ADENOVIRUS ARMED WITH BISPECIFIC T CELL ENGAGER (BITE)

Abstract

A modified adenovirus, in particular Enadenotucirev (EnAd), armed with at least two bispecific T cell engager (BiTE?) each comprising at least two binding domains, wherein at least one of the domains is specific for a surface antigen on an immune cell of interest, such as a T-cell of interest. Also provided are a composition, such as a pharmaceutical formulation comprising the virus, use of the virus and virus formulations for treatment, such as in the treatment of cancer. The disclosure also extends to processes for preparing the virus.

Claims

1. An adenovirus comprising a sequence of formula (I):
5ITR-B.sub.1-B.sub.A-B.sub.2-B.sub.X-B.sub.B-B.sub.Y-B.sub.3-3ITR(I) wherein: B.sub.1 is bond or comprises: E1A, E1B or E1A-E1B; B.sub.A comprises-E2B-L1-L2-L3-E2A-L4; B.sub.2 is a bond or comprises: E3; B.sub.X is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both; B.sub.B comprises L5; B.sub.Y is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both; B.sub.3 is a bond or comprises: E4; wherein the adenovirus encodes a first Bispecific T cell Engager (BiTE) comprising at least two binding domains, Bd1 and Bd2, wherein at least one of the said domains, such as Bd1, is specific to a surface antigen on an immune cell, such as a T cell and a second BiTE comprising at least two binding domains, Bd3 and Bd4, and at least one of said binding domains, such as Bd3, is specific to a surface antigen on an immune cell, such as a T cell; and wherein the adenovirus is EnAd or Ad11.

2. An adenovirus according to claim 1, wherein the adenovirus is EnAd.

3. An adenovirus according to claim 1 or 2, wherein the surface antigen is a component of the T-cell receptor complex (TCR), such as selected from CD3, TCR-? and TCR-?.

4. An adenovirus according to claim 3, wherein the surface antigen is CD3 such as CD3?, CD3? and CD3?, in particular CD3?.

5. An adenovirus according to claim 1 or 2, wherein one of the binding domains in the BiTE is specific to a non-TCR activating protein such as CD31, CD2 and CD277.

6. An adenovirus according to any one of claims 1 to 5, wherein binding domain in the first BiTE (such as Bd1) and a binding domain in the second BiTE (such as Bd3) are specific to the same surface antigen on the immune cell, such as a T cell.

7. An adenovirus according to any one of claims 1 to 5, wherein binding domain in the first BiTE (such as Bd1) and the binding domain in the second BiTE (such as Bd2) are specific to the different surface antigens, for example on the same or different immune cells.

8. An adenovirus according to any one of claims 1 to 7, wherein Bd2 in said first BiTE and Bd4 in said second BiTE bind the same antigen of interest.

9. An adenovirus according to any one of claims 1 to 7, wherein the Bd2 in said first BiTE and Bd4 in said second BiTE bind different antigens of interest.

10. An adenovirus according to any one of claims 1 to 9, wherein one of the binding domains is specific to a tumour antigen, such as CEA, MUC-1, EpCAM, HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigens Le.sup.y, Le.sup.x, Le.sup.b, PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3.

11. An adenovirus according to claim 10, wherein one of the binding domains, for example Bd2 and/or Bd4, is specific to EpCAM.

12. An adenovirus according to any one of claims 1 to 11, wherein one of the binding domains is specific to a tumour stromal antigen, for example fibroblast activation protein (FAP), TREM1, IGFBP7, FSP-1, platelet-derived growth factor-? receptor (PDGFR-?), platelet-derived growth factor-? receptor (PDGFR-?) and vimentin.

13. An adenovirus according to claim 12, wherein one of the binding domains, for example Bd2 and/or Bd4, is specific to FAP.

14. A adenovirus according to claim 12 or 13, wherein the stromal antigen is an antigen is selected from a myeloid derived suppressor cell antigen, a tumor associated macrophage, and combinations thereof.

15. An adenovirus according to claim 14, wherein the antigen is selected from CD163, CD206, CD68, CD11c, CD11b, CD14, CSF1 Receptor, CD15, CD33, CD66b and a combination of two or more of the same.

16. An adenovirus according to any one of claims 1 to 15, wherein at least one of B.sub.X or B.sub.Y is not a bond.

17. An adenovirus according to any one of claims 1 to 16, wherein the adenovirus is chimeric.

18. An adenovirus according to any one of claims 1 to 17, wherein the adenovirus is oncolytic.

19. An adenovirus according to any one of claims 1 to 18, wherein the adenovirus replication capable.

20. An adenovirus according to claim 19, wherein the adenovirus is replication competent.

21. An adenovirus according to any one of claims 1 to 18, wherein the adenovirus is replication deficient.

22. An adenovirus according to any one of claims 1 to 21, wherein the first BiTE and the second BiTE are encoded in the same location in the adenovirus.

23. An adenovirus according to any one of claims 1 to 21, wherein the first BiTE and the second BiTE are encoded in different locations within the adenovirus.

24. An adenovirus according to any one of claims 1 to 23, wherein at least one BiTE is encoded in a region selected from E1, E3, B.sub.X, B.sub.Y and combinations thereof.

25. An adenovirus according to claim 24, wherein a BiTE is encoded at least in position B.sub.X, for example under the control of an exogenous promoter such as a CMV promoter.

26. An adenovirus according to claim 24 or 25, wherein a BiTE is encoded at least in position By, for example under the control of an endogenous promoter, such as the major late promoter or under the control of an exogenous promoter, such as the CMV promoter.

27. An adenovirus according to any one of claims 1 to 26, for example wherein both BiTEs are encoded in position B.sub.Y.

28. An adenovirus according to claim 26 or 2, wherein a transgene or transgene cassette is under the control of an endogenous promoter, the major late promoter.

29. An adenovirus according to any one of claims 1 to 28, wherein the BiTE has a short half-life, for example 48 hours or less.

30. An adenovirus according to any one of claims 1 to 29, wherein a binding domain in the first BiTE (such as Bd2) is specific to a tumour antigen, for example a tumor antigen disclosed herein, and a binding domain in the second BiTE (such as Bd4) is specific to a tumour stromal antigen, for example a stromal antigen on a fibroblast or tumour associated macrophage or a myeloid derived suppressor cells, for example as described herein.

31. An adenovirus according to any one of the claims 1 to 29 wherein an encoded BiTE comprises a VH domain comprising an amino acid sequence as set forth in SEQ ID NOs: 8, or an amino acid sequence that is at least 95% identical thereto and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 9 or a sequence at least 95% identical thereto

32. An adenovirus according to claim 31, wherein the encoded BiTE comprises and scFv comprising an amino acid sequence as set forth in SEQ ID NO: 7 or a sequence at least 95% identical thereto.

33. An adenovirus according to any one of the claims 1 to 30, wherein the encoded BiTE comprises a VH domain comprising an amino acid sequence as set forth in SEQ ID NOs: 8, or an amino acid sequence that is at least 95% identical thereto and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 7 or a sequence at least 95% identical thereto.

34. An adenovirus according to claim 32, wherein the encoded BiTE comprises and scFv comprising an amino acid sequence as set forth in SEQ ID NO: 7 or a sequence at least 95% identical thereto

35. An adenovirus according to any one of the claims 1 to 33, wherein the encoded BiTE comprises a VH domain comprising an amino acid sequence as set forth in SEQ ID NOs: 13, or an amino acid sequence that is at least 95% identical thereto and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 12 or a sequence at least 95% identical thereto.

36. An adenovirus according to claim 34, wherein the encoded BiTE comprises and scFv comprising an amino acid sequence as set forth in SEQ ID NO: 11, 74, 75 or a sequence at least 95% identical to any one of the same.

37. An adenovirus according to any one of the claims 1 to 35, wherein the encoded BiTE comprises a VH domain comprising an amino acid sequence as set forth in SEQ ID NOs: 18, or an amino acid sequence that is at least 95% identical thereto and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 17 or a sequence at least 95% identical thereto.

38. An adenovirus according to claim 36, wherein the encoded BiTE comprises and scFv comprising an amino acid sequence as set forth in SEQ ID NO: 16, 72, 73 or a sequence at least 95% identical to any one of the same.

39. An adenovirus according to any one of claims 1 to 38, wherein the adenovirus comprises a sequence shown in SEQ ID NO: 120.

40. An adenovirus according to any one of claims 1 to 39, wherein the adenovirus encodes at least one further transgene, for example 1, 2, 3 or 4 further transgenes.

41. An adenovirus according to claim 39, wherein the further transgene(s) encodes a cytokine, chemokine and/or an immunomodulator, for example encoded in position B.sub.Y.

42. An adenoviruses according to claim 39 or 40, wherein at least one further transgene encodes a cytokine, for example selected from MIP1?, IL-1?, IL-1?, IL-6, IL-9, IL-12, IL-13, IL-17, IL-18, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-33, IL-35, IL-2, IL-4, IL-5, IL-7, IL-10, IL-15, IL-21, IL-25, IL-1RA, IFN?, IFN?, IFN?, TNF?, lymphotoxin ? (LTA), Flt3L, GM-CSF and IL-8.

43. An adenovirus according to any one of claims 39 to 41, wherein at least one further transgene encodes a chemokine, for example selected from CCL2, CCL3, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19 and CCL21.

44. A composition comprising an adenovirus according to any one of claims 1 to 42, and a diluent or carrier.

45. A composition according to claim 43, wherein the formulation comprises a second oncolytic virus, for example Ad11 or a derivative thereof, such as EnAd, in particular wherein the additional virus is according to the present disclosure.

46. A method of treating a patient comprising administering a therapeutically effective amount of an adenovirus of any one of claims 1 to 43 or a composition of claim according to claim 44 or 45.

47. A method according to claim 45, for the treatment of cancer, in particular a solid tumour.

Description

DESCRIPTION OF THE FIGURES

[0612] FIG. 1 (A) schematic representation of a BiTE antibody of the present disclosure comprising or lacking an optional decahistidine affinity tag. Ig SP: signal peptide; 10His: decahistidine affinity tag; L: GS linker; V.sub.L: variable light domain; V.sub.H variable heavy domain. (B) plasmid map for pSF-CMV-EpCAMBiTE. (C) plasmid map for pSF-CMV-FAPBiTE. (D) plasmid map for pSF-CMV-ControlBiTE.

[0613] FIG. 2 (A) dot blot showing the quantification of the recombinant BiTEs. (B) shows a graph showing the ELISA results for FAP. (C) graph showing the ELISA results for EpCAM.

[0614] FIG. 3 shows a graph showing the expression levels of CD69 (A) and CD25 (B) for T cells co-cultured alone or with NHDF cells in the presence of FAP BiTE and control BiTE measured using flow cytometry.

[0615] FIG. 4 (A) graph showing the levels of IFN ? expression for T cells co-cultured alone or with NHDF cells in the presence of FAP BiTE and control BiTE measured by intracellular cytokine staining. Graphs (B) & (C) show the expression levels of CD69 and CD25 for T cells co-cultured alone or with DLD cells in the presence of EpCAM BiTE and control BiTE measured using flow cytometry.

[0616] FIG. 5 (A) graph showing the levels of IFN ? expression for T cells co-cultured with DLD cells in the presence of EpCAM BiTE and control BiTE measured by intracellular cytokine staining. Graphs (B) & (C) showing the levels of CD69 and CD25 for PBMCs co-cultured with DLD cells in the presence of EpCAM BiTE and control BiTE measured by flow cytometry.

[0617] FIG. 6 (A) graph showing the results of a LDH assay showing the cytoxicity of NHDF cells which have been co-cultured with T cells and FAP BiTE or control BiTE. (B) graph showing the results of a LDH assay showing the cytoxicity of BTC100 cells which have been co-cultured with T cells and FAP BiTE or control BiTE. (C) Images of NHDF cells after co-culture with T cells and FAP BiTE vs control BiTE.

[0618] FIG. 7 (A) scatter plots showing FAP expression in multiple patient-derived cells. (B) graph showing the % of cells expressing EpCAM and FAP across multiple cells and cell lines.

[0619] FIG. 8 (A) graph showing the NHDF dose response for FAP BiTE with increasing BiTE concentration. Graph (B) & (C) showing the results of a LDH assay showing the cytoxicity of DLD cells which have been co-cultured with T cells and EpCAM BiTE or control BiTE.

[0620] FIG. 9 (A) graph showing the results of a LDH assay showing the cytoxicity of SKOV cells which have been co-cultured with T cells and EpCAM BiTE or control BiTE. (B) graph showing the results of a LDH assay showing the cytoxicity of MCF7 cells which have been co-cultured with T cells and EpCAM BiTE or control BiTE.

[0621] FIG. 10 shows a graph showing the NHDF dose response for EpCAM BiTE with increasing BiTE concentration.

[0622] FIG. 11 (A) graph showing FAP expression in CHO cells determined by FAP or isotope control antibody and analysed by flow cytometry. (B) shows a graph showing the results of a LDH assay showing the cytoxicity of CHO or CHO-FAP cells which have been co-cultured with T cells and FAP BiTE or control BiTE.

[0623] FIG. 12 shows a graph showing T-cell activation (based on CD69 and CD25 expression levels) by CHO vs CHO-FAP cells, analysed using flow cytometry.

[0624] FIG. 13 (A) graphs showing EpCAM expression of the parental cell lines vs stable transfected variant determined by staining with EpCAM or isotope control antibody and analysed using flow cytometry. (B) graph showing the results of a LDH assay showing the cytoxicity of CHO or CHO-EpCAM cells which have been co-cultured with T cells and EpCAM BiTE or control BiTE.

[0625] FIG. 14 shows graph showing T-cell activation (based on CD69 and CD25 expression levels) by CHO vs CHO-EpCAM cells, analysed using flow cytometry.

[0626] FIG. 15 (A) graph showing the ability of FAP BiTE to activate CD4+ or CD8+ T-cells (based on CD69 and CD25 expression levels), analysed using flow cytometry. (B) graph showing the results of a LDH assay showing the cytoxicity of NHDF cells which have been co-cultured with CD4+ or CD8+ T cells and FAP BiTE or control BiTE.

[0627] FIG. 16 (A) graph showing CD4+ and CD8+ T-cell activation (based on CD69 and CD25 expression levels) by DLD cells in the presence of EpCAM or control BiTE analysed using flow cytometry. (B) graph showing the results of a LDH assay showing the cytoxicity of DLD cells which have been co-cultured with CD4+ or CD8+ T cells and EpCAM BiTE or control BiTE.

[0628] FIG. 17 (A) graph showing the number of CD3+ T cells from ascites cultured with control or FAP BiTE. (B) graph showing the CD25 expression levels of T cells from ascites cultured with control or FAP BiTE. (C) graph showing the number of FAP+ cells from ascites cultured with control or FAP BiTE.

[0629] FIG. 18 (A) schematic representation of the genome of the adenoviruses of the present disclosure. (B) graphs comparing the kinetics of CMV vs SA promoter driven expression.

[0630] FIG. 19 (A) graph showing the quantification of the number of detected virus genomes per cell for NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 and EnAd. (B) graphs showing the oncolytic activity of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 or EnAd assessed by infection of A549 cells.

[0631] FIG. 20 (A) graphs showing T-cell activation (based on CD69 and CD25 expression levels) by NG-601, NG-602, NG-605 and NG-606 when co-cultured with CHO-FAP, analysed using flow cytometry. (B) graphs showing T-cell activation (based on CD69 and CD25 expression levels) by NG-601, NG-602, NG-605 and NG-606 when co-cultured with CHO-EpCAM, analysed using flow cytometry.

[0632] FIG. 21 shows graphs showing the results of experiments to determine the quantity of FAP BiTE produced from NG-605 and NG-606.

[0633] FIG. 22 shows graphs showing the results of experiments to determine the quantity of EpCAMBiTE produced from NG-601 and NG-602.

[0634] FIG. 23 shows microscopy images of Ad293 cells infected with NG-607, NG-608, NG-609 and NG-610.

[0635] FIG. 24 (A) graph indicating the cytotoxicity of DLD cells infected with EnAd, analysed using XCELLigence. (B) graph indicating the cytotoxicity of SKOV cells infected with EnAd, analysed using XCELLigence. (C) graph indicating the cytotoxicity of NHDF cells infected with EnAd, analysed using XCELLigence.

[0636] FIG. 25 (A) graph indicating the ability of NG-603, NG-604, NG-605, NG-606 and EnAd to kill NHDF cells, analysed using XCELLigence. (B) graph indicating the ability of NG-603, NG-604, NG-605, NG-606 and EnAd to kill NHDF cells, analysed using an LDH assay.

[0637] FIG. 26 shows graphs showing T-cell activation (based on CD69 and CD25 expression levels) by NG-603, NG-604, NG-605, NG-606 co-cultured with NHDF cells, SKOV and T cells, analysed using flow cytometry.

[0638] FIG. 27 (A) graph showing T-cell activation (based on CD69 and CD25 expression levels) by NG-603, NG-604, NG-605, NG-606 co-cultured with NHDF and SKOV cells vs. SKOV alone, analysed using flow cytometry. (B) graph indicating the cytotoxicity of NHDF cells infected with NG-605 and NG-606, analysed using an LDH assay

[0639] FIG. 28 shows still frame images from timelapse videos of lysis of NHDF cells by recombinant FAP BiTE, EnAd, NG-603 or NG-605.

[0640] FIG. 29 shows still frame images from timelapse videos of lysis of NHDF cells by NG-607, NG-608, NG-609 or NG-610.

[0641] FIG. 30 shows a graph indicating the cytotoxicity of DLD cells infected with EnAd, NG-601, NG-602, NG-603 and NG-604 in the presence of T cells or absence of T cells, analysed using XCELLigence.

[0642] FIG. 31 shows a graph indicating the cytotoxicity of DLD cells infected with EnAd, NG-601, NG-602, NG-603 and NG-604 in the presence of T cells or absence of T cells, analysed using an LDH assay.

[0643] FIG. 32 shows a graph showing T-cell activation (based on CD69 and CD25 expression levels) by EnAd, NG-601, NG-602, NG-603 and NG-604, analysed by flow cytometry.

[0644] FIG. 33 shows the results of experiments to determine the ability of NG-601 to kill DLD tumour cells at varying multiplicity of infection (MOI) in the presence or absence of CD3.sup.+ T-cells, assessed using xCELLigence.

[0645] FIG. 34 shows graphs indicating the ability of EnAd and NG-601, NG-602, NG-603 and NG-604 to kill SKOV tumour cells in the presence or absence of CD3.sup.+ T-cells, assessed using xCELLigence.

[0646] FIG. 35 shows graphs indicating the ability of EnAd and NG-601, NG-602, NG-603 and NG-604 to kill SKOV tumour cells in the presence or absence of CD3.sup.+ T-cells, assessed using an LDH assay.

[0647] FIG. 36 shows a graph showing T-cell activation (based on CD69 and CD25 expression levels) by EnAd, NG-601, NG-602, NG-603 and NG-604 co-cultured with SKOV tumour cells, analysed using flow cytometry.

[0648] FIG. 37 shows a graph showing T-cell activation (based on CD69 and CD25 expression levels) by EnAd, NG-601, NG-602, NG-603 and NG-604 co-cultured with ascites cells, analysed using flow cytometry.

[0649] FIG. 38 shows still frame images from timelapse videos of lysis of NHDF cells by EpCAM BiTE, EnAd, NG-601 or NG-603.

[0650] FIG. 39 shows microscopy images of ascites cells obtained from a patient, infected with viruses of the present disclosure and stained with EnAd-CMV-GFP and EnAd-SA-GFP as a reporters to determine infection and late stage viral gene expression.

[0651] FIG. 40 (A) graph indicating the expression levels of CD25 on CD3+ T cells in ascites samples which were infected with viruses of the present disclosure. (B) graph indicating the number of FAP+ cells in ascites samples which were infected with viruses of the present disclosure.

[0652] FIG. 41 shows microscopy images of ascites cells obtained from a cancer patient, infected with viruses of the present disclosure and stained with EnAd-CMV-GFP and EnAd-SA-GFP as a reporters to determine infection and late stage viral gene expression.

[0653] FIG. 42 shows a graph indicating the number of CD3+ T cells in ascites samples obtained from a cancer patient and infected with viruses of the present disclosure.

[0654] FIG. 43 shows a graph indicating the CD25 expression levels on CD3+ T cells in ascites samples obtained from a cancer patient and infected with viruses of the present disclosure.

[0655] FIG. 44 shows a graph indicating the number of FAP+ cells in ascites samples obtained from a cancer patient and infected with viruses of the present disclosure.

[0656] FIG. 45 Characterisation of EpCAM BiTE and its effects on PBMC-derived T cells (A) Schematic of the structure of the EpCAM-targeted BiTE and non-specific control BiTE. The VL and VH domains are connected with flexible peptide linkers (L) rich in serine and glycine for flexibility and solubility. Ig SP, Light chain immunoglobulin signal peptide; 10His, decahistidine affinity tag. (B) Induction of activation markers CD69 and (C) CD25 on CD3-purified PBMC cultured alone or with DLD cells (5:1) in the presence of BiTE-containing supernatants. CD69 and CD25 were measured by flow cytometry after 24 h of co-culture. Significance was assessed versus IgG isotype (D) Percent of IFN?-positive T-cells after 6 h in co-culture with DLD cells (5:1) and BiTE-containing supernatants. (E) Proliferation, represented by division index and percentage of parental T cell population entering proliferation, of CFSE-stained T-cells in co-culture with DLD cells (5:1) and BiTE-containing supernatants. Fluorescence was measured by flow cytometry 5 days after co-culture. Division index was modelled using FlowJo proliferation tool. (F) Degranulation of T-cells, measured by CD107a externalisation, in co-culture with DLD cells (5:1) and BiTE-containing supernatants. Externalisation was assessed by co-culture with a CD107a-specific antibody for 6 h followed by flow cytometry analysis. (G) Cytokine levels were measured by LEGENDplex human Th cytokine panel using supernatants from co-cultures of T-cells with DLD cells (5:1) in the presence of BiTE-containing supernatants for 48 h. Each condition was measured in biological triplicate and data represented as mean?SD. Significance was assessed versus untreated unless stated otherwise using a one-way ANOVA test with Tukey's Post Hoc analysis, *p<0.05, **p<0.01, ***p<0.001.

[0657] FIG. 46 Characterisation of recombinant EpCAM BiTE (A) Dot blot to estimate the quantity of EpCAM BiTE produced by transfected HEK293A cells. (B) ELISA measuring the level of EpCAM binding by controls or recombinant EpCAM or non-specific BiTE. Significance was assessed by comparison to empty vector control sample using a one-way ANOVA test with Tukey's Post Hoc analysis, ***p<0.001

[0658] FIG. 47 Assessment of antigen-specificity of EpCAM BiTE-mediated T cell cytotoxicity (A) Induction of activation marker CD25 on CD3+ T-cells in co-culture with CHO or CHO-EpCAM cells (5:1) and BiTE-containing supernatants, measured by FACS analysis after 24 h of co-culture. (B) Cytotoxicity of CHO or CHO-EpCAM cells cultured with BiTE-containing supernatants alone or in coculture with T-cells. Cytotoxicity was assessed by release of LDH into the culture supernatants after 24 h of incubation. (C) Cytotoxicity of multiple EpCAM-positive carcinoma cells after 24 h in co-culture with T-cells (1:5) and BiTE-containing supernatants. Viability was measured by MTS assay after 24 h of co-culture. (D) Levels of EpCAM expression (N=1) assessed by FACS analysis of EpCAM-positive cell lines in (C), compared to background fluorescence measured by using an isotype control antibody. (AC) Each condition was measured in biological triplicate and represented as mean?SD. Significance was assessed versus untreated or T-cell only controls using a one-way ANOVA test with Tukey's Post Hoc analysis, *p<0.05, **p<0.01, ***p<0.001.

[0659] FIG. 48 Cytotoxicity of EnAd-expressing EpCAM BiTE in SKOV3 cells SKOV3 cells were incubated with EnAd or recombinant viruses in the absence (A) or presence (B) of T cells and cytotoxicity was measured by LDH release at the specified time-points. Significance was assessed by comparison to uninfected control wells using a one-way ANOVA test with Tukey's Post Hoc analysis, ***p<0.001

[0660] FIG. 49 Identification of which T cells are responsible for BiTE-mediated cytotoxicity (A) 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. (B) 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 days incubation, by FACS analysis. (C) 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. (D) 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. 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<0.05, **p<0.01, ***p<0.001.

[0661] FIG. 50 Cytotoxicity and T cell activation by EnAd-expressing EpCAM BiTE in DLD cells Cytotoxicity for infected DLD cells in absence (A) or presence of T-cells (B). DLD cells were infected and co-cultured with T-cells and cytotoxicity was measured by LDH release at the specified timepoints. (C-D) T-cells from (B) were harvested and stained for activation markers CD69 (C) or CD25 (D) and analysed via flow cytometry. (E-F) Quantification of EpCAM BiTE-produced from DLD cells infected with recombinant viruses. Standard curve of LDH released (Abs) of DLD in co-culture with CD3+ cells and varying known quantities of recombinant EpCAM BiTE (E). In parallel, co-cultures were incubated with diluted supernatants (10,000-fold) from 3 day infected DLD cells (F). Standard curve allowed the approximate determination of EpCAM BiTE produced at 165 ?g and 50 ?g per million DLD cells for EnAd-CMV-EpCAMBiTE and EnAd-SA-EpCAMBiTE, respectively. Significance was assessed by comparison to uninfected control wells using a one-way ANOVA test with Tukey's Post Hoc analysis, ***p<0.001

[0662] FIG. 51 Characterisation of oncolytic virus EnAd expressing EpCAM BiTE using cell lines and PBMC derived T cells (A) DLD cells were infected with parental EnAd or recombinant virus (100 vp/cell) and wells harvested at 24 or 72 h. Replication was assessed by measuring genomes using qPCR against viral hexon. (B) Cytotoxicity of DLD cells infected with EnAd or recombinant virus at increasing concentrations of virus. Cytotoxicity was measured by MTS assay after 5 days infection. (C) Supernatants from day 3 uninfected or virus-infected HEK293A cells were assessed for transgene expression by immunoblot analysis and probed with an anti-His antibody. (D) Induction of activation marker CD25 of CD3-positive T-cells cultured with CHO or CHO-EpCAM (E:T 5:1) and diluted HEK293A supernatants from (D). Activation was measured by surface expression of CD25 by flow cytometry. (E) Cytotoxicity of CHO or CHO-EpCAM cells incubated with HEK293A supernatants from (D) alone or in co-culture with CD3-purified PBMC (E:T 5:1). HEK293A supernatants were diluted 300-fold. Cytotoxicity was assessed by LDH released into the supernatant after 24 h incubation. Each condition was measured in biological triplicate and represented as mean?SD. Significance was assessed using a one-way ANOVA test with Tukey's Post Hoc analysis with each condition compared to untreated, *p<0.05, **p<0.01, ***p<0.001.

[0663] FIG. 52 Cellular composition of the malignant exudates (A) Representative image (pleural effusion sample, Patient 3 from FIG. 57) demonstrating screening of ascites and exudate fluids for their cellular composition, as assessed by flow cytometry. (B) Absolute number of each cell type (in 10,000 cell sample size) is documented in the table.

[0664] FIG. 53 Superior potency of EnAd expressing EpCAM BiTE in partially EnAd-resistant cancer cell line (A-B) Viability of SKOV3 cells were monitored in real-time over 160 h by xCELLigence-based cytotoxicity assay. SKOV3 cells were seeded and infected with EnAd or BiTE-armed EnAd viruses at 0 h, with uninfected cells serving as a negative control. In (B) CD3-purified PBMC (5:1) were added 2 h post-infection and impedance was measured at 15 min intervals. (C-D) CD3-purified PBMC were cultured with SKOV3 cells (5:1) that were infected with parental EnAd or recombinant armed viruses. At each time-point, T cells were harvested and analysed for surface expression of CD69 (C) or CD25 (D) by flow cytometry. (E) Time-lapse sequences showing co-cultures of SKOV3 carcinoma cells (unstained), NHDF fibroblasts (red) and CD3-purified PBMC (blue), infected with EnAd, EnAd-CMVEpCAMBiTE or uninfected. Apoptosis was visualised using CellEvent Caspase 3/7 detection reagent (green). Images were taken on a Nikon TE 2000-E Eclipse inverted microscope at intervals of 15 min covering a period of 96 h. Representative images were recorded at the times displayed; original magnification ?10; scale bar 100 ?m. (A-D) Each condition was measured in biological triplicate and represented as mean?SD. Significance was assessed by comparison to uninfected control using a oneway ANOVA test with Tukey's Post Hoc analysis, *p<0.05, **p<0.01, ***p<0.001.

[0665] FIG. 54 Expression of PD1 and the effect of PD1 antibodies on BiTE-mediated T cell activation (A) The expression of PD1 by endogenous T cells following their initial isolation from pleural effusions was assessed by flow cytometry. (B-D) Unpurified total cells from pleural effusions (from three different patients) were incubated in 100% fluid from the same pleural exudate in the presence of free BiTE, EnAd or recombinant virus. After 5 days, the total cell population was harvested, and the number of (B) CD3+ T cells and those which were (C) CD25+ were quantified. (D) The number of EpCAM+ cells was measured using flow cytometry. Significance was assessed by comparison to untreated control wells using a one-way ANOVA test with Tukey's Post Hoc analysis, ***p<0.001

[0666] FIG. 55 EnAd expressing EpCAM BiTE can selectively kill primary human tumour cells from chemotherapy-pretreated patients (A) Cytotoxicity of EpCAM+ cells or (B) FAP+ fibroblasts, 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. (C) Induction of activation marker CD25 on CD3-positive T-cells cultured with ascites derived EpCAM+ and FAP+ cells from (A+B). 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.

[0667] FIG. 56 EpCAM BiTE can overcome immune suppressive effects of ascites fluid and activate endogenous T cells (A-B) PBMC-derived T cells were incubated with anti-CD3 antibodies in RPMI culture medium or the presence of 100% peritoneal ascites fluid from five ovarian cancer patients. (A) At 24 h induction of T cell activation markers CD69 and CD25 were analysed, and (B) degranulation of T-cells measured by CD107a externalisation, using flow cytometry. (C) Viability of MCF7 cells were monitored in real-time over 60 h by xCELLigence-based cytotoxicity assay. MCF7 cells were seeded and incubated with control or EpCAM BiTE at 25 h, in the presence of RPMI medium or 100% ascites fluid #1 or #2. Untreated cells served as a negative control. CD3-purified PBMC (5:1) were added at the same time and impedance was measured at 15 min intervals. (D) Endogenous unpurified total cells from peritoneal ascites were incubated in 100% ascites fluid in the presence of free EpCAM or control BiTE. After 24 h, the total cell population was harvested, and the number of CD3+/CD69+ and CD3+/CD25+ cells measured by flow cytometry. Each condition was measured in biological triplicate and represented as mean?SD. Significance was assessed by comparison to RPMI (A+B), untreated (D) or control BiTE (E) using a oneway ANOVA test with Tukey's Post Hoc analysis, *p<0.05, **p<0.01, ***p<0.001.

[0668] FIG. 57 EnAd expressing EpCAM BiTE can activate endogenous T cells to kill endogenous tumour cells within malignant pleural exudates Unpurified total cells from pleural effusions (from four different patients) were incubated in 100% fluid from the same pleural exudate 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+ were quantified. (C) The number of EpCAM+ cells was measured using flow cytometry. (D) Representative images (magnification ?10; scale bar 100 ?m) and flow cytometry analysis of pleural effusion cells of Patient 3 (cancer cells and lymphocytes) following treatment with EnAd or EnAd-CMVEpCAM BiTE. (E) At 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. 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.

[0669] FIG. 58 shows quantity of IL-10 measured in normal serum (NS) or patient malignant exudate fluids (A: peritoneal ascites, P: pleural effusions) using Human IL-10 ELISA MAX kit (Biolegend, 430604).

[0670] FIG. 59 shows CD3/28 bead-mediated PBMC T-cell activation (based on CD69/CD25 levels) in patient fluids vs normal serum measured by flow cytometry. A: patient exudate fluid, P: pleural fluid.

[0671] FIG. 60 shows CD3/28 bead-mediated PBMC T-cell degranulation (based on CD107a expression) in patient fluids. A: ascites, P: pleural fluid.

[0672] FIG. 61 shows the correlation between IL-10 levels in patient fluids and CD3/CD28 bead-mediated T-cell degranulation.

[0673] FIG. 62 shows EpCAM BiTE bead-mediated PBMC T-cell activation (based on CD69/CD25 expression) in patient fluids. A: ascites, P: pleural fluid.

[0674] FIG. 63 shows EpCAM BiTE bead-mediated PBMC T-cell degranulation (based on CD107a expression) in patient fluids. A: ascites, P: pleural fluid.

[0675] FIG. 64 shows EpCAM BiTE bead-mediated cytotoxicity of SKOV3 in patient fluids. A: ascites, P: pleural fluids.

[0676] FIG. 65 shows EpCAM BiTE-mediated T-cell activation (based on CD25/CD69 expression) in RPMI media vs ascites fluid.

[0677] FIG. 66 shows the ability of EnAd-SA-EpCAMBiTE and EnAd-SA-ControlBiTE to induce T cell-mediated target cell lysis in RPMI media vs ascites fluid. ((A) number of CD3+. (B) CD25 expression of T-cells. (C) number of EpCAM+ cells determined by flow cytometry.

[0678] FIG. 67 shows the ability of EnAd-SA-EpCAMBiTE and EnAd-SA-ControlBiTE to induce T cell-mediated target cell lysis in ascites fluid (7 patient samples). (A) number of CD3+. (B) CD25 expression of T-cells. (C) number of EpCAM+ cells determined by flow cytometry. See FIG. 67A for legend.

[0679] FIG. 68 shows a comparison of activation of T-cell cytokine production by recombinant FAP BiTE protein in the presence of human fibroblasts and by polyclonal activation with anti-CD3/CD28 beads. (A) IFN? levels measured by ELISA. (B) Cytokine levels measured by cytokine bead array.

[0680] FIG. 69 FAP-targeted BiTE induces T-cell degranulation and specific cytotoxicity of FAP+ cells (A) Degranulation of T-cells in culture with NHDF cells (5:1) and (B) BiTE-containing supernatants. Degranulation was assessed by externalisation of CD107a following 6 h culture with a CD107a-specific antibody and measured by flow cytometry. CD3/CD28 Dynabeads were used as a positive control. (C) Cytotoxicity of NHDF cells after 24 h in co-culture with T-cells (1:5) and 10-fold serial dilutions of BiTE-containing supernatants. Cytotoxicity was assessed by release of LDH into culture supernatants. (D) Lysis of NHDF by LDH release (left) and CD25 induction on T-cells (right) was assessed after 24 h co-culture with PBMC-derived T-cells (1:5) from six healthy donors and BiTE-containing supernatants.

[0681] FIG. 70 EnAd expressing FAP BiTE selectively kills FAP.sup.+ fibroblasts and decreases TGFb in peritoneal ascites samples (A,B) Number of FAP.sup.+ fibroblasts (A) and EpCAM.sup.+ tumour cells (B) after 72 h culture with PBMC-derived T-cells and EnAd or recombinant viruses. Ascites cells were first isolated from three patients ascites and expanded ex vivo. Cell number was measured at 72 h post-infection by flow cytometry. (C) Induction of activation marker CD25 on PBMC-derived CD3 cells from (A) was measured at 72 h post-infection. (D) Levels of TGFb were measured by ELISA using supernatants harvested from (A).

[0682] FIG. 71 shows the activation of endogenous tumor associated T-cells and associated killing of FAP+ cells in patient malignant ascites biopsy samples by FAP BiTE protein and EnAd-FAPBiTE viruses. (A) T cell activation measured by CD25 expression. (B) residual number of FAP+ cells measured by flow cytometry.

[0683] FIG. 72 Effect of PD-L1 blocking antibodies on BiTE-mediated T cell activation in patient sample (A) Expression of PD1 by endogenous T cells and PD-L1 on FAP+ cells following their initial isolation from peritoneal ascites was assessed by flow cytometry. (B) Unpurified total cells from peritoneal ascites were incubated in 50% fluid from the same exudate in the presence of free BiTE, EnAd or recombinant virus, with or without anti-PD-L1 blocking antibody. After 2 days, the total cell population was harvested, and the number of CD25+ T-cells was quantified by flow cytometry. (C) Quantity of interferon gamma in culture supernatants from (B, D) measured by ELISA. (D) The number of residual FAP+ cells in (B) was measured using flow cytometry.

[0684] FIG. 73 EnAd expressing BiTEs activate and redirect T-cells from patient biopsy samples to lyse NHDF fibroblasts (A) The expression of PD-1 by endogenous T cells following isolation from healthy donors or malignant exudate cancer biopsy samples. PD-1 expression was measured by flow cytometry. (B) The proportion of CD3+ cells within the unpurified cell population of PBMC and cancer biopsy samples as measured by flow cytometry. (C) Levels of interferon gamma measured by ELISA in culture supernatants harvested from (B) at 120 h post-treatment. (D) Viability of NHDF fibroblasts were monitored in real time over 130 h by xCELLigence cytotoxicity assay in co-culture with PBMC or total cancer biopsy cells (1:5) and BiTE-containing supernatant.

[0685] FIG. 74 shows the effect of immunosuppressive ascites fluid samples on FAP BiTE- and anti-CD3/CD28 bead-mediated activation of PBMC T-cells. (A) PBMC T cells activated with anti-CD3/Cd28 Dynabeads. (B) PBMC T cells activated with control or FAP BITEs in the presence of NHDF cells. NS: normal serum, A: peritoneal ascites.

