cd19 biotin  (Miltenyi Biotec)

Journal: Biomolecules

Article Title: Introducing CAR-T Therapy in Kazakhstan: Establishing Academic-Scale Lentiviral Vector and CAR-T Cell Production

doi: 10.3390/biom15081166

Figure Lengend Snippet: Workflow for lentiviral vector (LV) production and results with the control vector LV/CAR-GFP. ( a ) Schematic of the vector production process. Packaging cells were expanded in progressively larger culture vessels (100 mm dishes, T-175 flasks, 5-layer stacks) before calcium phosphate transfection with the LV packaging system (transfer vector, Gag/Pol helper, and VSV-G envelope plasmid). Conditioned medium was harvested 48 h post-transfection, clarified, and filtered. Viral particles from ~600 mL supernatant underwent concentration through tangential flow filtration (TFF), followed by ultracentrifugation, yielding 1 mL of high-titer preparation. These procedures provided a sufficient viral stock for multiple CAR-T cell manufacturing runs. ( b ) Comparison of the transgene in the control vector LV/CAR-GFP and the experimental vector NCB.LV.CD19-CAR. In LV/CAR-GFP, the CAR gene is fused to GFP via an uncleavable linker. In similar experiments, amounts of GFP-fluorescent cells served as an indicator of transfection efficiency. ( c – e ) Results of preliminary experiments with the CAR-GFP-expressing vector. The photographs on panels ( c ) and ( d ) show typical results from transfection for LV packaging. Calcium phosphate precipitation method allows the efficient transfection of high-density cultures (100,000 cells/cm 2 ). The photographs were taken at 48 h post-transfection. Magnification = 50×. Scale bar = 100 µm. ( e ) Determining the optimal acceleration for centrifugal concentration of LV particles. The average functional titers are shown as means ± SDs, expressed as a percentage of the maximum titer across all experiments. The titer obtained at 20,000× g was set to 100%.

Article Snippet: For CAR detection, cells were stained with 2 μL of biotinylated CD19 CAR Detection Reagent (Miltenyi Biotec 130-129-550) per 1 × 10 6 cells.

Techniques: Plasmid Preparation, Control, Transfection, Concentration Assay, Filtration, Comparison, Expressing, Functional Assay

Journal: Biomolecules

Article Title: Introducing CAR-T Therapy in Kazakhstan: Establishing Academic-Scale Lentiviral Vector and CAR-T Cell Production

doi: 10.3390/biom15081166

Figure Lengend Snippet: Functional characterization of CAR-T cells via cytokine secretion and cytotoxicity assays. ( a ) Key Th1 cytokine release profile. CAR + T-cells were cocultured with CD19 + targets (B cells) (Exp) and compared to controls: non-transduced T-cells (NT) cultured with targets, and CAR + T-cells without targets (baseline, BL). Coculture of CAR + effectors with targets induced significant production of IFN-γ, TNF-α, and IL-2, indicating antigen-specific activation. Data shown as means ± SDs (V1: n = 6; V2: n = 6); ( b ) cytotoxic activity of CAR + T-cells was assessed by measuring lysis of labeled target cells at effector-to-target (E:T) ratios of 10:1 and 5:1. Control cocultures contained non-transduced (NT) T-cells with labeled targets. Both V1- and V2-derived CAR + cells exhibited dose-dependent target cell lysis (increasing with higher E:T ratios). Each data point represents the mean of triplicate measurements per sample; ( c ) violin plots with connected medians show a trend toward enhanced cytotoxicity in V2- vs. V1-derived CAR + T-cells; ns = not significant, * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: For CAR detection, cells were stained with 2 μL of biotinylated CD19 CAR Detection Reagent (Miltenyi Biotec 130-129-550) per 1 × 10 6 cells.

