Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: Mesothelin and MUC16 are heterogeneously expressed in patient samples and human-derived cancer cell lines. A ) Dot plot of mesothelin (APC) and MUC16 (PE) expression in cells obtained from three patients diagnosed with ovarian cancer. Numbers in each quadrant represent percentages of the total “Alive” cell population as determined by DAPI staining via flow cytometry. ( B ) RNA expression of mesothelin and MUC16 in ovarian and pancreatic PDXs in Log2(TPM+1) units. Expression levels were obtained from the cBioPortal for Cancer Genomics, available through the Center for Patient Derived Models (CPDM) database at Dana-Farber, one-way ANOVA test was used to compare RNA expression levels of mesothelin and MUC16 between pancreatic and ovarian PDXs. ( C, D ) Tissue array of ovarian ( C ) and pancreatic ( D ) tumors together with adjacent normal tissue area, stained for mesothelin and MUC16 by IHC. Each magnified picture details the same tissue area for both stainings. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Adenoc., adenocarcinoma; ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; IHC, immunohistochemistry; meso, mesothelin; MUC16, Mucin16; PDX, patient-derived xenograft, TPM, transcripts per million.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: Derivative Assay, Expressing, Staining, Flow Cytometry, RNA Expression, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: Tandem scFv screening to target mesothelin and MUC16. ( A ) Schematic of the six tandem scFv designs. ( B ) MFI values of soluble FITC-labeled mesothelin (upper panel) and soluble His-tag-labeled MUC16 (lower panel) bound to each CAR construct expressed in Jurkat cells. MFI values were obtained from Jurkat-mCherry + cells. Bars represent the mean±SEM of three technical replicates. ( C, D ) MFI values of soluble mesothelin ( C ), and MUC16 ( D ) bound to each tandem CAR as single staining (empty dot) or sequential double staining (filled dot), where one antigen was added, the cells were washed, and the second antigen was added. For the sequential double staining we stained first with soluble mesothelin, washed, and then we added soluble MUC16. The MFI of the double staining for each antigen is relative to the MFI value of the corresponding single antigen staining. ( E ) Flow cytometry histograms showing mesothelin and MUC16ecto expression in ASPC-1 and OVCAR3 cancer cell lines that endogenously express (endo) or do not express (neg), are knocked out (KO) for, or are artificially transduced (TR) for mesothelin or MUC16ecto. (F) Heat map of GFP MFI values from CAR-Jurkat NFAP-GFP reporter cells exposed to tumor cells for 24 hours. MFI values were obtained from mCherry + CAR-Jurkat NFAT-eGFP cells. ( G and H) Multiparametric representation of the MFI of antigen bound to CAR-Jurkat cells (X and Y values) along with the MFI of the GFP (color scale) and the percentage of GFP + cells (circle size) in CAR-Jurkat NFAT-eGFP mCherry + cells in response to (G) ASPC-1 cells and (H) OVCAR3 cells. MFI values of GFP (color scale) and the percentage of GFP + cells (circle size) are the mean of each CAR-Jurkat NFAT-eGFP reporter cell co-cultures with the different ASPC-1 and OVCAR3 (E) tumor cells, excluding the double negative tumor cells. In B, significance between the different tandem scFv constructs was measured using one-way ANOVA test with Turkey’s multiple comparison test. In C and D, significance between the single and combinatorial staining for each tandem scFv was measured using two-way ANOVA test with Fisher LSD test. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. ANOVA, analysis of variance; CAR, chimeric antigen receptor; FITC, fluorescein Isothiocyanate; Fisher LSD test, Fisher least significant difference test; east Significance difference test; meso, mesothelin; MFI, mean fluorescence intensity; MUC16, Mucin16; MUC16ecto, Mucin16 ectodomain; ns, not significant; scFv, single-chain variable fragment, TanCAR, tandem CAR configuration; UTD, untransduced.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: Labeling, Construct, Staining, Double Staining, Flow Cytometry, Expressing, Comparison, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: TanCAR in vitro antitumor activity, cytokine production, and avidity . ( A, B ) Bar graph showing the percent cytotoxicity after 24 hours of ( A ) ASPC-1 or ( B ) OVCAR3 cancer cells co-culture with CAR-T cells (SS1, 4H11, TanCAR1 or TanCAR3) or UTD-T cells at an E:T ratio of 3:1, assessed by luciferase-based killing assay. Bars represent the mean±SEM of three healthy donors measured in triplicate or duplicate. Stars indicate significant differences between monospecific CAR-T cells and tandem CAR-T cells using a two-tailed Mann-Whitney test. ( C, D ) Heat maps showing the pg/mL concentration of IL-2, IFNγ, TNFα, or GM-CSF in supernatant collected from killing assays after 24 hours of co-culture of CAR-T cells with ( C ) ASPC-1 or ( D ) OVCAR3 cancer cells at an E:T ratio of 10:1. Data is the mean from two healthy donors. In A and B, significance between monospecific CAR-T cells and tandem CAR-T cells using a two-tailed Mann-Whitney test, only statistical significance is depicted on the graph. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. CAR, chimeric antigen receptor; ecto TR , transduced with the MUC16 ectodomain; endo , endogenous expression of the antigen; E:T, effector to target; IFNγ, interferon-gamma; IL, interleukin; KO , CRISPR KO of the endogenous antigen; meso, mesothelin; MUC16, Mucin16; MUC16ecto, Mucin16 ectodomain; neg , endogenously negative for the antigen; TanCAR, tandem CAR configuration; UTD, untransduced.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: In Vitro, Activity Assay, Co-Culture Assay, Luciferase, Two Tailed Test, MANN-WHITNEY, Concentration Assay, Transduction, Expressing, CRISPR

