Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Schematic representations of ligand-based conventional and split-design CAR approaches. The extracellular domains of BAFF and APRIL were used as target moieties to generate conventional CAR-T cells, referred to as APRIL CAR and BAFF CAR, respectively. b , d Representative images of cell‒cell conjugates captured at 100× oil objective magnification using a laser scanning confocal microscope (Nikon, A1R). APRIL or 9E10-IgG4m (pre-incubated with Myc-APRIL) CAR-T cells were co-cultured with RPMI8226-GFP cells ( b ), while BAFF or 9E10-IgG4m (pre-incubated with Myc-BAFF) CAR-T cells were co-cultured with IM9-GFP cells ( d ). Fluorescent labels included Hoechst (blue), anti-PKC-θ (red), and GFP (green) and a merged view of all stains. Scale bar = 10 μm. c , e Statistical analysis of the mean fluorescence intensity of PKC-θ at the IS in panels b and d, respectively. In panel c, sample sizes: APRIL CAR, n = 37; 9E10-IgG4m, n = 39. In panel e, BAFF CAR, n = 34; 9E10-IgG4m, n = 44. All n values represent individual cells. P values were determined by paired two-tailed t -tests. f , g Cytotoxicity assays of conventional and split-design CAR-T cells against the indicated target cells at various E:T ratios for 24 h in triplicate. h , i Inflammatory cytokine release assay. Conventional CAR-T cells or sCAR-T cells along with 1 nM corresponding switches were co-cultured with the specific target cells for 24 h at an E:T ratio of 1:1 in triplicate. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. j , l Schematic representations of ligand-based split-design CAR and FDA-approved CAR, referred to as BCMA CAR ( j ) and CD19 CAR ( l ), respectively. k , m Cytotoxicity assays of FDA-approved CAR-T cells and split-design CAR-T cells against the indicated target cells at various E:T ratios for 24 h in triplicate. Data in this figure are representative of three independent experiments. Error bars represent mean ± SD. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: Microscopy, Incubation, Cell Culture, Fluorescence, Two Tailed Test, Release Assay

Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). b Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). c , d Quantification of the average radiance (p/s/cm /sr) of the luminescence, related to APRIL- ( c ) and BAFF-( d )-based CAR-T-cell therapy. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. e Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Tukey correction were used to assess significance. f , g Survival curves of the mice subjected to the indicated treatments. Survival curves were compared using the log-rank (Mantel‒Cox) test. h Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). i Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). j Quantification of the average radiance (p/s/cm /sr) of the luminescence. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) and CD19/CD22 CAR-T. k Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. l Assessment of the presence of persistent human CD3 + (hCD3 + ) T cells in peripheral blood by flow cytometry over a 3-week follow-up period. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) with BAFF-CAR-T at each time point. m Survival curves of mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. All n represents biological replicates from different mice. Data in this figure are representative of one of two independent experiments. Error bars represent mean ± SEM. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). b Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). c Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. d Quantification of the average radiance (p/s/cm 2 /sr) of the luminescence. Two-way ANOVA multiple comparisons in Sidak correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-APRIL) with BCMA CAR-T. e Survival curves of mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. f Timeline of the in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). g Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). h Evaluation of serum inflammatory cytokine release by ELISA 24 hours after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. i Quantification of the average radiance (p/s/cm 2 /sr) of the luminescence. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) with CD19 CAR-T. j Assessment of the presence of tumor cells (GFP + CD19 + or GFP + CD19 - ) in peripheral blood by flow cytometry on the 18th day of the experiment. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. k Survival curves of the mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. All n represents biological replicates with different mice. Data are in this figure representative of one of two independent experiments. Error bars represent mean ± SEM. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry