x75  (Novus Biologicals)

Journal: Journal for Immunotherapy of Cancer

Article Title: Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

doi: 10.1136/jitc-2023-008179

Figure Lengend Snippet: MUC16 expression, CAR design and functionality in vitro. (A) Left —Gene expression of MUC16 , MSLN , MUC1 and FOLR1 in patient samples across clinical stages. Data were taken from a TCGA OC cohort of 375 patients. The color scale varies from the low expression (blue) to high expression (red). The different clinical stages are shown in a graphical colorful legend as indicated. Right —Volcano plot where the x- axis is the fold change in gene expression and y -axis is the statistical significance (-log10 p value). Genes with a high degree of significance (p values <0.05) and substantial fold changes are in red and genes with lower significance or smaller fold changes are in green. Non-significant genes are in gray. (B) Representation of MUC16 protein at the plasma membrane and recognition sites of the antibodies used in this study. K-series hybridoma supernatants staining of HEK, HeLa, and OVCAR3 cell lines. The commercial anti-CA125 antibody, X75, was used as a positive control, and staining with the secondary only (anti-mouse) was used as a background control. Samples were then analyzed by flow cytometry. (C) Expression of MUC16CAR constructs in J76-NFAT-GFP assessed by staining with biotinylated anti-mFab antibody and fluorescent streptavidin. Samples were then analyzed by flow cytometry. Left —CAR expression in J76-NFAT-GFP as percent mFab-positive cells (N=4–5 independent transductions, expression of CARs, data are mean±SD). Right —Representative staining of J76-NFAT-GFP transduced or not (Mock) with the indicated MUC16CAR construct. (D) J76-NFAT-GFP cells expressing the indicated MUC16CAR construct were co-cultured (E:T=1:2) overnight with MUC16 pos (HeLa, OVCAR3) or MUC16 neg (HEK) cell lines, with effector-only (E-only) condition used as a baseline. CAR reactivity was assessed on the basis of GFP expression, N=3 independent experiments, data are mean±SD. (E) As in (C) using primary T cells from N=3–4 healthy donors. (F) Functional activity of primary T cells incubated with the indicated target cells (top: HEK, middle: HeLa, bottom: OVCAR3). Left— Degranulation activity: CAR-transduced T cells were co-cultured with target cells at E:T=1:2 overnight, in the presence of anti-CD107a fluorescent antibody. Target cells were gated out and CD107a was detected on T cells. Data are mean±SD, n=2–4 donors, N=4–6 independent experiments, one-way ANOVA, *p<0.05, **p<0.01. Right —Cytotoxic activity: CAR T cells were tested in bioluminescence-based citotoxicity co-cultures at different E:T ratios, as indicated. Anti-CD19 CAR, FMC63, was included as an irrelevant CAR control. The time point displayed is at 10 hours of co-culture. Data are mean±SD, N=3 independent experiments, two-way ANOVAs with Dunnett’s multiple comparisons, **p<0.01, ***p<0.001. (G) Cytotoxic assay in three-dimensional (3D) cultures (spheroids). Left —Representation of the assay. Right —HeLa and HEK cells were established as spheroids for 5 days. T cells were added, and killing was measured 24 hours later (day 6). Annexin V was recorded in IncuCyte live-cell imaging, and fold increase relative to target cells alone was calculated. Data are mean of normalized values±SD, N=2 different donors, one-way ANOVA, *p<0.05. ANOVA, analysis of variance; bioluminescence-based citotoxicity, bioluminescence-based cytotoxicity; CAR, chimeric antigen receptor; E:T, effector:target ratio; HEK, human embryonic kidney; OC, ovarian cancer; TCGA, The Cancer Genome Atlas.

Article Snippet: To test the reactivity of CAR T cells against OC samples, the samples were labeled with commercial anti-CA125 antibody (X75 at 1/200 dilution, Thermo Fisher Scientific, #MA1-90039) followed by a secondary antibody (APC goat anti-mouse IgG, BioLegend #405308, at 1/200 dilution), and labeling intensity was normalized against the same samples labeled with the secondary antibody only.

Techniques: Expressing, In Vitro, Gene Expression, Clinical Proteomics, Membrane, Staining, Positive Control, Control, Flow Cytometry, Construct, Cell Culture, Functional Assay, Activity Assay, Incubation, Co-Culture Assay, Live Cell Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

doi: 10.1136/jitc-2023-008179

Figure Lengend Snippet: Cytokine production of MUC16CAR T cells and reactivity against primary patient samples. (A) Cytokine quantification. CAR T cells were co-cultured for 24 hours with target cells at an E:T ratio of 1:2, and the supernatant was collected for Bioplex cytokine assays. Selected cytokines are shown, data are mean±SD, N=3–4 independent measurements, two-way ANOVA. *p<0.05, **p<0.01. (B) Reactivity of CAR T cells against CA125-positive and CAR-negative samples. Tumor samples from patients with OC (effusions or material from debulking surgery) were labeled for CA125 (X75 antibody). Samples were categorized as CA125-positive (n>9), or CA125-negative (n>7), based on the percentage of cells labeling for CA125 (threshold 10%). These samples were co-cultured with CAR T cells overnight at E:T=1:2 in the presence of anti-CD107a. Effector cells were distinguished by prelabeling CAR T cells with Cell Trace Violet. CAR T cells cultured alone were used for normalization. Data are presented as mean±SD, one-way ANOVA. *p<0.05, **p<0.01. ANOVA, analysis of variance; CA125, cancer antigen 125; CAR, chimeric antigen receptor; E:T, effector:target ratio; HEK, human embryonic kidney; IL, interleukin.

Article Snippet: To test the reactivity of CAR T cells against OC samples, the samples were labeled with commercial anti-CA125 antibody (X75 at 1/200 dilution, Thermo Fisher Scientific, #MA1-90039) followed by a secondary antibody (APC goat anti-mouse IgG, BioLegend #405308, at 1/200 dilution), and labeling intensity was normalized against the same samples labeled with the secondary antibody only.

Techniques: Cell Culture, Labeling

Journal: Journal for Immunotherapy of Cancer

Article Title: Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

doi: 10.1136/jitc-2023-008179

Figure Lengend Snippet: MUC16CAR T cell activity is unaffected by soluble antigen. Bioluminescence-based cytotoxicity assays in the presence of recombinant CA125 (300 kU/L) were performed at an E:T ratio of 10:1. Data are mean±SD, N=5 donors, one-way analysis of variance, with Šidák’s multiple comparisons test. ****p<0.0001. CA125, cancer antigen 125; CAR, chimeric antigen receptor; E:T, effector:target ratio; HEK, human embryonic kidney; ns, not significant.

Article Snippet: To test the reactivity of CAR T cells against OC samples, the samples were labeled with commercial anti-CA125 antibody (X75 at 1/200 dilution, Thermo Fisher Scientific, #MA1-90039) followed by a secondary antibody (APC goat anti-mouse IgG, BioLegend #405308, at 1/200 dilution), and labeling intensity was normalized against the same samples labeled with the secondary antibody only.

Techniques: Activity Assay, Recombinant

Journal: Journal for Immunotherapy of Cancer

Article Title: Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

doi: 10.1136/jitc-2023-008179

Figure Lengend Snippet: MUC16 expression, CAR design and functionality in vitro. (A) Left —Gene expression of MUC16 , MSLN , MUC1 and FOLR1 in patient samples across clinical stages. Data were taken from a TCGA OC cohort of 375 patients. The color scale varies from the low expression (blue) to high expression (red). The different clinical stages are shown in a graphical colorful legend as indicated. Right —Volcano plot where the x- axis is the fold change in gene expression and y -axis is the statistical significance (-log10 p value). Genes with a high degree of significance (p values <0.05) and substantial fold changes are in red and genes with lower significance or smaller fold changes are in green. Non-significant genes are in gray. (B) Representation of MUC16 protein at the plasma membrane and recognition sites of the antibodies used in this study. K-series hybridoma supernatants staining of HEK, HeLa, and OVCAR3 cell lines. The commercial anti-CA125 antibody, X75, was used as a positive control, and staining with the secondary only (anti-mouse) was used as a background control. Samples were then analyzed by flow cytometry. (C) Expression of MUC16CAR constructs in J76-NFAT-GFP assessed by staining with biotinylated anti-mFab antibody and fluorescent streptavidin. Samples were then analyzed by flow cytometry. Left —CAR expression in J76-NFAT-GFP as percent mFab-positive cells (N=4–5 independent transductions, expression of CARs, data are mean±SD). Right —Representative staining of J76-NFAT-GFP transduced or not (Mock) with the indicated MUC16CAR construct. (D) J76-NFAT-GFP cells expressing the indicated MUC16CAR construct were co-cultured (E:T=1:2) overnight with MUC16 pos (HeLa, OVCAR3) or MUC16 neg (HEK) cell lines, with effector-only (E-only) condition used as a baseline. CAR reactivity was assessed on the basis of GFP expression, N=3 independent experiments, data are mean±SD. (E) As in (C) using primary T cells from N=3–4 healthy donors. (F) Functional activity of primary T cells incubated with the indicated target cells (top: HEK, middle: HeLa, bottom: OVCAR3). Left— Degranulation activity: CAR-transduced T cells were co-cultured with target cells at E:T=1:2 overnight, in the presence of anti-CD107a fluorescent antibody. Target cells were gated out and CD107a was detected on T cells. Data are mean±SD, n=2–4 donors, N=4–6 independent experiments, one-way ANOVA, *p<0.05, **p<0.01. Right —Cytotoxic activity: CAR T cells were tested in bioluminescence-based citotoxicity co-cultures at different E:T ratios, as indicated. Anti-CD19 CAR, FMC63, was included as an irrelevant CAR control. The time point displayed is at 10 hours of co-culture. Data are mean±SD, N=3 independent experiments, two-way ANOVAs with Dunnett’s multiple comparisons, **p<0.01, ***p<0.001. (G) Cytotoxic assay in three-dimensional (3D) cultures (spheroids). Left —Representation of the assay. Right —HeLa and HEK cells were established as spheroids for 5 days. T cells were added, and killing was measured 24 hours later (day 6). Annexin V was recorded in IncuCyte live-cell imaging, and fold increase relative to target cells alone was calculated. Data are mean of normalized values±SD, N=2 different donors, one-way ANOVA, *p<0.05. ANOVA, analysis of variance; bioluminescence-based citotoxicity, bioluminescence-based cytotoxicity; CAR, chimeric antigen receptor; E:T, effector:target ratio; HEK, human embryonic kidney; OC, ovarian cancer; TCGA, The Cancer Genome Atlas.

Article Snippet: As a positive control, we used an anti-CA125 antibody (clone X75, Thermo Fisher Scientific, #MA1-90039) at a dilution of 1/200.

Techniques: Expressing, In Vitro, Gene Expression, Clinical Proteomics, Membrane, Staining, Positive Control, Control, Flow Cytometry, Construct, Cell Culture, Functional Assay, Activity Assay, Incubation, Co-Culture Assay, Live Cell Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

doi: 10.1136/jitc-2023-008179

Figure Lengend Snippet: Cytokine production of MUC16CAR T cells and reactivity against primary patient samples. (A) Cytokine quantification. CAR T cells were co-cultured for 24 hours with target cells at an E:T ratio of 1:2, and the supernatant was collected for Bioplex cytokine assays. Selected cytokines are shown, data are mean±SD, N=3–4 independent measurements, two-way ANOVA. *p<0.05, **p<0.01. (B) Reactivity of CAR T cells against CA125-positive and CAR-negative samples. Tumor samples from patients with OC (effusions or material from debulking surgery) were labeled for CA125 (X75 antibody). Samples were categorized as CA125-positive (n>9), or CA125-negative (n>7), based on the percentage of cells labeling for CA125 (threshold 10%). These samples were co-cultured with CAR T cells overnight at E:T=1:2 in the presence of anti-CD107a. Effector cells were distinguished by prelabeling CAR T cells with Cell Trace Violet. CAR T cells cultured alone were used for normalization. Data are presented as mean±SD, one-way ANOVA. *p<0.05, **p<0.01. ANOVA, analysis of variance; CA125, cancer antigen 125; CAR, chimeric antigen receptor; E:T, effector:target ratio; HEK, human embryonic kidney; IL, interleukin.

Article Snippet: As a positive control, we used an anti-CA125 antibody (clone X75, Thermo Fisher Scientific, #MA1-90039) at a dilution of 1/200.

Techniques: Cell Culture, Labeling

Journal: Journal for Immunotherapy of Cancer

Article Title: Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

doi: 10.1136/jitc-2023-008179

Figure Lengend Snippet: MUC16CAR T cell activity is unaffected by soluble antigen. Bioluminescence-based cytotoxicity assays in the presence of recombinant CA125 (300 kU/L) were performed at an E:T ratio of 10:1. Data are mean±SD, N=5 donors, one-way analysis of variance, with Šidák’s multiple comparisons test. ****p<0.0001. CA125, cancer antigen 125; CAR, chimeric antigen receptor; E:T, effector:target ratio; HEK, human embryonic kidney; ns, not significant.

Article Snippet: As a positive control, we used an anti-CA125 antibody (clone X75, Thermo Fisher Scientific, #MA1-90039) at a dilution of 1/200.

Techniques: Activity Assay, Recombinant