established, advanced-stage tumors. (A) Schematic of the xenograft model used to evaluate CAR T-cell activity in vivo . NSG mice were inoculated with 1 × 10 5 Nalm-6 CD19 Low cells. Eleven days after tumor inoculation, mice received a single intravenous dose of CAR T cells (1 × 10 6 ). Tumor burden was monitored by bioluminescence imaging using the IVIS Spectrum in vivo imaging system every 7 days. Mice were euthanized according to predefined clinical criteria, and survival was recorded. (B) Kaplan-Meier survival curves and statistical analysis of the advanced disease model. Experimental groups are indicated as follows: vehicle control (brown), Mock (electroporated, non-transfected cells; gray), FMC63 CAR-T (black), H1 CAR-T (red), and H2 CAR-T (blue). (C) Longitudinal assessment of tumor burden and anatomical localization, quantified by bioluminescence intensity (Total Flux, photons/second). (D) Kinetics of tumor progression and regression monitored by representative bioluminescence imaging across all experimental cohorts.

Article Snippet: Molecular dynamics simulations were performed using homology models of FMC63 scFv (scFv FMC63 ) and its humanized variants (H1 and H2) named scFv H1 and scFv H2 , constructed based on the FMC63–CD19 crystal structure (PDB ID: 7URV).

Techniques: Activity Assay, In Vivo, Imaging, In Vivo Imaging, Control, Transfection

Journal: Nature Communications

Article Title: Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor

doi: 10.1038/s41467-026-72846-2

Figure Lengend Snippet: A Polyfunctional editing strategy for the epi-silencing of B2M and TET2 and insertion of the CD19–28ζ CAR:ΔLNGFR construct into TRAC . B Left: percentages of the indicated T cell populations at day 14 after poly-editing (mean ± SD of 7 blood donors). TCR + : TCR-positive cells (green bar). TCR - : TCR-negative cells (light blue bar). TCR - /ΔLNGFR + : cells negative for endogenous TCRs and positive for ΔLNGFR (lilac bar). B2M - : B2M-negative cells (gray bar). B2M + : B2M-positive cells (white bar). Right: representative flow cytometry dot plots of poly-edited T cells showing expression of the TCRs and ΔLNGFR (left plot) and, within the TCR - /ΔLNGFR + cells, of B2M (right plot). C Fold-change in TET2 (light blue bars) and B2M (gray bars) expression in poly-edited vs . mock-treated cells (mean ± SD of 7 blood donors). D Schematic of the in vivo experiment. NSG mice were injected with GLuc.NALM-6 cells and, 7 days later, with the indicated T cell populations. Peripheral blood was collected at the indicated time points to measure tumor growth, phenotype human T cells, and quantify cytokine release. Mock: mock-transfected T cells. CAR-T Δ LNGFR : T cells expressing CD19–28ζ CAR:ΔLNGFR from TRAC . Poly-edited cells: T cells expressing CD19–28ζ CAR:ΔLNGFR from TRAC with epi-silencing of B2M and TET2 . E Tumor growth (left; mean ± SEM) and survival (right) curves over 20 days post-transplantation of the indicated T cell populations. n = 6 mice for Mock; n = 12 mice for each other group. ** p = 0.0075, *** p = 0.0008 by Mantel-Cox (log-rank) test. F Circos plots from CAST-Seq analyses of triple KO (left) and poly-edited (right) T cells ( n = 2 experimental replicates). Triple-KO cells were transfected with mRNA encoding Cas9 and the TRAC gRNA, together with gRNA B#6 and gRNA TE#19 . For poly-editing, the TRAC gRNA and the selected guide combinations for B2M and TET2 were used. Aberrations at the TRAC locus are in violet; translocations between TRAC and either B2M or TET2 are in blue; translocations between TRAC and OT sites of all gRNAs are in gray. G Number of unique CAST-Seq reads corresponding to either aberrations (violet bars) or translocations (gray bars) at the TRAC locus in the indicated treatments. Graphs were generated using GraphPad Prism (GraphPad Software). Source data are provided as a file.

Article Snippet: LNGFR; 130-113-422, Miltenyi), CD3 (345763, BD Biosciences), HLA-ABC (565332, BD Biosciences), HLA-E (130-117-402, Miltenyi), and CD19 CAR FMC63 Idiotype (130-127-342, Miltenyi).