[0686] FIG. 75 FAP BiTE expressing EnAd polarises CD11b.sup.+ macrophage in patient ascites to a more inflammatory phenotype (A) Unpurified total cells from ascites sample were incubated in 50% ascites fluid in the presence of free BiTE or BiTE expressing virus. Interferon gamma treatment was used as a positive control. After 3 days, the total cell population was harvested and the induction of activation marker CD25 on CD3+ cells was measured by flow cytometry. (B) Levels of interferon gamma in culture supernatants from (A) were measured by ELISA. (C) At 3 days, the expression levels of CD68, CD86, CD206 and CD163 on CD11b+ cells from (A) were measured by flow cytometry. Representative flow cytometry spectra from triplicates is shown alongside the complete data set.

[0687] FIG. 76 Characterisation of architecture and cellular composition of solid prostate tumour (A) EpCAM staining, (B) CD8 staining, (C) FAP staining. (D) Representative immunohistochemistry images of CD25 induction within prostate tumour slices following treatment with BiTE expressing viruses. Tumour cores were sliced at 300 uM thickness with a Leica vibratome, cultured and infected in inserts and harvested after 7 days treatment. (E) Levels of IFNg in tissue slice culture medium measured by ELISA. Supernatants were harvested from slices cultures of malignant and benign tissue at the specified time-point. (F) Levels of IL-2 in tissue culture medium of malignant and benign tissue measured by ELISA.

[0688] FIG. 77A-C shows a schematic representation of the transgene cassettes used in Example 33.

[0689] FIG. 77D shows a graph indicating the number of viral genomes detected per cell in NG-611, NG-612 and NG-617 treated tumour cells.

[0690] FIG. 78 shows the percentage of T cells expressing CD69 (a), CD25 (b) HLA-DR (c), CD40L (d) or cell surface CD107a (e) following co-culture with EpCam expressing SKOV cells and supernatants harvested from A549 cells at 24, 48 or 72 hrs post-treatment with NG-611 virus particles compared to NG-612, enadenotucirev or untreated control supernatants.

[0691] FIG. 79 shows the percentage of T cells expressing CD69 (a), CD25 (b) HLA-DR (c), CD40L (d) or cell surface CD107a (e) following co-culture with FAP expressing MRC-5 cells and supernatants harvested from A549 cells at 24, 48, or 72 hrs post-treatment with NG-612 virus particles compared to NG-611, enadenotucirev or untreated control supernatants.

[0692] FIG. 80 shows the percentage of MRC-5 cells that express EpCAM and FAP

[0693] FIG. 81 shows IFN? expression in the supernatants of T cell co-cultures with SKOV cells (A) or MRC-5 cells (B) incubated with supernatants harvested from A549 cells at 24, 48 or 72 hrs post-treatment with NG-611, NG-612 or enadenotucirev virus particles, or untreated control supernatants.

[0694] FIG. 82 shows anti-tumour efficacy and immune activation of BiTE expressing viruses in vivo. (a) tumour volume in mice treated with saline, enadenotucirev or NG-611. (b) Ratio of CD8 to CD4 T cells in NG-611 treated tumours compared to enadenotucirev treated or untreated controls.

[0695] FIG. 83 shows schematic representation of transgene cassettes. (a) NG-615, (b) NG-640, (c) NG-641.

[0696] FIG. 84 shows a graph indicating the number of viral genomes detected per cell in NG-612 and NG-615 treated tumour cells

[0697] FIG. 85 shows the expression of IFN?, MIP1? and Flt3 L in the cellular supernatant of NG-615 vs the supernatant of enadenotucirev and untreated control tumour cells.

[0698] FIG. 86 shows the number of T cells expressing CD69 (a), CD25 (b) HLA-DR (c), CD40L (d) or cell surface CD107a (e)) following co-culture with FAP expressing MRC-5 cells and supernatants harvested from A549 cells at 24, 48 or 72 hrs post-treatment with NG-615 virus particles compared to NG-612, enadenotucirev or untreated control supernatants.

[0699] FIG. 87 shows IFN? expression in the supernatants of T cell co-cultures with MRC-5 cells incubated with supernatants harvested from A549 cells at 24, 48 or 72 hrs post-treatment with NG-612, NG-615 or enadenotucirev virus particles, or untreated control supernatants.

[0700] FIG. 88 shows schematic representation of the NG-618 transgene cassette

[0701] FIG. 89 shows the detection of surface FAP expression on MRC-5 cells (a) or EpCam expression on SKOV cells (b) following incubation with supernatants harvested from A549 cells at 72 hrs post-treatment with NG-611, NG-612, NG-615 or enadenotucirev virus particles.

[0702] FIG. 90 shows the percentage of T cells expressing CD24 (a), CD40L (b) or cell surface CD107a (c) following co-culture with FAP expressing MRC-5 cells and supernatants harvested from A549 cells at 72 hrs post-treatment with NG-618 virus particles compared to enadenotucirev or untreated controls.

[0703] FIG. 91 shows the percentage of T cells expressing CD24 (a), CD40L (b) or cell surface CD107a (c) following co-culture with EpCam expressing SKOV cells and supernatants harvested from A549 cells at 72 hrs post-treatment with NG-618 virus particles compared to enadenotucirev or untreated controls.

[0704] FIG. 92 shows the percentage of dead MRC-5 (a) or SKOV (b) cells following co-culture with T cells and supernatants harvested from A549 cells at 72 hrs post-treatment with NG-618 virus particles compared to enadenotucirev or untreated controls.

SEQUENCES

[0705] SEQ ID NO: 1 Anti-EpCAM BiTE DNA coding sequence, with N-terminal signal sequence and C-terminal deca-His affinity tag [0706] SEQ ID NO: 2 Anti-EpCAM BiTE protein sequence, with N-terminal signal sequence and C-terminal deca-His affinity tag [0707] SEQ ID NO: 3 Anti-FAP BiTE DNA coding sequence, with N-terminal signal sequence and C-terminal deca-His affinity tag [0708] SEQ ID NO: 4 Anti-FAP BiTE amino acid sequence, with N-terminal signal sequence and C-terminal deca-His affinity tag [0709] SEQ ID NO: 5: Control (Anti-FHA) BiTE DNA coding sequence, with N-terminal signal sequence and C-terminal deca-His affinity tag [0710] SEQ ID NO: 6: Control (Anti-FHA) BiTE amino acid sequence with N-terminal signal sequence and C-terminal deca-His affinity tag [0711] SEQ ID NO: 7: Anti-CD3 ScFv amino acid sequence [0712] SEQ ID NO: 8: Anti-CD3 VH [0713] SEQ ID NO: 9: Anti-CD3 VL [0714] SEQ ID NO: 10: Anti-CD3 ScFv linker sequence [0715] SEQ ID NO: 11: Anti-FAP ScFv [0716] SEQ ID NO: 12: Anti-FAP VL domain [0717] SEQ ID NO: 13: Anti-FAP VH domain [0718] SEQ ID NO: 14: Anti-FAP and Anti-EpCAM linker sequence [0719] SEQ ID NO: 15: BiTE leader sequence [0720] SEQ ID NO: 16: Anti-EpCAM ScFv [0721] SEQ ID NO: 17: Anti-EpCAM VL [0722] SEQ ID NO: 18: Anti-EpCAM VH [0723] SEQ ID NO: 19: Control BiTE (Anti-FHA) [0724] SEQ ID NO: 20: Control (Anti-FHA) ScFv [0725] SEQ ID NO: 21: Control (Anti-FHA) VL [0726] SEQ ID NO: 22: Control (Anti-FHA) VH [0727] SEQ ID NO: 23: Control (Anti-FHA) ScFv linker sequence [0728] SEQ ID NO: 24: Deca-His Tag sequence [0729] SEQ ID NO: 25: FAP BiTE-P2A-RFP (ITALICS=leader, BOLD=furin cleavage site, UNDERLINE=P2A sequence, lower case=RFP) [0730] SEQ ID NO: 26: Control (Anti-FHA) BiTE-P2A-RFP (ITALICS=leader, BOLD=furin cleavage site, UNDERLINE=P2A sequence, lower case=RFP) [0731] SEQ ID NO: 27: Human EpCAM DNA coding sequence [0732] SEQ ID NO: 28: Human EpCAM amino acid sequence [0733] SEQ ID NO: 29: Human FAP DNA coding sequence [0734] SEQ ID NO: 30: Human FAP amino acid sequence [0735] SEQ ID NO: 31: CMV promoter sequence [0736] SEQ ID NO: 32: SV40 late polyadenylation sequence [0737] SEQ ID NO: 33: Null sequence [0738] SEQ ID NO: 34: Null sequence [0739] SEQ ID NO: 35: Null sequence [0740] SEQ ID NO: 36: Null sequence [0741] SEQ ID NO: 37: Null sequence [0742] SEQ ID NO: 38 EnAd genome [0743] SEQ ID NO: 39 B.sub.X DNA sequence corresponding to and including bp 28166-28366 of the EnAd genome [0744] SEQ ID NO: 40 B.sub.Y DNA sequence corresponding to and including bp 29345-29379 of the EnAd genome [0745] SEQ ID NO: 41 HIS-Tag [0746] SEQ ID NO: 42 Splice acceptor sequence. [0747] SEQ ID NO: 43 SV40 poly Adenylation sequence [0748] SEQ ID NO: 44 EpCam BiTE nucleic acid sequence (OKT3) [0749] SEQ ID NO: 45 FAP BiTE nucleic acid sequence (OKT3) [0750] SEQ ID NO: 46 FAP BiTE nucleic acid sequence (aCD3) [0751] SEQ ID NO: 47 NG-611 Transgene cassette [0752] SEQ ID NO: 48 NG-612 Transgene cassette [0753] SEQ ID NO: 49 NG-613 Transgene cassette [0754] SEQ ID NO: 50 Restriction site insert (B.sub.X) [0755] SEQ ID NO: 51 Restriction site insert (B.sub.Y) [0756] SEQ ID NO: 52 CMV promoter sequence [0757] SEQ ID NO: 53 PGK promoter sequence [0758] SEQ ID NO: 54 CBA promoter sequence [0759] SEQ ID NO: 55 short splice acceptor (SSA) DNA sequence [0760] SEQ ID NO: 56 splice acceptor (SA) DNA sequence [0761] SEQ ID NO: 57 branched splice acceptor (bSA) DNA sequence [0762] SEQ ID NO: 58 Kozak sequence (null sequence) [0763] SEQ ID NO: 59 Example of start codon [0764] SEQ ID NO: 60 Internal Ribosome Entry Sequence (IRES) [0765] SEQ ID NO: 61 P2A peptide [0766] SEQ ID NO: 62 F2A peptide [0767] SEQ ID NO: 63 E2A peptide [0768] SEQ ID NO: 64 T2A peptide [0769] SEQ ID NO: 65 polyadenylation (polyA) sequence [0770] SEQ ID NO: 66 Leader sequence [0771] SEQ ID NO: 67 Leader sequence [0772] SEQ ID NO: 68 IFN? amino acid sequence [0773] SEQ ID NO: 69 IFN? amino acid sequence [0774] SEQ ID NO: 70 TNF? amino acid sequence [0775] SEQ ID NO: 71 DNA sequence corresponding to E2B region of the EnAd genome (bp 10355-5068) [0776] SEQ ID NO: 72: Anti-EpCAM BiTE DNA coding sequence, with N-terminal signal sequence and C-terminal deca-His affinity tag [0777] SEQ ID NO: 73: Anti-EpCAM BiTE protein sequence, with N-terminal signal sequence without C-terminal deca-His affinity tag [0778] SEQ ID NO: 74: Anti-FAP BiTE DNA coding sequence, with N-terminal signal sequence without C-terminal deca-His affinity tag [0779] SEQ ID NO: 75: Anti-FAP BiTE amino acid sequence, with N-terminal signal sequence without C-terminal deca-His affinity tag [0780] SEQ ID NO: 76: Control (Anti-FHA) BiTE DNA coding sequence, with N-terminal signal sequence without C-terminal deca-His affinity tag [0781] SEQ ID NO: 77: Control (Anti-FHA) BiTE amino acid sequence with N-terminal signal sequence without C-terminal deca-His affinity tag [0782] SEQ ID NO: 78: Control BiTe (Anti-FHA) without C-terminal deca-His affinity tag [0783] SEQ ID NO: 79: Null sequence [0784] SEQ ID NO: 80: Null sequence [0785] SEQ ID NO: 81: Null sequence [0786] SEQ ID NO: 82: Null sequence [0787] SEQ ID NO: 83: EpCam BiTE nucleic acid sequence (OKT3) [0788] SEQ ID NO: 84: Null sequence [0789] SEQ ID NO: 85: FAP BiTE nucleic acid sequence (OKT3) [0790] SEQ ID NO: 86: Null sequence [0791] SEQ ID NO: 87: FAP BiTE nucleic acid sequence (aCD3) [0792] SEQ ID NO: 88: NG-611 Transgene cassette [0793] SEQ ID NO: 89: NG-612 Transgene cassette [0794] SEQ ID NO: 90: NG-613 Transgene cassette [0795] SEQ ID NO: 91: NG-614 Transgene cassette [0796] SEQ ID NO: 92: NG-617 Transgene cassette [0797] SEQ ID NO: 93: EpCam BiTE amino acid sequence (OKT3) [0798] SEQ ID NO: 94: FAP BiTE amino acid sequence (OKT3) [0799] SEQ ID NO: 95: FAP BiTE amino acid sequence (aCD3) [0800] SEQ ID NO: 96: Null sequence [0801] SEQ ID NO: 97: Null sequence [0802] SEQ ID NO: 98: Null sequence [0803] SEQ ID NO: 99: Null sequence [0804] SEQ ID NO: 100: Null sequence [0805] SEQ ID NO: 101: Null sequence [0806] SEQ ID NO: 102: Null sequence [0807] SEQ ID NO: 103: Null sequence [0808] SEQ ID NO: 104: Null sequence [0809] SEQ ID NO: 105: Flt3L nucleic acid sequence [0810] SEQ ID NO: 106: Null sequence [0811] SEQ ID NO: 107: MIP1? nucleic acid sequence [0812] SEQ ID NO: 108: Flexible linker sequence [0813] SEQ ID NO: 109: IFN? nucleic acid sequence [0814] SEQ ID NO: 110: CXCL10 nucleic acid sequence [0815] SEQ ID NO: 111: CXCL9 nucleic acid sequence [0816] SEQ ID NO: 112: NG-615 Transgene cassette [0817] SEQ ID NO: 113: NG-640 Transgene cassette [0818] SEQ ID NO: 114: NG-641 Transgene cassette [0819] SEQ ID NO: 115: FLT3L amino acid sequence [0820] SEQ ID NO: 116: MIP1? amino acid sequence [0821] SEQ ID NO: 117: IFN? amino acid sequence [0822] SEQ ID NO: 118: CXCL9 amino acid sequence [0823] SEQ ID NO: 119: CXCL10 amino acid sequence [0824] SEQ ID NO: 120: NG-618 Genome [0825] SEQ ID NO: 121: NG-618 EpCam BiTE nucleic acid sequence [0826] SEQ ID NO: 122: NG-618 FAP BiTE nucleic acid sequence [0827] SEQ ID NO: 123: NG-618 Transgene cassette [0828] SEQ ID NO: 124 to 297 are linker sequences

EXAMPLES

[0829] NG-601 adenovirus encoding a EpCam BiTE in position BY under the control of a CMV promoter [0830] NG-602 adenovirus encoding a EpCam BiTE and splice acceptor in position BY [0831] NG-605 adenovirus encoding a FAP BiTE in position BY under the control of a CMV promoter [0832] NG-606 adenovirus encoding a FAP BiTE and splice acceptor in position BY [0833] NG-611 adenovirus encoding a EpCam BiTE and SSA in position BY [0834] NG-612 adenovirus encoding a FAP BiTE and SSA in position BY [0835] NG-613 adenovirus encoding a FAP BiTE and SA in position BY [0836] NG-614 adenovirus encoding a FAP BiTE and SA in position BY (with different CD3 specificity to NG-613) [0837] NG-615 adenovirus encoding a FAP BiTE, FLt3 Ligand, interferon alpha, MIP alpha, ans SSA in position BY. [0838] NG-616 adenovirus encoding a FAP BiTE, FLt3 Ligand, interferon alpha, MIP alpha, ans SA in position BY. [0839] NG-617 adenovirus encoding FAP BiTE and SSA in position BY [0840] NG-640 adenovirus encoding FAP BiTE, CXCL10, CXCL9 and SSA in position BY. [0841] NG-641 adenovirus encoding FAP BiTE, CXCL10, CXCL9 and interferon alphap in position BY.

Example 1

[0842] Recombinant BiTEs were designed and proteins produced as described in this example.

BITE Engineering

[0843] BiTEs are generated by joining two single chain antibody fragments (ScFv) of different specificities with a flexible Gly.sub.4Ser linker. ScFv's are created by the joining of V.sub.H and V.sub.L domains from parental monoclonal antibodies by a linker. Each BiTE was designed with an N-terminal signal sequence for mammalian secretion and a C-terminal decahistidine affinity tag for detection and purification. BiTEs were engineered by standard DNA cloning techniques and inserted into protein expression vectors (FIG. 1). The anti-EpCAM BiTE is that from patent WO 2005040220 (SEQ ID NO: 63 therein), with a signal sequence and affinity tag added. The anti-FAP BiTE was created de novo using the anti-FAP ScFv from patent WO2010037835A2 and the anti-CD3 ScFv from patent WO 2005040220 (SEQ ID 63 therein), with a signal sequence and affinity tag added. A control BiTE used the anti-FHA (filamentous hemagglutinin from Bordetella pertussis) ScFv from Hussein et al, 2007 (Hussein A H et al (2007) Construction and characterization of single-chain variable fragment antibodies directed against the Bordetella pertussis surface adhesins filamentous hemagglutinin and pertactin. Infect Immunity 75, 5476-5482) and the anti-CD3 ScFv from patent WO 2005040220 (SEQ ID NO: 63 therein), with a signal sequence and affinity tag added. The DNA coding and amino acid sequences for these BiTEs are SEQ ID NOs: 1-6.

Recombinant BiTE Production

[0844] Recombinant BiTE proteins were produced by cloning the respective sequences into the pSF-CMV vector using a CMV promoter (SEQ ID NO: 31) to drive protein expression (FIG. 1). The concentration of plasmid DNA for plasmids, pSF-CMV-EpCAMBiTE, pSF-CMV-FAPBiTE and pSF-CMV-ControlBiTE (Table 2), were measured via NanoDrop. Empty pSF-CMV vector is included as a negative control. 54.7 ?g of each was diluted with 4 mL OptiMEM. 109.2 ug PEI (linear, MW 25000, Polysciences, USA) were diluted in 4 mL OptiMEM medium and mixed with the 4 ml of diluted DNA to generate DNA-PEI complexes (DNA:PEI ratio of 1:2 (w/w)). After incubation at room temperature for 20 minutes, the complex mixture was topped up to 18 mL with OptiMEM and this transfection mixture was added to a T175 flask containing Ad293 cells at 90% confluency. After incubation of the cells with the transfection mix for 4 hrs at 37? C., 5% CO.sub.2, 30 mL of cell media (DMEM high glucose with glutamine supplemented, phenol red-free) was added to the cells and the flasks was incubated 37? C., 5% CO.sub.2 for 48 hours. Another flask of cells was transfected in parallel with pSF-CMV-GFP to ensure efficient transfection efficiency. In order to harvest secreted protein, the supernatant of transfected cells was collected and centrifuged at 350 g at 4? C. for 5 minutes to remove cell components (Allegra X-15R, Beckman Coulter). Supernatants were transferred to 10 k MWCO Amicon Ultra-15 Centrifugal Filter Units (Millipore). After spinning at 4750 rpm and 4? C., the volume of the retentate was adjusted with the flow through to obtain a 50-fold higher concentration. Aliquots of concentrated protein were stored at ?80? C.

TABLE-US-00002 TABLE 2 p employed as a prefix in naming constructs indicates that the construct is a plasmid. Coding Sequence [plasmid DNA] Plasmid ID SEQ ID NO: ng/ml pSF-CMV-EpCAMBiTE SEQ ID NO: 1 3717 pSF-CMV-FAPBiTE SEQ ID NO: 3 6700 pSF-CMV-ControlBiTE SEQ ID NO: 5 5300 pSF-Lenti-EpCAM SEQ ID NO: 27 2529.3 pSF-Lenti-FAP SEQ ID NO: 29 659.6

Recombinant BiTE Detection

[0845] To detect the BiTE, the C-terminal decahistidine affinity tag can be probed with an anti-His antibody using the technique of western blotting. Protein samples were adjusted with lysis buffer to a final volume of 15 ?L including 2.5 ?L 6? Laemmli SDS Sample Buffer which contains ?-mercaptoethanol and SDS. Samples were incubated for 5 minutes at 95? C. to denature proteins and loaded onto 15-well 10% precast polyacrylamide gels (Mini-PROTEAN TGX Precast Gels, BioRad, UK). Gels were run at 180 V for 45 minutes in 1? running buffer within a Mini-PROTEAN Tetra System (BioRad, UK). Proteins from the SDS gels were transferred onto nitrocellulose membranes by wet electroblotting at 300 mA and 4? C. for 90 minutes in 1? transfer buffer within a Mini Trans-Blot Cell (BioRad, UK). Transfer was performed in presence of an ice pack to limit heat. The nitrocellulose membrane was then blocked with 5% milk in PBS-T on a shaker for 1 hour at room temperature, and probed with anti-His (C-term) antibody (mouse ?-6?His, clone 3D5, Invitrogen, UK, #46-0693), diluted 1:5000 in PBS/5% milk. After incubation on a shaker overnight at 4? C., the membrane was washed and probed with HRP-labelled polyclonal secondary ?-mouse-immunoglobulin-antibody (1:10.000 in PBS/5% milk, Dako, #P0161) for 1 hour at room temperature. For visualization, SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, UK) was applied, following manufacturer's instructions and exposed to X-ray film and developed in an automatic film processor. The results demonstrated the expression and secretion of BiTE protein from Ad293 cells transfected with the BiTE expression plasmids, but not the parental vector.

Recombinant BiTE Quantification

[0846] To measure the quantity of recombinant BiTE protein, the technique of dot blot was used to compare the BiTE signal to a His-tagged (C-term 10His) protein standard (10?His-tagged human Cathepsin D, Biolegend, #556704). Two-fold serial dilutions of BiTE samples and protein standard were prepared, and 1.5 uL of each directly applied to a nitrocellulose membrane and air-dried for 20 minutes. The blocking and staining protocol described above for western blotting was then performed. The molar concentration of the protein standard was adjusted to represent a BiTE concentration of 250 ?g/mL. The results (FIG. 2A) demonstrated the expression and secretion of BiTE protein from Ad293 cells transfected with the BiTE expression plasmids.

FAP Binding ELISA

[0847] The FAP-binding activity of the FAP BiTE and control (anti-FHA) BiTE (SEQ ID NOs: 4 and 6) secreted from cells transfected with pSF-CMV-FAPBiTE or pSF-CMV-ControlBiTE was assessed by enzyme-linked immunosorbent assay (ELISA). Empty pSF-CMV vector supernatants were included as a negative control. ELISA plates (Nunc Immuno MaxiSorp 96 well microplate) were prepared by coating overnight at 4? C. with human FAP/seprase protein (100 ng/well, Sino Biological Inc, 10464-H07H-10) in PBS buffer. Plates were washed between all subsequent binding steps with PBS 0.05% Tween 20. The plates were blocked for 1 hour at room temperature with 5% BSA in PBS 0.05% Tween 20. Aliquots of BiTE protein, or protein harvested from empty pSF-CMV vector-transfected wells, were diluted 10-fold into PBS/5% BSA/0.05% Tween 20. All samples were added to the FAP coated plates and incubated for 2 hr at room temperature. The detection antibody, anti-His (C-term) antibody (mouse anti-6?His, clone 3D5, Invitrogen, UK, #46-0693), was diluted 1:1000 and applied for 1 hour at room temperature. HRP conjugated anti-mouse-Fc (1:1000 in PBS/5% milk, Dako) was then applied for 1 hr at room temperature before HRP detection was performed with HRP substrate solution 3.3.5.5-teramethylethylenediamine (TMB, Thermo-Fisher). Stop solution was used for terminating the reaction and the developed colour was measured at 450 nm on a plate reader. Absorbance at 450 nm was plotted for FAP BiTE, control BiTE and empty vector supernatants, demonstrating specific binding of the FAP BiTE to FAP protein. The results (FIG. 2B) show the specific binding of the FAP BiTE and not control BiTE to recombinant FAP protein.

EpCAM Binding ELISA

[0848] The EpCAM-binding activity of the EpCAM BiTE and control BiTE (SEQ ID NOs: 2 and 6) secreted from cells transfected with pSF-CMV-EpCAMBiTE or pSF-CMV-ControlBiTE was assessed by enzyme-linked immunosorbent assay (ELISA). Empty pSF-CMV vector supernatants are included as a negative control. ELISA plates (A Nunc Immuno MaxiSorp 96 well microplate) were prepared by coating overnight at 4? C. with human EpCAM/TROP-1 protein (50 ng/well, Sino Biological Inc, #10694-H02H-50) in PBS buffer. Plates were washed between all subsequent binding steps with PBS 0.05% Tween 20. The plates were blocked for 1 hour at room temperature with 5% BSA in PBS 0.05% Tween 20. Aliquots of BiTE protein, or protein harvested from empty pSF-CMV vector-transfected wells, were diluted 10-fold into PBS/5% BSA/0.05% Tween 20. All samples were added to the EpCAM coated plates and incubated for 2 hr at room temperature. The detection antibody anti-His (C-term) antibody (mouse anti-6?His, clone 3D5, Invitrogen, UK, #46-0693) was diluted 1:5000 and applied for 1 hour at room temperature. HRP conjugated anti-mouse-Fc (1:1000 in PBS/5% milk, Dako,) was then applied for 1 hr at room temperature before HRP detection was performed with HRP substrate solution 3.3.5.5-teramethylethylenediamine (TMB, Thermo-Fisher). Stop solution was used for terminating the reaction and the developed colour was measured at 450 nm on a plate reader. Absorbance at 450 nm was plotted for EpCAM BiTE, control BiTE and empty vector supernatants demonstrating specific binding of EpCAM BiTE to recombinant EpCAM. The results (FIG. 2C) show the specific binding of the EpCAM BiTE and not control BiTE to recombinant EpCAM protein.

Example 2

[0849] The functional activities of recombinant BiTE proteins were assessed in a number of different assays prior to constructing BiTE transgene-bearing EnAd viruses.

Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)

[0850] Human PBMCs were isolated by density gradient centrifugation either from fresh human blood samples of healthy donors or from whole blood leukocyte cones, obtained from the NHS Blood and Transplant UK in Oxford. In either case, the samples were diluted 1:2 with PBS and 25 mL of this mixture was layered onto 13 mL Ficoll (1.079 g/mL, Ficoll-Paque Plus, GE Healthcare) in a 50 mL Falcon tube. Samples were centrifuged (Allegra X-15R, Beckman Coulter) at 1600 rpm for 30 minutes at 22? C. with the lowest deceleration setting to preserve phase separation. After centrifugation, 4 layers could be observed which included a plasma layer at the top, followed by an interface containing PBMCs, a Ficoll layer and a layer of red blood cells and granulocytes at the bottom. The PBMCs were collected using a Pasteur pipette and washed twice with PBS (1200 rpm for 10 minutes at room temperature) and re-suspended in RPMI medium supplemented with 10% FBS.

Isolation of CD3-Positive T-Cells

[0851] CD3-positive (CD3+) T-cells were extracted from PBMCs by depletion of non-CD3 cells using a Pan T Cell Isolation Kit (Miltenyi Biotec, #130-096-535), according to the manufacturer's protocol.

Processing Primary Ascites Samples

[0852] Primary human ascites samples were received from the oncology ward of the Churchill Hospital (Oxford University Hospitals) from patients with multiple indications, including but not limited to ovarian, pancreatic, breast and gastric cancer. Upon receipt, cellular and fluid fractions were separated, with aliquots of fluid frozen at ?20? C. for storage and future analysis. The cellular fraction was treated with red blood cell lysis buffer (Roche, #11814389001) to remove red blood cells, following the manufacturer's instructions. Cell types present in each sample was determined by staining for EpCAM, EGFR, FAP, CD45, CD11b, CD56, CD3, CD4, CD8, PD1 and CTLA4 and analysed by flow cytometry. Cells were then used fresh for ex vivo T-cell activation and target cell lysis experiments. In some cases, the cells were passaged in DMEM supplemented with 10% FBS for use in later experiments.

Cell Line Maintenance

[0853] All cell lines were maintained in DMEM (Sigma-Aldrich, UK) or RPMI medium (Sigma-Aldrich, UK) as specified in Table 3, supplemented with 10% (v/v) foetal bovine serum (FBS, Gibco?) and 1% (v/v) Penicillin/Streptomycin (10 mg/mL, Sigma-Aldrich, UK), in a humidified incubator (MCO-17AIC, Sanyo) at 37? C. and 5% CO.sub.2, unless otherwise specified. Cells were split every 2 to 3 days before reaching confluency by enzymatic dissociation with Trypsin/EDTA (0.05% trypsin 0.02% EDTA, Sigma-Aldrich, UK). In this process, culture medium was aspirated and cells were washed with 15 ml of PBS and subsequently cells were treated with 2 mL of Trypsin/EDTA for 2-10 minutes at 37? C. Trypsin was neutralized with 10 mL of DMEM containing 10% FBS and a portion of the cells was transferred into new flasks containing fresh medium. For routine cell culture, media was supplemented with 10% FBS, for infections and virus plasmid transfections with 2% FBS and for recombinant BiTE plasmid transfections with no FBS supplement.

TABLE-US-00003 TABLE 3 Culturing Cell line Origin of cells Media Source Ascites-derived Human primary ascites DMEM NHS Blood & cell lines Transplant UK BTC100 Human primary lung DMEM University of cancer-associated Oxford fibroblasts (CAF) CHO-K1 Chinese hamster ovary, RPMI ATCC adherent CHO-K1 stable Chinese hamster ovary, RPMI cell lines adherent DLD1 Human colorectal RPMI ATCC adenocarcinoma HEK 293A Human embryonic DMEM ATCC kidney, adherent HEK 293A Human embryonic DMEM stable cell lines kidney, adherent HEK 293T Human embryonic DMEM ATCC kidney, adherent MCF-7 Human, mammary DMEM ATCC gland, breast, adherent Normal human Normal adult human DMEM ATCC dermal primary dermal fibroblasts fibroblasts (NHDF) SKOV3 Human ovarian DMEM ATCC adenocarcinoma

Statistics

[0854] In cases where two conditions were being compared, statistical analyses were performed using a t-test. In all other cases, statistical analyses were performed by using a One-way ANOVA.

Characterisation of Human T-Cell Activation by Recombinant FAP BiTE

[0855] The ability of the FAP BiTE to induce T-cell activation in the presence or absence of normal human dermal fibroblast (NHDF) cells was compared. Human CD3+ T-cells (70,000 cells per well in 96-well U-bottom plates) were co-cultured alone or with NHDF cells (10:1 T:NHDF) in the presence of media alone or 300 ng/mL FAP or control BiTE. Cells were co-cultured for 24 hours at 37? C. and subsequently harvested with enzyme-free cell dissociation buffer (Thermo, #13151014). The expression levels of CD69 (FIG. 3A) and CD25 (FIG. 3B) on CD45+ T-cells were then analysed by antibody staining and flow cytometry and represented as geometric mean fluorescence (gMFI) values. Plate-immobilised anti-CD3 antibody (7.5 ?g/mL) was used as positive control for T cell activation. The FAP BiTE selectively induced the expression of activation markers CD69 and CD25 on T-cells, indicating that it was able to activate T cells.

[0856] In a second similar experiment, T-cells were assessed by intracellular cytokine staining 6 hr after co-culture with NHDF cells (200,000 CD3+ cells plus 40,000 NHDF in wells of a 96-well plate) and 300 ng/mL FAP or control BiTE. CD45+ T-cells were intracellularly stained for IFN? expression with Brefeldin A added into the culture medium 5 hours before harvest. As a positive control, T-cells were stimulated with soluble PMA (10 ng/mL) and ionomycin (1 ?g/mL). The results shown in FIG. 4A indicate that the FAP BiTE in the presence of NHDF resulted in a significantly higher number of IFN? expressing T-cells compared to the control BiTE.

Example 3

[0857] A similar set of experiments to those in example 2 were run to characterize the recombinant EpCAM BiTE protein.

Characterisation of Human T-Cell Activation by Recombinant EpCAM BiTE

[0858] The ability of the EpCAM BiTE to induce T-cell activation in the presence or absence of the EpCAM-positive DLD cell line was compared. Human CD3+ T-cells (70,000 cells per well in 96-well U-bottom plates) were co-cultured alone or with DLD cells (10:1 T:DLD) in the presence of media alone or 600 ng/mL EpCAM or control BiTE. Cells were co-cultured for 24 hours at 37? C. and subsequently harvested with enzyme-free cell dissociation buffer. The expression levels of CD69 and CD25 on CD45+ T-cells were then analysed by antibody staining and flow cytometry and data represented as geometric mean fluorescence (gMFI) values. Plate-immobilised anti-CD3 antibody (7.5 ?g/mL) was used as positive control for T cell activation. The EpCAM BiTE selectively induced the expression of activation markers CD69 and CD25 on T-cells, indicating that it was able to activate T cells (FIGS. 4B & C).

[0859] In a similar experiment, T-cells were assessed by intracellular cytokine staining 6 hr after co-culture with DLD cells (200,000 CD3+ T-cells plus 40,000 DLD cells per well of a 96-well plate) and 300 ng/mL EpCAM or control BiTE. CD45+ T-cells were intracellularly stained for IFN? expression with Brefeldin A added into the culture medium 5 hours before harvest. As a positive control, T cells were stimulated with soluble PMA (10 ng/mL) and ionomycin (1 ?g/mL). The results showed that the EpCAM BiTE in the presence of DLD resulted in a significantly higher number of IFN? expressing T-cells compared to the control BiTE (FIG. 5A).

[0860] In another similar experiment, PBMCs from 8 different blood donors were used to evaluate donor-dependent variations in BiTE-mediated T-cell activation. DLD (7,000 cells) were co-cultured with 100,000 PBMC in a U-bottom 96 well plate in the presence of media alone or 300 ng/mL of control or EpCAM BiTE. Cells were co-cultured for 24 hours at 37? C. and subsequently harvested. The expression levels of CD69 and CD25 on CD45+ T-cells were then analysed by antibody staining and flow cytometry and data represented as geometric mean fluorescence (gMFI) values. The results showed that the EpCAM BiTE induced the expression of activation markers CD69 and CD25 in CD3.sup.+ T-cells from all 8 donors (FIGS. 5B & C).

Example 4

[0861] In this example, the ability of recombinant FAP BiTE-activated T-cells to induce death of the fibroblast target cells was evaluated.

FAP BiTE Induces T Cell-Mediated Lysis of FAP-Positive Cell Lines and Primary Cells

[0862] NHDF (7,000 cells) were co-cultured with 70,000 T-cells in wells of a U-bottom 96 well plate in the presence of media alone or 300 ng/mL of control or FAP BiTE. After 24 hours of co-culture, supernatants were harvested and cytotoxicity determined by LDH assay following the manufacturer's instructions. The results are in FIG. 6A show that the FAP BiTE significantly increased lysis of NHDF cells.

[0863] In a similar experiment, 7,000 primary lung fibroblast cells (BTC100) were co-cultured with 70,000 CD3+ T-cells with or without 300 ng/mL of control or FAP BiTE. After 24 hours of co-culture, supernatants were harvested and cytotoxicity determined by LDH assay. The results in FIGS. 6B & C show that the FAP BiTE significantly increased lysis of primary human cancer associated fibroblast (CAF) cells. Expression of FAP by these and other patient-derived cell lines is shown in FIG. 7. The dose-response relationship for FAP BiTE-mediated cell lysis was evaluated by co-culturing 8,000 NHDF cells with 40,000 T-cells and BiTE concentrations ranging from 2?10.sup.3 to 2?10.sup.?2 ng/mL. After co-culture for 24 hours at 37? C., an LDH assay was performed on supernatants to determine target cell cytotoxicity. Dose response curves were fitted using a four parameter non-linear fit model integrated into GraphPad Prism, generating an EC50 value for the FAP BiTE of 3.2 ng/mL. The results (FIG. 8A) show a dose-dependent relationship between FAP BiTE concentration and cytotoxicity as measured by LDH assay (shown as Abs.sub.490).

Example 5

[0864] Similar studies to those in example 4 were used to demonstrate the ability of recombinant EpCAM BiTE-activated T-cells to induce death of target tumour cells was evaluated.

EpCAM BiTE Induces T Cell-Mediated Lysis of EpCAM-Positive Cell Lines

[0865] DLD tumour cells (7,000 cells) were co-cultured with 70,000 T-cells in wells of a U-bottom 96 well plate in the presence of media alone or 300 ng/mL of control or EpCAM BiTE. After 24 hours of co-culture, supernatants were harvested and cytotoxicity determined by LDH assay. The results in FIG. 8B show that the EpCAM BiTE significantly increased lysis of DLD cells (EpCAM expression on DLD cells is shown in FIG. 8C).

[0866] In a similar experiment, 4,000 SKOV cells were co-cultured with 40,000 CD3+ T-cells with or without 300 ng/mL of control or EpCAM BiTE. After 24 hours of co-culture, supernatants were harvested and cytotoxicity determined by LDH assay. The results in FIG. 9A show that the EpCAM BiTE significantly increased lysis of SKOV cells.

[0867] In another similar experiment, 5,000 MCF7 cells were co-cultured with 50,000 CD3+ T-cells with or without 300 ng/mL of control or EpCAM BiTE. After 24 hours of co-culture, supernatants were harvested and cytotoxicity determined by LDH assay. The results in FIG. 9B show that the EpCAM BiTE also significantly increased lysis of MCF7 cells.

[0868] The dose-response relationship for EpCAM BiTE-mediated cell lysis was evaluated by co-culturing 8,000 DLD with 40,000 T-cells and EpCAM or control BiTE concentrations ranging from 2?10.sup.3 to 2?10.sup.?2 ng/mL. After co-culture for 24 hours at 37? C., an LDH assay was performed on supernatants to determine target cell cytotoxicity. Dose response curves were fitted using a four parameter non-linear fit model integrated into GraphPad Prism, generating an EC50 value for the EpCAM BiTE of 7.4 ng/mL. The results in FIG. 10 show a dose dependent relationship between EpCAM BiTE concentration and cytotoxicity.

[0869] In conclusion, the results of this example demonstrate that the EpCAM BiTE was able to induce T-cell mediated lysis of multiple EpCAM-positive tumour cell lines.

Example 6

[0870] Stable FAP expressing CHO and Ad293 cell lines were generated as a means to demonstrate the FAP antigen specificity of the FAP BiTE by comparing to parental untransfected cells.

Generation of FAP-Expressing Stable-Transfected Cell Lines

[0871] The protein sequence of the FAP gene was obtained from the NCBI database (SEQ ID 30), reverse transcribed to generate a DNA coding sequence that was synthesised by Oxford Genetics Ltd (Oxford, UK). The FAP gene was cloned into pSF-Lenti vector by standard cloning techniques producing the pSF-Lenti-FAP vector. HEK293T cells were transfected with the lentivirus FAP expression vector alongside pSF-CMV-HIV-Gag-Pol, pSF-CMV-VSV-G, pSF-CMV-HIV-Rev. Lipofectamine 2000 was used as a transfection reagent and was added to the vector DNA at a DNA:lipofectamine ratio of 1:2, and incubated with the cells at 37? C. Supernatant containing lentivirus was harvested 48 hours later and mixed with polybrene (final concentration, 8 ?g/mL). The Lentivirus/polybrene mixture was added to seeded Ad293 or CHO cells and incubated at 37? C. On day 4, the supernatant was exchanged for media containing puromycin (2 ?g/mL for Ad293 and 7.5 ?g/mL for CHO). Stable variants were then clonally selected and FAP expression of the parental cell lines or stable-transfected variant was determined by staining with FAP or isotope control antibody and analysed by flow cytometry (FIG. 11A).

FAP BiTE-Mediated Target Cell Lysis is Specific to FAP-Expressing Cells

[0872] CHO or CHO-FAP cells (7,000 cells) were co-cultured alone or with human T-cells (70,000) in the presence of media alone or 2 ?g/mL control or FAP BiTE in wells of a U-bottom 96-well plate. After 24 hours incubation, supernatants were harvested and target cell cytotoxicity measured by LDH cytotoxicity assay as described in example 4 (FIG. 11B). T-cell activation was also determined by analysing the expression levels of CD69 and CD25 via flow cytometry (FIG. 12). Cytotoxicity was only observed when CHO-FAP cells were cultured with T-cells and FAP BiTE. This indicates that FAP BiTE mediated T-cell activation and target cell lysis is highly specific and limited to FAP-expressing cells, and not the FAP-negative parental cell line.

Example 7

[0873] Stable EpCAM expressing CHO amd Ad293 cell lines were generated as a means to demonstrate the EpCAM antigen specificity of the EpCAM BiTE by comparing to parental untransfected cells.

Generation of EpCAM-Expressing Stable-Transfected Cell Lines

[0874] The protein sequence of the EpCAM gene was obtained from NCBI database (SEQ ID 28), reverse transcribed to generate a DNA coding sequence that was synthesised by Oxford Genetics Ltd (Oxford, UK). The EpCAM gene was cloned into pSF-Lenti vector by standard cloning techniques producing the pSF-Lenti-EpCAM vector. HEK293T cells were transfected with lentivirus EpCAM expression vector alongside pSF-CMV-HIV-Gag-Pol, pSF-CMV-VSV-G, pSF-CMV-HIV-Rev. Lipofectamine 2000 was used as a transfection reagent and was added to the vector DNA at a DNA:lipofectamine ratio of 1:2, and incubated with the cells at 37? C. Supernatant containing lentivirus was harvested 48 hours later and mixed with polybrene (final concentration, 8 ?g/mL). The Lentivirus/polybrene mixture was added to seeded Ad293 or CHO cells and incubated at 37? C. On day 4, the supernatant was exchanged for media containing puromycin (2 ?g/mL for Ad293 and 7.5 ?g/mL for CHO). Stable variants were then clonally selected and EpCAM expression of the parental cell lines or stable-transfected variant was determined by staining with EpCAM or isotope control antibody and analysed by flow cytometry (FIG. 13A).

EpCAM BiTE-Mediated Target Cell Lysis is Specific to EpCAM-Expressing Cells

[0875] CHO or CHO-EpCAM cells (7,000 cells) were co-cultured alone or with human T-cells (70,000) in the presence of media alone or 2 ?g/mL control or EpCAM BiTE in wells of a U-bottom 96-well plate. After 24 hours incubation, supernatants were harvested and target cell cytotoxicity measured by LDH cytotoxicity assay (FIG. 13B). T-cell activation was also determined by analysing the expressions levels of CD69 and CD25 via flow cytometry (FIG. 14). Cytotoxicity was only observed when CHO-EpCAM cells were cultured with T-cells and EpCAM BiTE. This indicates that EpCAM BiTE mediated T-cell activation and target cell lysis is highly specific and limited to EpCAM-expressing cells, and not the EpCAM-negative parental cell line.

Example 8

[0876] In a further experiment, the ability of the recombinant FAP BiTE protein to activate CD4 or CD8 T-cells and the ability of each of these T-cell subsets to lyse NHDF cells was assessed. CD3+ T-cells (35,000) were co-cultured with 7,000 NHDF cells in the presence of 300 ng/mL control or FAP BiTE in wells of a U-bottom 96 well plate, and incubated at 37? C. for 24 hours. Cells were harvested and stained with antibodies to CD4 or CD8 and CD69 and CD25, and analysed by flow cytometry. The results (FIG. 15A) demonstrated that the FAP BiTE induced an increase in activation markers CD69 and CD25 in both CD4.sup.+ and CD8+ T-cells.

[0877] In a similar experiment, the ability of each T-cell subset (CD4 and CD8) to kill target cells was assessed. CD4+ T-cells were extracted from CD3-purified cells by positive selection using a CD4 T Cell Isolation Kit (Miltenyi Biotec, #130-045-101), according to the manufacturer's protocol, with the CD8 cells within non-isolated flow-through. In wells of a U-bottom 96-well plate, 7,000 NHDF were co-cultured with 35,000 CD4.sup.+ or CD8.sup.+ T-cells together with 300 ng/mL of control or FAP BiTE and incubated at 37? C. After 24 hours, supernatants were harvested and target cell cytotoxicity measured by LDH cytotoxicity assay. The results (FIG. 15B) show that the FAP BiTE induced both CD4+ and CD8+ T-cells to kill NHDF cells.

Example 9

[0878] The ability of the EpCAM BiTE to activate CD4+ or CD8+ T-cells and the ability of each subset to lyse DLD tumour cells was assessed. CD3+ T-cells (35,000) were co-cultured with 7,000 DLD cells in the presence of 300 ng/mL control or EpCAM BiTE in wells of a U-bottom 96 well plate, and incubated at 37? C. for 24 hours. Cells were harvested and stained with antibodies for CD4 or CD8 and CD69 and CD25, and analysed by flow cytometry. The results (FIG. 16A) demonstrated that the EpCAM BiTE induced an increase in activation markers CD69 and CD25 in both CD4+ and CD8+ T-cells.

[0879] In a similar experiment, the ability of each T-cell subset (CD4 and CD8) to kill target cells was assessed. CD4+ T-cells were extracted from CD3-purified cells by positive selection using CD4 T Cell Isolation Kit according to the manufacturer's protocol, with the CD8 cells within non-selected flow-through. In wells of a U-bottom 96-well plate, 7,000 DLD were co-cultured with 35,000 CD4+ or CD8.sup.+ T-cells with 300 ng/mL of control or EpCAM BiTE and incubated at 37? C. After 24 hours, supernatants were harvested and target cell cytotoxicity measured by LDH cytotoxicity assay (FIG. 16B). The results show that the EpCAM BiTE induced both CD4+ and CD8+ T-cells to kill DLD cells.

Example 10

[0880] Characterising FAP BiTE-Mediated Activation of Autologous Tumour-Associated Lymphocytes from Primary Malignant Ascites

[0881] To evaluate the activity of BiTE proteins using cancer patient derived cells, samples of primary malignant ascetic fluids containing both CD3+ T-cells and FAP+ cells were obtained for testing. Unpurified ascites cells (therefore unchanged from when received) were seeded at 250,000 cells per well of a U-bottom 96-well plate in either 100% ascites fluid or medium supplemented with 1% human serum in the presence of 500 ng/mL control or FAP BiTE. Untreated wells served as negative controls. After incubation at 37? C. for 5 days, the total cell population was harvested and the numbers of CD3+ T-cells (FIG. 17A) and expression levels of CD25 on CD3+ T-cells were determined (FIG. 17B). Total cell numbers per well were determined using precision counting beads. The results demonstrate that the FAP BiTE resulted in significant increase in T-cell activation of the tumour-associated T-cells from cancer patients.

[0882] As an extension of the experiment above, replicate wells were harvested and the number of FAP.sup.+ cells determined by flow cytometry (FIG. 17C). Total cell numbers per well were determined using precision counting beads. The results show that the FAP BiTE resulted in a significant decrease in numbers of autologous FAP-expressing cells in the ascites sample.

Example 11

[0883] Recombinant BiTE-expressing EnAd viruses were engineered, produced and purified using the methods described below.

Generation of BiTE-Expressing Enadenotucirev

[0884] EnAd is a replication competent chimeric group B adenovirus that contains frequent non-homologous nucleotide substitutions of Ad3 for Ad11p in the E2B region, a nearly complete E3 deletion and a smaller E4 deletion mapped to E4orf4 (Kuhn et al, Directed evolution generates a novel oncolytic virus for the treatment of colon cancer, PLoS One, 2008 Jun. 18; 3(6): e2409). A schematic representation of the genome of the adenoviruses used in this study is shown in FIG. 18A.

[0885] The plasmid pEnAd2.4 was used to generate the plasmids pEnAd2.4-CMV-EpCAMBiTE, pEnAd2.4-SA-EpCAMBiTE, pEnAd2.4-CMV-FAPBiTE, pEnAd2.4-SA-FAPBiTE, pEnAd2.4-CMV-ControlBiTE, pEnAd2.4-SA-ControlBiTE (Table 4) by direct insertion of a cassette encoding the EpCAM BiTE (SEQ ID NO: 1), FAP BiTE (SEQ ID NO: 3) or Control BiTE (SEQ ID NO: 5). The transgene cassette contained a 5 short splice acceptor sequence (SEQ ID NO: 33) or an exogenous CMV promoter (SEQ ID NO: 31), the EpCAM, FAP or control BiTE cDNA sequence and a 3 polyadenylation sequence (SEQ ID NO: 32). Construction of the plasmid was confirmed by DNA sequencing. The exogenous CMV promoter is constitutively active and thus leads to early expression of transgenes. The splice acceptor sequence drives expression under the control of the viral major late promoter and leads to later transgene expression following initiation of virus genome replication. The kinetics of this promoter-driven expression can be observed in FIG. 18B, in which GFP was used as the transgene.

