cd19 antigen constructs  (Sino Biological)

Journal: Turkish Journal of Hematology

Article Title: Preclinical Assessment of Efficacy and Safety Analysis of CAR-T Cells (ISIKOK-19) Targeting CD19-Expressing B-Cells for the First Turkish Academic Clinical Trial with Relapsed/Refractory ALL and NHL Patients

doi: 10.4274/tjh.galenos.2020.2020.0070

Figure Lengend Snippet: Construction of the anti-CD19 CAR-EGFRt gene and process optimization of CAR-LV production. A) The αCD19-CAR construct in the pCDCAR1 lentiviral vector was transcribed under the EF-1α promoter, which consists of the αCD19 scFv (FMC63 clone) head domain conjugated with CD8 hinge, CD28 transmembrane (TM), and co-stimulatory domains (CD), and the CD3ζ pro-activator signaling domain along with the P2A auto-cleavage peptide and a truncated form of EGFR (EGFRt) cell surface domain as a co-expression marker. B) Representative electron micrographs of lentiviruses showing a group of virus particles (arrow) in the sections (scale bars: 200 nm, 100 nm, 50 nm). C) The flow histogram showing the CAR-expressing control and untransduced cells that were stained with the Fab region of the CAR-specific antibody as a two-step antibody staining protocol. D) Flow cytometer plots showing Jurkat cells that were either untransduced or transduced with αCD19-CAR-EGFRt that were probed with the αEGFR-A488 antibody. E) Plot showing the CAR expression of Jurkat cells transduced with CAR-LV in a dose-dependent manner at day 4 of transduction. Viral particles were produced with FuGENE or PEI transfection reagent in HEK293T. The viral titer was concentrated to 100x using the Lenti-X concentrator reagent or TFF device. F) Bar graph showing titers of the CAR-LV virus that were produced with FuGENE + Lenti-X concentrator (blue bar), FuGENE + TFF concentration system (red bar), or PEI + Lenti-X concentrator (green bar) as x106 IFU/mL. Means and standard deviation ranges of at least three independent experiments are shown. *: p<0.05.

Article Snippet: A CD19 antigen-recognizing CAR synthetic gene construct was established in the second generation of the lentiviral vector by Creative Biolabs as a CD19 antigen-specific antibody head (clone FMC63) along with a CD8 hinge, CD28 TM, and co-stimulatory domains (CD), and the CD3ζ pro-activator signaling domain ( ).

Techniques: Construct, Plasmid Preparation, Expressing, Marker, Virus, Control, Staining, Flow Cytometry, Transduction, Produced, Transfection, Concentration Assay, Standard Deviation

Journal: Turkish Journal of Hematology

Article Title: Preclinical Assessment of Efficacy and Safety Analysis of CAR-T Cells (ISIKOK-19) Targeting CD19-Expressing B-Cells for the First Turkish Academic Clinical Trial with Relapsed/Refractory ALL and NHL Patients

doi: 10.4274/tjh.galenos.2020.2020.0070

Figure Lengend Snippet: Efficacy and cytotoxicity capacity of the CAR-T cells produced from ALL/NHL patients’ PBMCs. A) Bar graph (left) showing proportions of CD4+ and CD8+ T cells following CD3+ T cell isolation from PBMCs of seven patients. Bar graph (right) showing CAR expression in the T cells isolated from blood of 10 patients. The lines across the bars show the lowest and highest CAR expression achieved during CAR-T cell production. B) Histogram of CD19 expression in cell lines including RAJI, DAUDI, and K562, which were later used in cytotoxicity experiments as target cells. C) Flow cytometer plots of the CAR-T cells and the CTV-labeled target cells cultured at 1:1, 5:1, and 10:1 ratios (effector:target cell) for 24 h. Activation of the CAR-T cells was determined in flow cytometer analysis with the upregulation of CD25 and CD107a. D) Activation level (CD25+CD107a+) of the CAR-T cells that were cultured for 24 h either with the target cells at different ratios or without the target cells. E) IFNγ secretion capacity of the CAR-T cells upon co-culturing (dashed bar, only CAR-T; light gray bar, 1:1; black bar, 5:1; dotted bar, 10:1) for one day. F) Cytotoxicity capacity of the CAR-T cells against the target cells after 24 h of co-culturing. G) CAR (EGFR+) expression flow cytometer charts of the CAR-T cells cultured with or without RAJI cells for up to 7 days. Means, standard deviation ranges, and the data of at least three independent experiments with PBMCs from different ALL/NHL patients are shown.

Article Snippet: A CD19 antigen-recognizing CAR synthetic gene construct was established in the second generation of the lentiviral vector by Creative Biolabs as a CD19 antigen-specific antibody head (clone FMC63) along with a CD8 hinge, CD28 TM, and co-stimulatory domains (CD), and the CD3ζ pro-activator signaling domain ( ).

Techniques: Produced, Cell Isolation, Expressing, Isolation, Flow Cytometry, Labeling, Cell Culture, Activation Assay, Standard Deviation