Techniques: Construct, Flow Cytometry, Expressing, In Vivo, Injection, Transfection, Transplantation Assay, Generated, Software

Journal: Nature Communications

Article Title: Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor

doi: 10.1038/s41467-026-72846-2

Figure Lengend Snippet: A Scatter plot comparing whole-transcriptome analyses of T cells treated with the tripartite ETR and either full-length or truncated gRNAs targeting CD3D (left) or TGFBR2 (right) ( n = 2 experimental replicates). Data are expressed as log 2 TPM of mapped reads. B Polyfunctional editing strategy used to epi-silence CD3D and TGFBR2 and insert the scHLA-E:CD19–28ζ CAR cassette into exon 1 of B2M . C Left: percentages of the indicated T cell populations as measured by flow cytometry 14 days after polyfunctional editing (mean ± SD of 7 blood donors). HLA-ABC + : HLA-ABC-positive cells (light yellow bars); HLA-ABC - : HLA-ABC-negative cells (blue bars); CD19-CAR - : cells negative for the expression of the CD19–28ζ CAR (gray bars); CD19-CAR + : cells positive for the expression of the CD19–28ζ CAR (lilac bars); CD3 + : CD3-positive cells; CD3 - : CD3-negative cells; HLA-E - : HLA-E-negative cells; HLA-E + : HLA-E-positive cells. Right: representative flow cytometry dot plots of poly-edited T cells showing the expression levels of the HLA-ABC and CD3 (left plot) and, within the double-negative cells, of HLA-E and CD19-CAR (right plot). D Fold-change in the expression levels of CD3D (green bars) and TGFBR2 (orange bars) in poly-edited cells relative to mock-treated cells, 14 days post-editing (mean ± SD of 7 blood donors). Graphs were generated using GraphPad Prism (GraphPad Software). Source data are provided as a file.

Article Snippet: LNGFR; 130-113-422, Miltenyi), CD3 (345763, BD Biosciences), HLA-ABC (565332, BD Biosciences), HLA-E (130-117-402, Miltenyi), and CD19 CAR FMC63 Idiotype (130-127-342, Miltenyi).

Techniques: Flow Cytometry, Expressing, Generated, Software

Journal: Frontiers in Oncology

Article Title: Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells

doi: 10.3389/fonc.2026.1774431

Figure Lengend Snippet: Determination of the limit of detection (LOD) and lower limit of quantification (LLOQ) for the two-step method. CAR-T cell absolute counts were measured in 31 negative control samples from patients not treated with CD19 CAR-T cells. The mean background signal is represented by the blue line. LOD was defined as mean + 3 standard deviations (SD), and LLOQ as mean + 10 SD.

Article Snippet: New CAR detection reagent (CDR) directly coupled to fluorochrome, either BCMA (BCMA CDR-PE, Miltenyi Biotec 130-133-888) or CD19 (CD19 CDR α-FMC63-PE, Miltenyi Biotec 130-127-342) was added extemporaneously in the analytical tube.

Techniques: Negative Control

Journal: Frontiers in Oncology

Article Title: Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells

doi: 10.3389/fonc.2026.1774431

Figure Lengend Snippet: Gating strategy for the single-step method. Absolute counting beads were excluded based on scatter and fluorescence properties. Dead cells were excluded using 7-aminoactinomycin D (7-AAD). CD45-positive leukocytes were selected, and lymphocytes were identified according to side scatter (SSC) characteristics. CD3-positive T cells were gated, and CAR-T cells were defined as viable CD45+/CD3+/CAR+ events using directly fluorochrome-conjugated CAR detection reagents (CD19 or BCMA). CD4 and CD8 subpopulations were subsequently identified within the CAR-positive T-cell compartment. Absolute quantification was calculated using TruCount beads according to the manufacturer’s formula.

Article Snippet: New CAR detection reagent (CDR) directly coupled to fluorochrome, either BCMA (BCMA CDR-PE, Miltenyi Biotec 130-133-888) or CD19 (CD19 CDR α-FMC63-PE, Miltenyi Biotec 130-127-342) was added extemporaneously in the analytical tube.

Techniques: Fluorescence, Quantitative Proteomics

Journal: Frontiers in Oncology

Article Title: Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells

doi: 10.3389/fonc.2026.1774431

Figure Lengend Snippet: Determination of LOD and LLOQ for the single-step method. CAR-T cell absolute counts were measured in 10 negative control samples from patients not treated with CD19 or BCMA CAR-T cells. The blue line represents the mean background signal. LOD was defined as mean + 3 SD and LLOQ (green dashed line) as mean + 10 SD.

Article Snippet: New CAR detection reagent (CDR) directly coupled to fluorochrome, either BCMA (BCMA CDR-PE, Miltenyi Biotec 130-133-888) or CD19 (CD19 CDR α-FMC63-PE, Miltenyi Biotec 130-127-342) was added extemporaneously in the analytical tube.

Techniques: Negative Control