siglec 2 fc chimera protein  (R&D Systems)

Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a, Schematics of the hybrid AAV-SB construct, SB100X mRNA electroporation and CAR T/NK/macrophage/iPSC generation. b, Representative flow cytometry plots of human CD4 (gated for CD3+CD8−cells) AAV-SB-CD22.CAR T cells. Human CD3 T cells were first electroporated with SB100X mRNA, then transduced with a titration series of AAV-SB-CD22.CAR virus. CAR expression levels were evaluated at various time points from day 3 to day 14. c, Representative flow cytometry plots of human CD8 (gated for CD3+CD8+ cells) AAV-SB-CD22.CAR T cells. Human CD3 T cells were first electroporated with SB100X mRNA, then transduced with a titration series of AAV-SB-CD22.CAR virus. Data were collected at several time points from day 3 to day 14. d, Representative flow cytometry plots of AAV-SB-CD22.CAR and AAV-SB-BCMA.CAR T cells after cancer stimulation. e, Schematic representation of various AAV-SB-CD22.CAR transduction time points relative to SB100X mRNA electroporation. f, Representative flow cytometry plots of CD22.CAR T cells quantifying CAR-percentages of CD4 (top) and CD8 (bottom) T cells which were transduced with AAV-SB-CD22.CAR virus at various time points relative to when SB100X mRNA electroporation occurred (at 0 h). In this figure, for optimization of conditions, each assay was done with one donor with three technical replicates. Donor 2 T cells were used in this figure. Cells were not purified for CD8/CD4 populations before electroporation.

Article Snippet: Cells were incubated with CD22-Fc protein (R&D Systems) in PBS for 30 min on ice and washed with PBS twice to remove the unbonded protein.

Techniques: Construct, Electroporation, Flow Cytometry, Transduction, Titration, Virus, Expressing, Purification

Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a, Schematic of AAV-SB-CD22.CAR, AAV-SB-BCMA.CAR, and SB100X constructs and key procedures: mRNA in vitro transcription, AAV production, mRNA electroporation, flow cytometry, and kill assay. b, Representative flow cytometry plots of AAV-SB-BCMA.CAR CD4 (left) and CD8 (right) T cells show the percentage of CAR-expressing cells. CD4 cells are defined as CD3+ and CD8− cells. c, Quantification of CD22.CAR T cell ratio of (Fig. 1b, ​,c).c). d, Quantification of BCMA.CAR T ratio in human CD4 and CD8 T cells. In this figure, for optimization of conditions, each assay was done with one donor with three technical replicates. Donor 2 T cells were used in this figure.

Article Snippet: Cells were incubated with CD22-Fc protein (R&D Systems) in PBS for 30 min on ice and washed with PBS twice to remove the unbonded protein.

Techniques: Construct, In Vitro, Electroporation, Flow Cytometry, Expressing

Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a-c, Vector copy number (VCN) quantification of MAJESTIC-manufactured CAR-T cells. Purified CAR T cells were collected for DNA extraction after three weeks of mRNA electroporation and viral transduction. (a) left arm probe, (b) right arm probe, (c) left panel: left arm probe, right panel: right arm probe. d, SB100X transposase excision efficiency evaluation. Left panel: left arm probe, right panel: right arm probe. e, Cytolysis analysis of NAML6-GL (NAML6 with GFP and luciferase reporters) cancer cells that were co-cultured with Lenti-CD22.CAR and AAV-SB-CD22.CAR T cells. CAR-Ts were seeded at various effector:target (E:T) ratios, and luciferase imaging was performed at two time points (16h and 40h). f, Cytolysis analysis of MM.1R-GL (MM.1R with GFP and luciferase reporters) cancer cells that were co-cultured with Lenti-BCMA.CAR and AAV-SB-BCMA.CAR T cells. CAR-Ts were seeded at various effector : target (E:T) ratios, and luciferase imaging was performed at two time points (16h and 40h). g, Exhaustion and memory marker expression in CD22-CAR and HER2-CAR T cells before and post transfection. Unpaired t tests were performed to evaluate statistical significance. h, Bioluminescent density of NSG mice that were injected with NALM6-GL cancer cells and with CD22-CAR therapy (n = 7 mice per group). i, Quantification of total luminescence for (h). n = 7 mice. Two-way ANOVA with multiple comparisons tests was performed to evaluate statistical significance. j, Survival curve of NALM6-GL-induced leukemia-bearing NSG mice that treated with PBS, untreated CD8 T cells, and AAV-SB-CD22.CAR T cells. Log-rank (Mantel-Cox) tests were performed to evaluate statistical significance. Donor 0007, 4003, 5003, 003C, 0286 T cells were used in this figure. Significance notes: ns - not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were incubated with CD22-Fc protein (R&D Systems) in PBS for 30 min on ice and washed with PBS twice to remove the unbonded protein.

Techniques: Plasmid Preparation, Marker, Produced, Purification, DNA Extraction, Electroporation, Transduction, Luciferase, Cell Culture, Imaging, Expressing, Transfection, Injection

Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a, Representative flow cytometry plots of NY-ESO-1 T cells. b, Quantification of (a). c, Quantification of the CD22.CAR.iCasp T cell viability. d, Representative flow cytometry plots of CD22.CAR.iCasp9 T cells. e, Quantification of (d). f, Quantification of the CD22.CAR.iCasp9 T cells post antigen-specific cancer cells stimulation. In this figure, each assay has three technical replicates, donor 601c and donor 02 T cells were used in this figure.

Article Snippet: Cells were incubated with CD22-Fc protein (R&D Systems) in PBS for 30 min on ice and washed with PBS twice to remove the unbonded protein.

Techniques: Flow Cytometry

Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a, Flow cytometry of CD22.CAR ratio in donor 6760 CD4 T cells. b, Flow cytometry of CD22.CAR ratio in donor 6760 CD8 T cells. c,d, Quantification of flow cytometry data of CD22.CAR T cells from human primary CD4 (c) and CD8 (d) T cells, from an independent donor, with CAR T cells produced by plasmid transposon plasmid, transposon MC, transposon MC with mRNA-transposase and MAJESTIC (AAV-SB-CD22.CAR + SB100X mRNA). e,f, Quantification of time-course flow cytometry data of CD22.CAR T cells produced by MAJESTIC and other systems, generated from PBMCs of four independent healthy human donors (n = 4) (e, showing mean ± s.e.m., n = 4 for all time points; f, showing individual donors in separate panels). In this figure, data in a–d were sourced from one donor with three technical replicates; data in e and f were sourced from four independent donors (donor 0007, donor 4003, donor 5003 and donor 003C). Two-way ANOVA with multiple comparisons tests was used to evaluate statistical significance, ****P < 0.0001.

Article Snippet: Cells were incubated with CD22-Fc protein (R&D Systems) in PBS for 30 min on ice and washed with PBS twice to remove the unbonded protein.

Techniques: Flow Cytometry, Produced, Plasmid Preparation, Generated

Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a, Representative flow-cytometry plots of THP-1 cells (human monocytic cell line) transduced with AAV-SB-HER2.CAR virus (MOI = 3 × 105), transduced with HER2.CAR lentivirus (MOI = 5) or electroporated with plasmid DNA (4 μg total). b, Quantification of a. c, Representative flow-cytometry plots of human CD14+ macrophages transduced with AAV-SB-CD22.CAR virus (MOI = 5 × 105), transduced with CD22.CAR lentivirus (MOI = 10) or electroporated with plasmid DNA (3 μg). d, Quantification of c. Error bars, mean ± s.e.m. of three biological replicates.

Article Snippet: Cells were incubated with CD22-Fc protein (R&D Systems) in PBS for 30 min on ice and washed with PBS twice to remove the unbonded protein.

Techniques: Flow Cytometry, Transduction, Virus, Plasmid Preparation

Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a, Quantification of CD22.CAR T cell ratio for CD4 T cells that were transduced with AAV-SB-CD22.CAR virus at various time points. b, Quantification of CD22.CAR T cell ratio for CD8 T cells that were transduced with AAV-SB-CD22.CAR virus at various time points. c-d, Representative flow cytometry plots of CD22.CAR T cells produced via AAV-SB and a titrated serial of SB100X mRNA. (c) CD8 T cells. (d) CD4 T cells. e, Quantification of the cell viability of CD8 T cells. f, Quantification of CAR T cells. g, Flow cytometry plots of CAR T cell ratios before and after sorting. h, CD22.CAR T cell yield quantification (yield = total viable cell count × CAR-positive percentage). Cells were split into 3 technical replicates after electroporation. Yield is calculated for each technical replicate separately. i, CAR+ T cell generation efficiency (CAR+%) of MAJESTIC using Neon and Maxcyte approaches. In this figure, for optimization of conditions, each assay was done with one donor with three technical replicates. Donor 2 and donor 0286 T cells were used in this figure.

Article Snippet: Cells were incubated with CD22-Fc protein (R&D Systems) in PBS for 30 min on ice and washed with PBS twice to remove the unbonded protein.

Techniques: Titration, Transduction, Virus, Flow Cytometry, Produced, Cell Counting, Electroporation

Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a, Representative flow cytometry plots of CD8 and CD4 CD22.CAR T cells were produced via AAV-SB and a titrated series of SB100X mRNA. b, Representative flow cytometry plots measuring the viability of CD8 T cells. 7-AAD staining was performed to measure cell viability after mRNA electroporation, plasmid DNA electroporation and lentivirus transduction. Media were changed 1 day after electroporation to remove the virus. c, Representative flow cytometry plots of human CD8 T cells transduced with AAV-SB-CD22.CAR virus (MOI = 1 × 105 and 5 × 105), transduced with CD22.CAR lentivirus (MOI = 1 and 10) or electroporated with plasmid DNA (1 μg = 0.5 μg transposon plasmid + 0.5 μg transposase plasmid) at three time points. In this figure, each assay was done with one donor with at least three technical replicates, donor 0286 T cells were used in this figure.

Article Snippet: Cells were incubated with CD22-Fc protein (R&D Systems) in PBS for 30 min on ice and washed with PBS twice to remove the unbonded protein.

Techniques: Flow Cytometry, Produced, Staining, Electroporation, Plasmid Preparation, Transduction, Virus

Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a, Schematic of various therapeutic cells (that is, single scFv CAR, tandem scFv CAR, TCR-T and kill-switch CAR) that can be generated via the AAV-SB system. b, Flow cytometry and histogram overlays of cell viability post-electroporation as measured with 7-AAD staining. c, Representative flow cytometry plots of HER2.CAR T cells. d, Schematic representation of the AAV-SB-CD19.20.CAR construct. CD19 scFv and CD20 scFv CAR sequences are joined by a linker and are expressed together as a tandem scFv CAR. e, Representative flow cytometry plots to evaluate CAR expression of CD19.20.CAR T cells. f, Schematic representation of the AAV-SB-HER2.CAR. iCasp9 construct. g, Flow cytometry and histogram overlays of cell viability post-electroporation as measured with 7-AAD staining. h, Representative flow cytometry plots of CD22.CAR.iCasp9 T cells post antigen-specific cancer cells stimulation. In this figure, each assay was done with one donor with three to five technical replicates; donor 2 T cells were used for b and c; donor VP2 T cells were used for d and e; donor 2 T cells were used for g and h.

Article Snippet: Cells were incubated with CD22-Fc protein (R&D Systems) in PBS for 30 min on ice and washed with PBS twice to remove the unbonded protein.

Techniques: Generated, Flow Cytometry, Electroporation, Staining, Construct, Expressing

Journal: Nature biomedical engineering

Article Title: AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells

doi: 10.1038/s41551-023-01058-6

Figure Lengend Snippet: a, Flow-cytometry histogram overlays and bar plots of cell viability post-electroporation as measured with 7-AAD staining. b, Flow-cytometry data of CD22.CAR T cells from human primary CD3 T cells produced by plasmid transposon plasmid, transposon MC, transposon MC with mRNA-transposase, and MAJESTIC. c, Quantification of (b). d, Yield calculations (yield = CAR% * total viable cell count). All conditions started with an equal amount of primary T cells per replicate. Three CAR% replicates were averaged and then multiplied by the average of 2 cell count replicates. Left panel, total T cell count; Right panel, total CAR+ T cell count. e, Quantification of flow-cytometry data of CD22.CAR T cells from four human PBMCs (same data as Fig. 4e–f, plotted in dot-whisker plots).