Techniques: Functional Assay, Cell Culture, Activation Assay, Activity Assay, Lysis, Labeling, Control, Derivative Assay

Journal: eBioMedicine

Article Title: Tandem CD19/CD22 CAR T-cells as potential therapy for children and young adults with high-risk r/r B-ALL

doi: 10.1016/j.ebiom.2025.105872

Figure Lengend Snippet: Tandem anti-CD19/CD22 CAR T-cell therapy response. A) Schematic diagram of the tandem anti-CD19/CD22 CAR structure. B) Flow chart of the study. C) Swimmer plot showing clinical responses after tandem anti-CD19/CD22 CAR T-cell product infusion. D) PET-TC imaging of patient P9 before (A.1 and A.2) and 28 days after (B.1 and B.2) tandem anti-CD19/CD22 CAR T-cell infusion. E) Event free survival (EFS) Kaplan–Meier curve in all patients (n = 10). F) Overall survival (OS) Kaplan–Meier curve in all patients (n = 10). For E-F, black dots on the curve represent censored observations. HSCT, haematopoietic stem cell transplantation. MRD, minimal residual disease. EMR, extramedullary relapse. PD, progression of disease. ∗ For P6, 6 reinfusions were performed (every two weeks), the last with 3 doses. ∗∗P4 joined a clinical trial with carfilzomib after relapse, with PD shortly after.

Article Snippet: The CD19 (130-129-550) and CD22 (130-126-727) CAR detection reagents, anti-CD19-Viogreen (130-113-649), anti-CD3-Viogreen (130-113-142), and anti-biotin-PE (130-110-951) were from Miltenyi Biotec.

Techniques: Imaging, Transplantation Assay

Journal: eBioMedicine

Article Title: Tandem CD19/CD22 CAR T-cells as potential therapy for children and young adults with high-risk r/r B-ALL

doi: 10.1016/j.ebiom.2025.105872

Figure Lengend Snippet: Tandem anti-CD19/CD22 CAR T-cell product immunophenotype and analysis. A) Tandem anti-CD19/CD22 CAR T-cell expansion in all products manufactured in the CliniMACS Prodigy closed system. B) CD19-CAR and CD22-CAR expression in all products manufactured by flow cytometry. C) CD4 + and CD8 + cell populations in all 10 products manufactured. D) PD-1 receptor expression on product cells. In C and D, graphs show box and whisker plots (vertical bars, min to max points; box, first to third quartile, with median as horizontal bar). E) Memory subpopulations determined by flow cytometry. Central memory cells (CD45RA − CD27 + ), effector memory cells (CD45RA − CD27 − ), naïve cells (CD45RA + CD27 + ) and TEMRA cells (CD45RA + CD27 − ) are represented. F) Specific-lysis by tandem anti-CD19/CD22 CAR T-cells against SEM cell line determined by 4-h Europium-BATDA assay at different E:T ratios. G) Degranulation assay against SEM cell line determined by CD107a expression after 4 h of co-culture at 1:1 or 1:2 E:T ratios (see Methods). A–D and F and G , in green, living patients; in purple, relapsed patient; in black, patients who died.

Article Snippet: The CD19 (130-129-550) and CD22 (130-126-727) CAR detection reagents, anti-CD19-Viogreen (130-113-649), anti-CD3-Viogreen (130-113-142), and anti-biotin-PE (130-110-951) were from Miltenyi Biotec.

Techniques: Expressing, Flow Cytometry, Whisker Assay, Lysis, Degranulation Assay, Co-Culture Assay

Journal: eBioMedicine

Article Title: Tandem CD19/CD22 CAR T-cells as potential therapy for children and young adults with high-risk r/r B-ALL

doi: 10.1016/j.ebiom.2025.105872

Figure Lengend Snippet: Tandem anti-CD19/CD22 CAR T-cell persistence in patients after product infusion. A) Tandem anti-CD19/CD22 CAR expression was determined with anti-CD19 CAR gated on CD3 + cells. Left panel, absolute numbers of peripheral blood CAR T-cells per μl in infused patients detected by flow cytometry. Right panel, percentage of CAR + cells within the T cell compartment. B) Copies per ml as detected using real-time qPCR (see Methods) (16). C) IL-6 levels from serum after CAR T-cell infusion. D) Peak IL-6 levels were upregulated in patients with ICANS or severe CRS. Box and whisker plot shows all points (vertical bars, min to max points; box, first to third quartile, with median as horizontal bar). # No CRS group included patients with mild phenotype (I-II grade); CRS group included patients with III-IV grade. In green, living patients; in purple, relapsed patient; in black, patients who died.

Article Snippet: The CD19 (130-129-550) and CD22 (130-126-727) CAR detection reagents, anti-CD19-Viogreen (130-113-649), anti-CD3-Viogreen (130-113-142), and anti-biotin-PE (130-110-951) were from Miltenyi Biotec.

Techniques: Expressing, Flow Cytometry, Whisker Assay