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: TanCAR-T cell binding avidity to tumor cells expressing one or both antigens. ( A ) Schematic of one antigen at a time (left) or two antigens at a time (right panel) hypotheses for the binding of tandem CAR to two antigens ( B ) Bar graph of mesothelin and MUC16ecto MFI values in different cell lines and isotype controls, where H/H stands for Meso H MUC16ecto H ; H/L, Meso H MUC16ecto L ; M/M, Meso M MUC16ecto M ; L/H, Meso L MUC16ecto H ; and L/L, Meso L MUC16ecto L . ( C–G ) Avidity curves showing the ratio of T cells bound relative to UTD to ASPC-1 tumor cells (C, Meso H MUC16ecto H ; D, Meso H MUC16ecto L ; E, Meso M MUC16ecto M ; F, Meso L MUC16ecto H ; G, Meso L MUC16ecto L ) per acoustic force unit applied in picoNewtons (pN). ( H ) Schematic of one antigen-driven (left) or two antigen-driven (right) tandem CAR avidity profile. ( I ) Multiparametric representation of the MFI of mesothelin and MUC16ecto expression in different ASPC-1 shown in 4B (X and Y values) along with the mean ratio of bound TanCAR1-T cells relative to UTD to each ASPC-1 cell type (data from 4C–G) (color scale). ( J ) Avidity curves showing the ratio of TanCAR1-T cells bound relative to UTD to each ASPC-1 tumor cell line, per pN of acoustic force applied. ( K ) Dot plot graph of the ratio of bound TanCAR-T cells at 1000 pN in each ASPC-1 cell line. Stars indicate significant differences as measured by a two-way ANOVA with Fisher’s LSD test in C–G, and as measured by one-way ANOVA with Fisher’s LSD test in K. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, non-significant. ANOVA, analysis of variance; CAR, chimeric antigen receptor; Fisher LSD test, Fisher least significat difference test; meso, mesothelin; MFI, mean fluorescence intensity; MUC16ecto, Mucin16 ectodomain; TanCAR, tandem CAR configuration; UTD, untransduced.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: Binding Assay, Expressing, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: Tandem CAR-T cells overcome tumor heterogeneity. ( A ) Schematic of a mixed tumor cell culture with ASPC-1 Meso endo MUC16 neg , Meso KO MUC16ecto TR , and Meso endo MUC16ecto TR cells. ( B ) Violin plot of the tumor area 96 hours after co-culture with CAR-T or UTD-T cells, relative to time 0, measured via real-time killing assays using an IncuCyte Live-Cell Analysis system. The dotted line at y=1 represents the threshold for tumor clearance (below 1). Each dot in the violin plot represents a replicate of the experiment, with four technical replicates of three healthy donor T cells. ( C ) Bar graph showing the mean absolute number of each tumor cell population at the end of the real-time killing assay in ( B ), per CAR-T cell treatment group. Each color of the stacked bars represents the tumor populations depicted in ( A ). D ) Dot plot of the absolute number of each tumor cell population shown in ( C ), per CAR-T cell treatment group. Each dot represents a replicate of the experiment, with two technical replicates of three healthy donor T cells. ( E, F ) Schematic of the mixture culture of ASPC-1 Meso endo MUC16 neg iRFP+ cells and Meso KO MUC16ecto TR GFP+cells and their meso and MUC16ecto MFI values. ( G–I ) Total tumor area relative to time 0 (start of the co-culture) ( G ), measured using an IncuCyte Live-Cell Analysis system of a real-time cytotoxicity assay with CAR-T cells or UTD-T cells co-cultured for 96 hours 1:1 with a mixture of ASPC-1 Meso endo MUC16 neg iRFP+ cells and Meso KO MUC16ecto TR GFP+cells shown in E; and blue ( H ) and green ( I ) cell area relative to time 0. Curves represent the mean±SEM of three technical replicates of two healthy donors for each treatment group. ( J ) Dot plot of blue (Meso endo MUC16 neg ) and green (Meso KO MUC16ecto TR ) tumor cell area at 96 hours of co-culture with TanCAR1 relative to time 0 from ( H ) and ( I ). ( K–M ) Mixed tumor spheroids were created with Meso endo MUC16 neg iRFP+ (blue) and Meso KO MUC16ecto TR GFP+ (green) cells and treated with CAR T cells. ( I ) Representative images from different time points of mixed-tumor spheroids treated with CAR-T cells. ( L–M ) Violin plots of the ( L ) blue and ( M ) green tumor cell area at 106 hours relative to time 0, measured using an IncuCyte Live-Cell Analysis system. Dots represent technical triplicates and two healthy donors for each treatment group. Differences measured by Mann-Whitney test for each comparison in ( B, J, L, M ), Kruskal-Wallis test for ( D ) and a two-way ANOVA with Fisher’s LSD test for (G–I). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; CAR, chimeric antigen receptor; Fisher LSD test, Fisher least significat difference test; meso, mesothelin; MUC16, Mucin16; MUC16ecto, Mucin16 ectodomain; ns, non-significant; TanCAR, tandem CAR configuration; UTD, untransduced.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: Cell Culture, Co-Culture Assay, Cell Analysis, Cytotoxicity Assay, MANN-WHITNEY, Comparison

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: In vivo antitumor activity of TanCAR1-T in double-positive tumor cells heterogeneous tumor models. ( A ) Schematic of the in vivo experiments in NSG mice engrafted intraperitoneally with OVCAR3 tumor cells and treated 14 days later with CAR-T or UTD-T cells or left untreated (tumor alone). ( B ) Quantification of the flux (photons/s 2 ) from mice treated as indicated. Curves represent the median+IQR of each treatment group, with 14 mice per group treated with T cells from two healthy donors. ( C ) Schematic of the in vivo experiments in NSG mice engrafted subcutaneously with mixed ASPC-1 tumor cells and treated 14 days later with CAR-T or UTD-T cells or left untreated (tumor alone). ( D ) Caliper measurements from mice treated as indicated. Curves represent the mean±SEM of each treatment group, with 14 mice per group, repeated with T cells from two healthy donors. ( E ) Percentages of each tumor cell population from mice in ( D ), at the time of injection compared with the time of tumor collection from the mice. Each dot represents the mean±SEM of each treatment group treated with T cells from one healthy donor: SS1 CAR group contains primary tumor and lung metastasis from one mouse, 4H11 CAR group contains primary tumors from three mice, and TanCAR1 group shows primary tumors from six mice. The statistical analysis from 4H11 and TanCAR1 groups is shown. ( F ) Diagram explaining the skewed killing of TanCAR1-T cells towards tumor cells expressing high levels of one of the cognate antigens. ( G ) Schematic of the in vivo experiment in NSG mice engrafted subcutaneously with mixed ASPC-1 tumor cells, treated 14 days later with CAR-T or UTD-T cells, and euthanized at day 21 after CAR/UTD-T cells injection. ( H ) Representative images of IHC slides of tumors from mice after 21 days of CAR/UTD-T cell administration. Lower panels are magnifications of highlighted area in the upper panels. ( I ) Dot plot graph showing CD3 + cells per mm 2 in each group of treated mice. Differences measured by a two-way ANOVA test with Fisher’s LSD test for B and D, a Mann-Whitney test for E and a one-way ANOVA with Holm-Šídák’s multiple comparisons test for I. Only comparisons between CAR-T cells (TanCAR1, SS1CAR, and 4H11CAR) are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; CAR, chimeric antigen receptor; IHC, immunohistochemistry; IP, intraperitoneal; IV, intravenous; Fisher LSD test, Fisher least significat difference test; meso, mesothelin; MUC16, Mucin16; MUC16ecto, Mucin16 ectodomain; ns, non-significant; TanCAR, tandem CAR configuration; UTD, untransduced.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: In Vivo, Activity Assay, Injection, Expressing, MANN-WHITNEY, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Hemizygous nonsense variant in the moesin gene (MSN) leads to a new autoimmune phenotype of Immunodeficiency 50

doi: 10.3389/fimmu.2022.919411

Figure Lengend Snippet: Identical MSN gene variants in P1 and P2: c.650G>A p.Trp217Ter, W217X (sixth exon). The father (F) , maternal grandmother, and uncles carry wild-type alleles. The mother (M) is heterozygote. Family tree: white, wild-type; dotted, heterozygote, black, hemizygote.

Article Snippet: The expression level of moesin (Hs01085682_g1) was determined by RT-qPCR on QuantSudioTM 1 (Applied Biosystems, Waltham, MA, USA), and the samples were normalized to β-actin (Hs99999903_m1) mRNA level.

Techniques:

Journal: Frontiers in Immunology

Article Title: Hemizygous nonsense variant in the moesin gene (MSN) leads to a new autoimmune phenotype of Immunodeficiency 50

doi: 10.3389/fimmu.2022.919411

Figure Lengend Snippet: Relative quantity of MSN mRNA in PBMCs of healthy controls, mothers, and patients. Data are mean ± SEM for n = 4/healthy control and triplicates/mother, patients. * p < 0.05 control vs. mother, P1, and P2, based on one-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: The expression level of moesin (Hs01085682_g1) was determined by RT-qPCR on QuantSudioTM 1 (Applied Biosystems, Waltham, MA, USA), and the samples were normalized to β-actin (Hs99999903_m1) mRNA level.

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Hemizygous nonsense variant in the moesin gene (MSN) leads to a new autoimmune phenotype of Immunodeficiency 50

doi: 10.3389/fimmu.2022.919411

Figure Lengend Snippet: MSN protein detection. Western blot analysis showing the presence of moesin in PMBCs of healthy controls (C) and the patients’ mothers (M) . Beta-actin was used as the loading control.

Article Snippet: The expression level of moesin (Hs01085682_g1) was determined by RT-qPCR on QuantSudioTM 1 (Applied Biosystems, Waltham, MA, USA), and the samples were normalized to β-actin (Hs99999903_m1) mRNA level.

Techniques: Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Hemizygous nonsense variant in the moesin gene (MSN) leads to a new autoimmune phenotype of Immunodeficiency 50

doi: 10.3389/fimmu.2022.919411

Figure Lengend Snippet: Clinical and laboratory comparison of previously studied MSN deficiencies (PP1–PP9) vs. current subjects (P1 and P2).

Article Snippet: The expression level of moesin (Hs01085682_g1) was determined by RT-qPCR on QuantSudioTM 1 (Applied Biosystems, Waltham, MA, USA), and the samples were normalized to β-actin (Hs99999903_m1) mRNA level.

Techniques: Comparison, Mutagenesis