Journal: Journal of translational medicine

Article Title: CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia.

doi: 10.1186/s12967-024-04990-6

Figure Lengend Snippet: Fig. 1 Preparation and mechanism of CD19/CD20 dual-targeted CAR-NK cell. ①: Ex-vivo expanded natural killer cells are derived from umbilical cord blood. ②: The mRNA encoding anti-CD19 CARs (FMC63 scFv-CD8α-4-1BB-CD3ζ) and anti-CD20 CARs (LEU16 scFv-CD8α-4-1BB-CD3ζ) were constructed by IVT. ③-④: CD19/CD20 dual-targeted CAR-NK cells were generated by simultaneous electroporation of CAR-mRNA into UCB-NK cells derived from umbilical cord blood, which specifically recognizes CD19+ and/or CD20+ ALL cells. ⑤: Once activated, CAR-NK binds to the target antigen and then lyses tumor cells by releasing perforin and IFN-γ. CAR chimeric antigen receptor, IVT in vitro transcription, UCB umbilical cord blood, NK natural killer cells

Article Snippet: To assess the cell surface expression of CAR, electroporated cells were stained with Biotinylated Human CD20 Full-Length protein (stock 50 μg/mL, 1:25 dilution, ACRO Biosystems, Beijing, CHN) for 60 min at 4 °C.

Techniques: Ex Vivo, Derivative Assay, Construct, Generated, Electroporation, In Vitro

Journal: Journal of translational medicine

Article Title: CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia.

doi: 10.1186/s12967-024-04990-6

Figure Lengend Snippet: Fig. 2 Ex vivo expansion and identification of UCB-NK. A Schematic diagram of ex vivo-expanded UCB-NK cells. B Morphology of NK cells at different cultured times observed via an optical microscope (Scale Bar = 200 μm). C Growth curves of UCB-NK cells from different donors. D Purity of UCB-NK cells as determined by flow cytometry. E Expression of CD19 and CD20 antigens on the surface of different acute leukemia cells. F Cytotoxic effects of UCB-NK cells on different acute lymphoblastic leukemia cells. Data are presented as mean values and S.D. of triplicate samples (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3). UCB-NK, NK cells derived from umbilical cord blood

Article Snippet: To assess the cell surface expression of CAR, electroporated cells were stained with Biotinylated Human CD20 Full-Length protein (stock 50 μg/mL, 1:25 dilution, ACRO Biosystems, Beijing, CHN) for 60 min at 4 °C.

Techniques: Ex Vivo, Cell Culture, Microscopy, Flow Cytometry, Expressing, Derivative Assay

Journal: Journal of translational medicine

Article Title: CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia.

doi: 10.1186/s12967-024-04990-6

Figure Lengend Snippet: Fig. 3 Construction and identification of the second-generation CARs targeting CD19 and CD20 antigen. A Schematic illustration of the construction of mRNA encoding CD19 CARs and CD20 CARs utilized IVT. B Schematic diagrams of the CD19 CAR-mRNA and the CD20CAR-mRNA. FMC63(anti-CD19) scFv and LEU16 (anti-CD20) scFv were linked to the CD8α hinge and transmembrane domain, the 4-1BB (CD137) signaling domain, and the CD3 zeta signaling domain, respectively. C Denatured agarose gel electrophoresis of the CAR-mRNA. Lane (1) represents the CD19 CAR-mRNA group, lane (3) represents the CD20 CAR-mRNA group, and lane (2) represents mRNA Marker (6000nt). D Detection of CAR expression on NK cells by flow cytometry. CAR expression was detected by using one or two of four staining reagents: PE-Labeled monoclonal anti-FMC63 Antibody (column 1), Biotinylated Human CD20/MS4A1 full length protein followed by Streptavidin Protein-Alexa Fluor 647 (column 2), or FITC-Labeled Monoclonal Anti-FMC63 Antibody (column 3). The expression percentage of CAR on NK cells is noted on the right of each histogram. EGFP-transduced cells served as an additional negative control. E CD19 CARs and CD20 CARs expression kinetics on dual-target CAR-NK over 4 days. IVT in vitro transcription, scFv single-chain fragment variable regions, Cap1 Cap1 structure, UTR untranslated region

Article Snippet: To assess the cell surface expression of CAR, electroporated cells were stained with Biotinylated Human CD20 Full-Length protein (stock 50 μg/mL, 1:25 dilution, ACRO Biosystems, Beijing, CHN) for 60 min at 4 °C.

Techniques: Agarose Gel Electrophoresis, Marker, Expressing, Flow Cytometry, Staining, Labeling, Negative Control, In Vitro

Journal: Journal of translational medicine

Article Title: CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia.

doi: 10.1186/s12967-024-04990-6

Figure Lengend Snippet: Fig. 4 Specific cytotoxicity of CD19/CD20 dual-targeted CAR-NK cells against CD19 positive/CD20 positive acute lymphoma cells. A Detection of cytotoxicity of dual-target CAR-NK cells and single-target CAR-NK cells on acute lymphoma cells (BALL-1, REH, and Jurkat) at different E:T ratios. Data are presented as mean values and S.D. of triplicate samples (*p < 0.05, **p < 0.01, n = 3). B Death of BALL-1, REH, and Jurkat cell lines was detected by flow cytometry (CFSE/PI). Nucleated cell gates were gated according to the size and complexity of the samples (FCS-A and SSC-A, respectively). Nucleated cells were further gated in FSC-A and FSC-H to screen for single cells and exclude doublets. From the single cell gate, tumor cells were defined as CFSE+. The CFSE+PI+ population was defined as dead tumor cells from the CFSE+ gate

Article Snippet: To assess the cell surface expression of CAR, electroporated cells were stained with Biotinylated Human CD20 Full-Length protein (stock 50 μg/mL, 1:25 dilution, ACRO Biosystems, Beijing, CHN) for 60 min at 4 °C.

Techniques: Flow Cytometry