Article Snippet: Cells were incubated with CD22-Fc protein (R&D Systems) in PBS for 30 min on ice and washed with PBS twice to remove the unbonded protein.

Techniques: Flow Cytometry, Electroporation, Staining, Produced, Plasmid Preparation, Cell Counting, Whisker Assay

Journal: Nature biotechnology

Article Title: Massively parallel knock-in engineering of human T cells

doi: 10.1038/s41587-022-01639-x

Figure Lengend Snippet: a, Schematic of CLASH-mediated simultaneous CAR-T and Descartes library knock-in. b, Left, flow cytometry plots showing representative CAR22 knock-in into TRAC locus after CLASH vector-AAV6 transduction for 5 days (AAV6 g-MOI = 1 × 105). Right, quantification of CD3−CAR22+ percentages (infection replicates, n = 3). c, Representative flow cytometry analysis of CLASH knockout efficiency on surface makers (infection replicates, n = 3). d, Schematic of Descartes library design. Not drawn to scale. e, Sanger sequencing results of Descartes-Lib AAV plasmid and pooled Descartes-Lib CD22 CAR-T cells gDNA at TRAC locus. f, Schematic of the CD8 or CD4 CAR-T in vitro CLASH. CAR-T cells were co-cultured with NALM6 at E:T ratio = 0.2:1 for 7–8 rounds after electroporation. g, Left, representative flow cytometry plots of memory marker expression on CD8 or CD4 vector and Descartes-Lib CAR-T cells at endpoint of in vitro co-culture. Tcm = CD45RO+CCR7+ or CD45RO+CD62L+. Right, quantification of Tcm percentages (infection replicates, n = 3). h, Empirical CDF of crRNA representations in the genomic readouts of CD8 and CD4 in vitro Descartes-Lib CLASH knock-in CAR-T pool samples. TPM, transcripts per million. Statistics: in b and g, unpaired two-sided t-test was used to assess significance. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± s.e.m.

Article Snippet: For CAR staining, the CD22BBz CAR transduced T cells were incubated with 0.2 μg of CD22-Fc (R&D Systems) in 100 μl of staining buffer for 30 minutes and then stained with PE-IgG-Fc (BioLegend).

Techniques: Knock-In, Flow Cytometry, Plasmid Preparation, Transduction, Infection, Knock-Out, Sequencing, In Vitro, Cell Culture, Electroporation, Marker, Expressing, Co-Culture Assay

Journal: Nature biotechnology

Article Title: Massively parallel knock-in engineering of human T cells

doi: 10.1038/s41587-022-01639-x

Figure Lengend Snippet: a, T7E1 assay for individual crRNAs in CAR-T cells. Predicted cleaved bands, blue arrows. b–e, Flow analysis of mutant CAR-T cells in two donors (infection replicates, n = 3). b, Quantification of Tcm (CD45RO+CCR7+ or CD45RO+CD62L+). c, Quantification of exhaustion markers (LAG3+). d, Representative histograms of CellTrace Violet in mutant CAR-T cells, measured at day 0 (gray peak) or day 3 (cyan peak). Yellow dashed line, MFI for vector CAR-T. e, CellTrace Violet MFI quantification. f, T7E1 for PRDM1-cr3 to cr8 from donor 2 CAR-T cells. g, T7E1 for PRDM1-cr1and cr2 on another donor and different CAR-T construct. Note for a, f and g, gel is representative of three repeated experiments. h, Indel quantification of PRDM1-cr1 to cr8 in donor 2 CAR22 T cells (technical replicates, n = 3 for most samples, except cr4_control and c5_control (n = 1)). i, Top, schematics of PRDM1 protein primary structure. Bottom, PRDM1 protein western blot in vector and PRDM1 mutant CAR-T cells. Blot is representative of three repeated experiments. Blue arrowheads, WT protein. Blue stars, truncated mutant protein generated by PRDM1-cr1. j, Exon3 skipping after PRDM1-cr1 editing in CAR-T cells verified by Sanger sequencing of RT–PCR products. Note for f–j, cutting sites of crRNAs, vertical arrowheads on PRDM1 exon diagram. Predicted cleaved bands, horizontal arrowheads. Gel is representative of three repeated experiments. k, Protein structure alignment of PR domain (blue, PDB: 3DAL) and PR Δexon3 domain (gold, predicted via AlphaFold). Exon3 peptide, highlighted pink. l, Proliferation of PRDM1 mutant and control CD22 CAR-T cells with NALM6 stimulation (cell culture replicates, n = 3). m, Cytotoxicity of vector and PRDM1 mutant CAR-T cells for different donors (cell culture replicates, n = 3). Statistics: b, c and e, unpaired two-sided t-test; h, l and m, two-way ANOVA. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± s.e.m. LOF, loss of function.

Article Snippet: For CAR staining, the CD22BBz CAR transduced T cells were incubated with 0.2 μg of CD22-Fc (R&D Systems) in 100 μl of staining buffer for 30 minutes and then stained with PE-IgG-Fc (BioLegend).

Techniques: Mutagenesis, Infection, Plasmid Preparation, Construct, Control, Western Blot, Generated, Sequencing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: Nature biotechnology

Article Title: Massively parallel knock-in engineering of human T cells

doi: 10.1038/s41587-022-01639-x

Figure Lengend Snippet: a, Left, schematics of liquid cancer models. Right, FACS plot of CD19+CD22+ NALM6-GL cells. b,c, Bioluminescence imaging (b) and quantification (c) of NALM-6 leukemia-bearing NSG mice treated with the following: CD8 T (n = 7), vector CAR22 (n = 11) and PRDM1 Δexon3 CAR22 (n = 11). d,e, Bioluminescence imaging (d) and quantification (e) of NALM-6 leukemia-bearing NSG mice treated with the following: CD8 T (n = 5), vector CAR19 (n = 10) and PRDM1 Δexon3 CAR19 (n = 9). f, FACS histogram of HER2+ HT29-GL cells. g, Schematics of a HER2+ HT29 solid tumor model with two different doses of CAR-T treatments. Non-transduced CD3+ T (2 M, n = 5), vector HER2 CAR-T (2 M, n = 5; 0.5 M n = 5) and PRDM1 Δexon3 HER2 CAR-T (2 M, n = 5; 0.5 M, n = 5), all infused at day 13. h, Tumor growth curves of experiment in g. Black arrow, CAR-T treatment. i, Schematics of a rechallenge HT2 model with CAR-T treatment with the following: non-transduced CD3+ T (n = 6), vector HER2 CAR-T (n = 6) and Δexon3 HER2 CAR-T (n = 6). j–l, Quantification of whole-body bioluminescence (j) and tumor volume (k) over time, and percentage of relapsed animals, of experiment in g. Black arrow, CAR-T injection; blue arrow, tumor rechallenging. m,n, Quantification of cancer cells, tumor-infiltrating CAR-T cells and Tcm in tumor samples, from two cohorts with endpoints at day 14 (m) and day 18 (n) after infusion, respectively. Vector (n = 4) and PRDM1 Δexon3 CAR22 (n = 5). Statistics: c, e, h, j and k, two-way ANOVA; m and n, unpaired two-sided t-test. *P < 0.05, **P < 0.01 and ***P < 0.001 and NS, not significant. Data are shown as mean ± s.e.m. M, million.

Article Snippet: For CAR staining, the CD22BBz CAR transduced T cells were incubated with 0.2 μg of CD22-Fc (R&D Systems) in 100 μl of staining buffer for 30 minutes and then stained with PE-IgG-Fc (BioLegend).

Techniques: Imaging, Plasmid Preparation, Injection