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: International Journal of Molecular Sciences

Article Title: Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes

doi: 10.3390/ijms241310891

Figure Lengend Snippet: Kinetic binding constants of various immobilized anti-CD19 antibody ligands binding to the CD19-AD2 analyte in solution. Values are obtained after the global fitting of the binding signals obtained by BLI measurements. Signals detected above the level of non-specific binding were listed.

Article Snippet: Binding kinetics of CD19-AD2 to various commercially available biotinylated anti-CD19 antibodies, including FMC63 (Ab00613-2.0, Absolute Antibody Ltd., Oxford, Oxfordshire, UK), HIB19 (302204, BioLegend, USA), 4G7 (Ab00219-1.1, Absolute Antibody Ltd.), as well as 3B10 (TA506236AM, Origene Tech.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes

doi: 10.3390/ijms241310891

Figure Lengend Snippet: Real-time binding sensorgrams of the immobilized biotinylated anti-CD19 antibodies (ligand concentration 10 µg/mL) and CD19-AD2 construct (analyte). ( A ) FMC63-btn; ( B ) HIB19-btn; ( C ) 3B10-btn; ( D ) 4G7-btn were captured on SA biosensors and dipped in wells containing the analyte CD19-AD2 at different concentrations between 10 and 600 nM. The spectral shift, corresponding to the thickness of the biolayer, differs for individual analyses. The binding signals (blue sensorgrams) were obtained by subtracting the signals from ligand-coated SA biosensors dipped in wells with buffer (non-specific binding control). Fitted curves are depicted as red lines and were obtained by global fitting using a 1:1 ligand model.

Article Snippet: Binding kinetics of CD19-AD2 to various commercially available biotinylated anti-CD19 antibodies, including FMC63 (Ab00613-2.0, Absolute Antibody Ltd., Oxford, Oxfordshire, UK), HIB19 (302204, BioLegend, USA), 4G7 (Ab00219-1.1, Absolute Antibody Ltd.), as well as 3B10 (TA506236AM, Origene Tech.

Techniques: Binding Assay, Concentration Assay, Construct

Journal: Nature Medicine

Article Title: BCMA CAR T cells in a patient with relapsing idiopathic inflammatory myositis after initial and repeat therapy with CD19 CAR T cells

doi: 10.1038/s41591-025-03718-3

Figure Lengend Snippet: a , Serum CK levels after CD19 CAR T cell therapy and at relapse. The horizontal dotted line indicates the upper limit of normal range (<170 U per l). b , Muscle strength as indicated by MMT8 score. c , CD19 CAR + CD3 + T cell counts in blood. d , CD19 + B cell counts in blood. e – h , Counts of leukocytes ( e ); neutrophils ( f ); CD4 + T cells ( g ) and CD8 + T cells ( h ) in blood after lymphodepletion (in cells per µl). i , ELISA quantification of the humoral response to CAR (FMC63). j , Quantification of the anti-CAR T cell response in control donors and the index patient. k , Sequence of events demonstrating the onset of the anti-CAR T cell response and humoral response. Unstim, no T cell stimulation; +, stimulated T cells.

Article Snippet: Anti-FMC63 antibodies were detected using the ACROBIO Anti-CD19 (FMC63) CAR Immunogenicity ELISA Kit following kit instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Sequencing, Cell Stimulation