Fig 2: EnAd expressing EpCAM BiTE can selectively kill primary human tumour cells from chemotherapy-pretreated patients A, BCytotoxicity of EpCAM+ cells (A) or FAP+ fibroblasts (B), first isolated from three patients' ascites and expanded ex vivo, then incubated with recombinant BiTE, or infected with EnAd or recombinant virus. Cytotoxicity was measured by flow cytometry after 5 days.CInduction of activation marker CD25 on CD3-positive T cells cultured with ascites-derived EpCAM+ and FAP+ cells from (A and B).Data information: Each condition was measured in biological triplicate and represented as mean ± SD. Significance was assessed by comparison to untreated using a one-way ANOVA test with Tukey's post hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure.

Fig 3: Identification of which T cells are responsible for BiTE-mediated cytotoxicity BiTE-mediated T-cell activation of CD4 and CD8 cells 24 h after co-culture of CD3 T cells with DLD cells (5:1) and BiTE-containing supernatant. Activation was assessed by surface expression of CD69 and CD25 and measured by flow cytometry.Proliferative response of CFSE-stained CD4 and CD8 T cells in co-culture with DLD cells and incubated with BiTE-containing supernatants. Fluorescence was measured after 5-day incubation, by FACS analysis.Degranulation of CD4 and CD8 cells following 6-h co-culture with DLD cells and BiTE-containing supernatants. A CD107a-specific antibody is added to the culture media for the duration of the co-culture, and degranulation is assessed by flow cytometry.Cytotoxicity by either the CD4 or CD8 T-cell subset is assessed by LDH release into supernatant, following 24-h incubation of DLD cells with CD4- or CD8-purified T cells (1:5) and BiTE-containing supernatant.Data information: Each condition was measured in biological triplicate and represented as mean ± SD. EpCAM BiTE treatment was compared to control BiTE unless stated otherwise, and significance was assessed using a one-way ANOVA test with Tukey's post hoc analysis, **P < 0.01, ***P < 0.001. Source data are available online for this figure.

Fig 4: EnAd expressing EpCAM BiTE can overcome immune-suppressive effects of pleural effusion and ascites fluid AQuantity of human IL-10 in primary malignant exudates. NS, normal serum; A, ascites fluid; P, pleural effusion.B, CPBMC-derived T cells were incubated with anti-CD3/CD28 beads in normal serum or exudate fluid (50%) from 12 cancer patients. Induction of T-cell activation markers CD69 and CD25 at 24 h (B) and degranulation (CD107a externalisation) at 6 h (C) were analysed using flow cytometry.D, ECD3-purified PBMCs were co-cultured with SKOV3 (5:1) and EpCAM or control BiTE in the presence of normal serum or either pleural effusion or ascites fluid (50%) from seven patients. CD69 and CD25 dual positivity (D) and CD107a externalisation (E) on CD3+ T cells were measured by flow cytometry at 24 and 6 h, respectively.FViability of SKOV3 cells was monitored in real time over 130 h by xCELLigence-based cytotoxicity assay. SKOV3 cells were seeded and incubated with EnAd-SA-ControlBiTE or EnAd-SA-EpCAMBiTE at 16 h. Uninfected cells served as a negative control. CD3-purified PBMCs (5:1) were added 2 h post-infection in the presence of culture medium or either pleural effusion or ascites fluid. Impedance was measured at 10-min intervals.Data information: (A–E) Each condition was measured in biological triplicate and represented as mean ± SD. Significance was assessed by comparison to normal serum using a one-way ANOVA test with Tukey's post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001.Source data are available online for this figure.

Fig 5: EnAd-EpCAMBiTE shows reproducible activity within an expanded cohort of patient malignant peritoneal and pleural exudates A–CUnpurified total cells from ascites or pleural effusions (from seven different patients; pleural effusion, blue; peritoneal ascites, red) were incubated in 50% fluid from the same exudate sample in the presence of free BiTE, EnAd or recombinant virus. After 5 days, the total cell population was harvested, and the number of (A) CD3+ T cells and those which were (B) CD25+ was quantified. (C) The number of EpCAM+ cells was measured using flow cytometry.DRepresentative microscopy images (magnification ×10; scale bar, 100 µm) and flow cytometry analysis of pleural effusion cells of patient 3 (cancer cells and lymphocytes) following treatment with EnAd or EnAd-SA-EpCAM BiTE. Pink, total cells; blue, CD3+ cells; red, CD3+CD25+ cells; black, EpCAM+ cells.EAt 5 days, cytokine levels were measured by LEGENDplex human Th cytokine panel using pleural effusion cultures following incubation with free recombinant BiTE or infection with EnAd or recombinant virus. E:T (CD3+:EpCAM+) ratio upon receipt of patient samples were 0.51 (patient 1), 37.6 (patient 2), 0.39 (patient 3), 4.44 (patient 4), 0.14 (patient 5), 6.12 (patient 6) and 30.6 (patient 7).Data information: Each condition was measured in biological triplicate and represented as mean ± SD. Significance was assessed by comparison to untreated control samples using a one-way ANOVA test with Tukey's post hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure.