Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Selective B cell depletion upon intravenous infusion of replication-incompetent anti-CD19 CAR lentivirus

doi: 10.1016/j.omtm.2022.05.006

Figure Lengend Snippet: In vitro proliferation and cytolytic activity of murine CAR-T cells (A) 2.5 × 10 5 C57BL/6 splenocytes were transduced with 1D3-CD19CAR-GFP or GFP-only lentivirus at an MOI of 5. The transduced splenocytes were then co-cultured with an excess of irradiated C57BL/6 splenocytes, which provided a source of B cells expressing the CD19 antigen. The 1D3-CD19CAR transduced splenocytes showed a 12.2-fold ± 0.09-fold (mean ± SD) expansion (red, square), compared with GFP transduced (green, triangle) (p < 0.002, paired two-tailed t test). (B) 2.5 × 10 5 C57BL/6 splenocytes were transduced with the FMC63-CD19CAR-GFP or GFP-only lentivirus at an MOI of 5. Splenocytes were then co-cultured with irradiated C57BL/6 splenocytes, which provided a source of B cells expressing the CD19 antigen. The FMC63-CD19CAR transduced splenocytes showed an 8.8-fold ± 0.03-fold expansion (orange, square), compared with GFP transduced (cyan, triangle), (p < 0.004, paired two-tailed t test). (C) 1 × 10 5 FMC63-CD19CAR-GFP or 1D3-CD19CAR murine T cells from day 63 of the expansion assays were co-cultured for 5 h with target B cells, isolated from C57BL/6 splenocytes, at an E:T ratio of 1:1. Unviable target murine B cells were measured through the shift in the FVS780 signal, measured by flow cytometry ( Figure S4 ). Expanded murine 1D3-CAR-T cells showed robust and selective targeting of murine B cells, with 24.8% ± 1.69% of B cells being unviable after a 5-h co-culture. The FMC63-CD19CAR T cells also showed robust and selective targeting of murine B cells, with 18.5% ± 1.08% of B cells being unviable after a 5-h co-culture. Two-way ANOVA, Sidak’s multiple comparison ∗∗∗p < 0.002. (D) 1D3-, FMC63-CD19CAR-, and GFP-transduced and expanded murine T cells were co-cultured with target A20 cells for 4 h. The CD19 antigen on the A20 cells were blocked with either the CD19 1D3 or FMC63-blocking antibody at a starting concentration of 10 μg/mL decreasing 10-fold down to 0 μg/mL, at an E:T ratio of 4:1. Using flow cytometry, target cell death was measured through the shift in the FVS780 signal ( Figure S3 and Table S1 ). The murine CAR-T cells showed robust and selective response when co-cultured with A20 cells. This response decreased with increasing CD19-blocking antibody concentration. 38.8% ± 0.71% of unblocked A20 cells were unviable when co-cultured with the 1D3-CD19CAR T cells, which decreased to 19.7% ± 0.28% of A20 cells blocked with 10 μg/mL of 1D3-CD19-blocking antibody. 41.1% ± 0.92% of unblocked A20 cells were unviable when co-cultured with the FMC63-CD19CAR T cells, which decreased to 20.8% ± 0.57% of A20 cells blocked with 10 μg/mL of FMC63-CD19. Two-way ANOVA p = 0.04. Nonlinear regression analysis (sigmoidal) analysis of 1D3-CD19CAR T cells versus A20 CD19-blocking antibody dose response, R 2 = 0.95. FMC63-CD19CAR T cells versus A20 CD19-blocking antibody dose response, R 2 = 0.96. GFP versus A20 CD19-blocking antibody dose response, R 2 = 0.01.

Article Snippet: In order to verify the ability for both the 1D3-and FMC63-CD19CAR to target the mouse CD19 antigen, the A20 cells were incubated with 10, 1, 0.1, 0.001, or 0 μg/mL of either the 1D3-CD19-blocking antibody (152402, BioLegend) or the FMC63-CD19-blocking antibody (NBP2-52716, Novus Biologicals).

Techniques: In Vitro, Activity Assay, Transduction, Cell Culture, Irradiation, Expressing, Two Tailed Test, Isolation, Flow Cytometry, Co-Culture Assay, Comparison, Blocking Assay, Concentration Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Selective B cell depletion upon intravenous infusion of replication-incompetent anti-CD19 CAR lentivirus

doi: 10.1016/j.omtm.2022.05.006

Figure Lengend Snippet: The abundance of immune cell subsets in treated mice over time, as a percentage of total PBMCs C57BL/6 wild-type mice were treated with a single intravenous injection of lentivirus (3.6–4.0 × 10 6 IU in 200 μL of PBS), to deliver either the 1D3-CD19CAR-GFP CAR, FMC63-CD19CAR, or control (GFP only). N equals eight mice per group and error bars represent the standard deviation (SD) from the mean value reported for each group. T cells, but not other immune cell subsets tested, show a substantial transduced cell population. (A) Representative flowgrams showing increasing GFP-positive T cells in PBMCs of treated mice, over time. The x axis of the flowgrams is CD3 positivity and GFP positivity on the y axis. (B–E). (B) Total T cells versus 1D3-, FMC63-CD19CAR, and GFP-transduced T cells (CD3 + , CD90.2 + T cells). (C) Total B cells versus 1D3-, FMC63-CD19CAR, and GFP-transduced B cells (CD20 + , CD45R/B220 + B cells). (D) Total macrophage versus 1D3-, FMC63-CD19CAR, and GFP-transduced macrophage (CD11b + macrophage and other non-T cells). (E) Total NK/NK-T cells versus 1D3-, FMC63-CD19CAR, and GFP-transduced NK/NK-T cells (CD335 + NK/NKT cells). Flow gating strategy outlined in Figure S4 and data in Table S3 and .

Article Snippet: In order to verify the ability for both the 1D3-and FMC63-CD19CAR to target the mouse CD19 antigen, the A20 cells were incubated with 10, 1, 0.1, 0.001, or 0 μg/mL of either the 1D3-CD19-blocking antibody (152402, BioLegend) or the FMC63-CD19-blocking antibody (NBP2-52716, Novus Biologicals).

Techniques: Injection, Control, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Selective B cell depletion upon intravenous infusion of replication-incompetent anti-CD19 CAR lentivirus

doi: 10.1016/j.omtm.2022.05.006

Figure Lengend Snippet: Immunophenotyping of 1D3-CD19CAR-GFP (blue, circle), FMC63-CD19CAR-GFP (red, square), or control (GFP only, green, triangle) transduced T cells in peripheral blood of treated mice Values shown are the percentage of total CD3 + , CD90.2 + T cells represented by each subtype (effector = CD44 high , CD62L low CD127 low ; effector memory = CD44 high , CD62L low CD127 high ; central memory = CD44 high , CD62L high CD127 high ; intermediate phenotype = CD44 high CD62L high CD127 low ; naive = CD44 low , CD62L high CD127 high ; regulatory = CD4 + FOXP3 + ). N equals eight mice per group and error bars represent the SD from the mean value reported for each group. (A) At week 5, the 1D3-CD19CAR-GFP T cells were predominantly CD8 + effector memory T cells (5.1% ± 0.9% of total CD3 + T cells), effector T cells (1.7% ± 0.2% of total CD3 + T cells), and CD4 + intermediate T cells (1.3% ± 0.2% of total CD3 + T cells). At week 5, the majority of FMC63-CD19CAR-GFP T cells were CD8 + effector memory T cells (2.0% ± 0.2% of total CD3 + T cells) and effector T cells (0.7% ± 0.1% of total CD3 + T cells). (B) At week 8, the majority of 1D3-CD19CAR-GFP T cells were effector memory T cells (0.7% ± 0.6% of total CD3 + T cells) and CD8 + effector T cells (1.3% ± 0.4% of total CD3 + T cells). The majority of FMC63-CD19CAR-GFP T cells at week 8 were CD8 + effector memory T cells (2.60% ± 0.538% of total CD3 + T cells) and effector T cells (1.1% ± 0.6% of total CD3 + T cells). GFP-expressing CD4 + FOXP3 + regulator T cells were present at both time points but comprised a small percentage (<1%) of total T cells. Flow gating strategy outlined in Figure S5 and data in Table S5 .

Article Snippet: In order to verify the ability for both the 1D3-and FMC63-CD19CAR to target the mouse CD19 antigen, the A20 cells were incubated with 10, 1, 0.1, 0.001, or 0 μg/mL of either the 1D3-CD19-blocking antibody (152402, BioLegend) or the FMC63-CD19-blocking antibody (NBP2-52716, Novus Biologicals).

Techniques: Control, Expressing

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: 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: This was followed by two washes in FACS buffer (PBA solution with 2% bovine serum albumin) and subsequent staining with allophycocyanin (APC)-conjugated Streptavidin Protein (stock: 200 μg/mL, 1:200 dilution, ACRO Biosystems) and FITC-Labeled Monoclonal Anti-FMC63 Antibody (stock: 50 μL, 1:50 dilution, ACRO Biosystems, clone: Y45) (or PE-Labeled Monoclonal Anti-FMC63 Antibody (stock: 50 μL, 1:50 dilution, ACRO Biosystems, clone: Y45)) 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: 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: This was followed by two washes in FACS buffer (PBA solution with 2% bovine serum albumin) and subsequent staining with allophycocyanin (APC)-conjugated Streptavidin Protein (stock: 200 μg/mL, 1:200 dilution, ACRO Biosystems) and FITC-Labeled Monoclonal Anti-FMC63 Antibody (stock: 50 μL, 1:50 dilution, ACRO Biosystems, clone: Y45) (or PE-Labeled Monoclonal Anti-FMC63 Antibody (stock: 50 μL, 1:50 dilution, ACRO Biosystems, clone: Y45)) for 60 min at 4 °C.

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