TABLE-US-00004 TABLE 4 [plasmid DNA] Plasmid ID ng/ml pEnAd2.4-CMV-EpCAMBiTE 205.3 pEnAd2.4-SA-EpCAMBiTE 325.2 pEnAd2.4-CMV-FAPBiTE 1322.8 pEnAd2.4-SA-FAPBiTE 3918.3 pEnAd2.4-CMV-ControlBiTE 189.1 pEnAd2.4-SA-ControlBiTE 236.2 pEnAd2.4-CMV-FAPBiTE-RFP 1599 pEnAd2.4-SA-FAPBiTE-RFP 1872 pEnAd2.4-CMV-ControlBiTE-RFP 1294 pEnAd2.4-SA-ControlBiTE-RFP 2082

Virus Production and Characterisation

[0886] The plasmids EnAd2.4-CMV-EpCAMBiTE, pEnAd2.4-SA-EpCAMBiTE, pEnAd2.4-CMV-FAPBiTE, pEnAd2.4-SA-FAPBiTE, pEnAd2.4-CMV-ControlBiTE, pEnAd2.4-SA-ControlBiTE were linearised by restriction digestion with the enzyme AscI to produce the liner virus genome. Digested DNA was purified by isopropanol extraction and precipitated for 16 hrs, ?20? C. in 300 ?l>95% molecular biology grade ethanol and 10 ?l 3 M Sodium Acetate. The precipitated DNA was pelleted by centrifuging at 14000 rpm, 5 mins and was washed in 50 ?l 70% ethanol, before centrifuging again, 14000 rpm, 5 mins. The clean DNA pellet was air dried and resuspended in 1004 water. 6.25 ?g DNA was mixed with 15.64 lipofectamine transfection reagent in OptiMEM and incubated for 20 mins, RT. The transfection mixture was then added to a T-25 flask containing Ad293 cells grown to 80% confluency. After incubation of the cells with the transfection mix for 4 hrs at 37? C., 5% CO.sub.24 mls of cell media (DMEM high glucose with glutamine supplemented with 10% FBS) was added to the cells and the flasks was incubated 37? C., 5% CO.sub.2. The transfected Ad293 cells were monitored every 24 hrs and were supplemented with additional media every 48-72 hrs. The production of virus was monitored by observation of a significant cytopathic effect (CPE) in the cell monolayer. Once extensive CPE was observed the virus was harvested from Ad293 cells by three freeze-thaw cycles. Single virus clones were selected by serial diluting harvested lysate and re-infecting Ad293 cells, and harvesting wells containing single plaques. Serial infections of Ad293 cells were performed once an infection had reached full CPE in order to amplify the virus stocks. Viable virus production during amplification was confirmed by observation of significant CPE in the cell monolayer.

Virus Purification

[0887] Once potent virus stocks were amplified the viruses were purified by double caesium chloride density gradient centrifugation (banding) to produce NG-601, NG-602, NG-603, NG-604, NG-605 and NG-606 virus stocks. These stocks were titred by micoBCA assay (Life Technologies), following manufacturer's instructions (Table 5).

TABLE-US-00005 TABLE 5 Virus NG ID Genome TCID50/ EnAd ID NO: SEQ ID vp/mL mL EnAd-CMV-EpCAMBiTE NG-601 SEQ ID NO: 34 2.2494 ? 10.sup.12 1.26 ? 10.sup.11 EnAd-SA-EpCAMBiTE NG-602 SEQ ID NO: 35 4.21746 ? 10.sup.12 1.58 ? 10.sup.11 EnAd-CMV-ControlBiTE NG-603 1.42607 ? 10.sup.12 5.01 ? 10.sup.10 EnAd-SA-ControlBiTE NG-604 3.31073 ? 10.sup.12 2.00 ? 10.sup.11 EnAd-CMV-FAPBiTE NG-605 SEQ ID NO: 36 1.64653 ? 10.sup.12 1.58 ? 10.sup.11 EnAd-SA-FAPBiTE NG-606 SEQ ID NO: 37 1.28148 ? 10.sup.12 3.98 ? 10.sup.19 EnAd-CMV-ControlBiTE-P2A-RFP NG-607 5.963 ? 10.sup.12 1.26 ? 10.sup.9 EnAd-SA-ControlBiTE-P2A-RFP NG-608 1.51848 ? 10.sup.12 6.31 ? 10.sup.9 EnAd-CMV-FAPBiTE-P2A-RFP NG-609 1.57517 ? 10.sup.12 7.94 ? 10.sup.9 EnAd-SA-FAPBiTE-P2A-RFP NG-610 7.74881 ? 10.sup.11 5.01 ? 10.sup.10

Example 12

[0888] The activities of NG-601, NG-602, NG-603, NG-604, NG-605 and NG-606 viruses were characterised using the methods described below.

Characterisation of BiTE Encoding EnAd Activity Compared to EnAd in Carcinoma Cell Lines

[0889] The ability NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 or EnAd to replicate was analysed by infection of A549 lung carcinoma cells and assessed by qPCR. A549 cells were seeded in wells of a 24-well plate at a cell density of 2?10.sup.5 cells/well. Plates were incubated for 18 hrs, 37? C., 5% CO.sub.2, before cells were either infected with 100 virus particles per cell (ppc) or were left uninfected. Wells were harvested 24, 48 or 72 hrs post infection and DNA purified using PureLink genomic DNA mini kit (Invitrogen) according to the manufacturer's protocol. Total viral genomes were quantified by qPCR with each extracted sample or standard using an EnAd hexon gene specific primer-probe set in the reaction mix detailed in Table 6. qPCR was performed as per the programme in Table 7.

TABLE-US-00006 TABLE 6 Reagent Volume/well (?l) 2 ? qPCRBIO Probe Mix (PCRBiosystems) 10 EnAd Forward primer 0.08 EnAd Reverse primer 0.08 EnAd Probe 0.8 NFW 4.04 Sample 5 Well Volume 20

TABLE-US-00007 TABLE 7 No. Cycles Temperature (? C.) Duration (secs) 1 95 120 40 95 5 60-65 20-30

[0890] Quantification of the number of detected virus genomes per cell demonstrated that NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 and EnAd virus replication were comparable in the A549 cell line (FIG. 19A).

[0891] Oncolytic activity of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 or EnAd was assessed by infection of A549 (FIG. 19B). A549 cells were seeded in 96-well plate at a cell density of 1.5?10.sup.4 cells/well. Plates were incubated for 18 hrs, 37? C., 5% CO.sub.2, before cells were infected with increasing ppc of virus (5-fold serial dilution, 4.1?10.sup.?7 to 5000 virus ppc) or were left uninfected. A549 cytotoxicity was measured on day 5 by CellTiter 96? AQueous One Solution Cell Proliferation Assay (MTS) (Promega, # G3582). Dose response curves were fitted using a four parameter non-linear fit model integrated into GraphPad Prism. IC50 values generated for each virus demonstrated that the oncolytic activities of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 and EnAd was comparable for each virus.

Confirmation of Functional BiTE Transgene Expression from NG-601, NG-602, NG-603, NG-604, NG-605, NG-606

[0892] To determine whether the viruses NG-601, NG-602, NG-605, NG-606 produced functional BiTEs, T-cell activation assays using CHO, CHO-EpCAM and CHO-FAP cell lines as target cells were performed. 10,000 target cells were co-cultured with 50,000 CD3+ T-cells in wells of a U-bottom 96-well plate with Ad293 viral supernatants diluted 100-fold in culture medium and incubated for 24 hrs, 37? C., 5% CO.sub.2. T-cells were harvested and stained with antibodies specific for CD25 and CD69 and analysed by flow cytometry. The results (FIGS. 20A and 20B) indicated that the viruses NG-601 and NG-602 expressed a functional BiTE transgene that activated T cells when co-cultured with CHO-EpCAM cells, and NG-605 and NG-606 expressed a functional BiTE transgene that activated T cells when co-cultured with CHO-FAP cells, but not when co-cultured with CHO cells.

Quantification of BiTE Expression in a Colon Carcinoma Cell Line

[0893] The quantity of BiTE expression by NG-601, NG-602, NG-605, NG-606 infection of the human colon carcinoma cell line DLD was assessed. DLD cells were seeded in 6 well culture plates at a density of 1.2?10.sup.6 cells per well. 18 hrs post-seeding, DLD cells were infected with EnAd, NG-601, NG-602, NG-603, NG-604, NG-605, NG-606 at 100 ppc. Cells were cultured for 72 hrs before the supernatants were collected from the wells and centrifuged for 5 mins, 1200 rpm to remove cell debris. The clarified supernatants were then used for a killing assay, with cytotoxicity compared to a standard curve generated with a recombinant BiTE of known concentration, allowing determination of quantity of BiTE in viral supernatants.

[0894] To determine the quantity of FAP BiTE produced from NG-605 and NG-606, a cytotoxicity assay was performed in which 8,000 NHDF were co-cultured with 40,000 CD3+ T-cells and DLD viral supernatants diluted 1 in 10.sup.3, 1 in 10.sup.4 and 1 in 10.sup.5. A standard curve was generated by incubating NHDF and CD3+ T-cells with FAP or control BiTE at 10-fold serial dilutions from 3333 to 3.33?10.sup.?4 ng/?L. Supernatants were harvested 24 hour post-treatment and cytotoxicity measured by LDH assay. Quantity of BiTE expressed was determined by comparing cytotoxicity of viral supernatants to that of the recombinant BiTE standard curve. The results (FIG. 21) indicated that the viruses NG-605 and NG-606 produced 9.8 and 49.2 ?g FAP BiTE per million DLD cells, respectively. To determine the quantity of EpCAM BiTE produced from NG-601 and NG-602, a cytotoxicity assay was performed in which 8,000 DLD cells were co-cultured with 40,000 CD3+ T-cells and DLD viral supernatants diluted 1 in 10.sup.3, 1 in 10.sup.4 and 1 in 10.sup.5. A standard curve was generated by incubating DLD and CD3+ T-cells with EpCAM or control BiTE at 10-fold serial dilutions from 3333 to 3.33?10.sup.?4 ng/?L. Supernatants were harvested 24 hour post-treatment and cytotoxicity measured by LDH assay (FIG. 22). Quantity of BiTE expressed was determined by comparing cytotoxicity of viral supernatants to that of the recombinant BiTE standard curve. The results indicated that the viruses NG-601 and NG-602 produced 165 and 50.3 ?g EpCAM BiTE per million DLD cells, respectively.

Example 13

[0895] In addition to encoding a FAP or Control BiTE, the NG-607, NG-608, NG-609, NG-610 viruses also carry a red fluorescent protein (RFP) transgene for visualization of infected cells using fluorescent microscopy methods (SEQ ID NOS: 25 & 26, Table 4). The functional activities of these viruses were characterised using the methods described below.

Confirmation of Transgene Expression from NG-607, NG-608, NG-609, NG-610

[0896] The ability of viruses NG-607, NG-608, NG-609 and NG-610 to produce their BiTE transgene was assessed by infection of Ad293 cells. Ad293 cells were plated in a 6-well plate at 1?10.sup.6 cells/well. Plates were incubated for 24 hrs, 37? C., 5% CO.sub.2, before cells were infected with viruses at 100 ppc or were left uninfected. At 48 hours post-infection, plaques were irradiated with a fluorescent mercury lamp and photographed (FIG. 23). The results suggested that the viruses NG-607, NG-608, NG-609 and NG-610 express the RFP transgene.

Example 14

[0897] In the next series of experiments, the ability of EnAd and FAP or control BiTE viruses NG-603, NG-604, NG-605, NG-606, NG-607, NG-608, NG-609, NG-610 to kill target cells, including tumour cells and fibroblasts, was evaluated.

[0898] In the first study, the ability of EnAd to kill DLD cells was assessed using xCELLigence technology. DLD cells were plated in a 48-well E-plate at 1.2?10.sup.4 cells/well and incubated for 18 hrs, 37? C., 5% CO.sub.2, before cells were either infected with 100 EnAd ppc or were left uninfected. XCELLigence was used to measure target cell cytotoxicity every 15 minutes over an 8 day incubation period. The results (FIG. 24A) suggest that EnAd was able to kill DLD cells effectively over the time period.

[0899] In a similar experiment, the ability of EnAd to kill SKOV cells was assessed using xCELLigence technology. SKOV cells were plated in a 48-well E-plate at 1?10.sup.4 cells/well and incubated for 18 hrs, 37? C., 5% CO.sub.2, before cells were either infected with 100 EnAd ppc or were left uninfected. xCELLigence was used to measure target cell cytotoxicity every 15 minutes for a period of 8 days. The results (FIG. 24B) suggest that SKOV cells are resistant to EnAd-mediated cytotoxicity over this time frame.

[0900] In a similar experiment, the ability of EnAd to kill NHDF cells was also assessed using xCELLigence technology. NHDF cells were plated in a 48-well E-plate at 4?10.sup.3 cells/well and incubated for 18 hrs, 37? C., 5% CO.sub.2, before cells were either infected with 100 EnAd ppc or were left uninfected. xCELLigence was used to measure target cell cytotoxicity every 15 minutes over the same time period as for A549 and SKOV cells. The results (FIG. 24C) suggest that EnAd is unable to kill NHDF cells in the period of time observed.

[0901] In a similar experiment, the ability of NG-603, NG-604, NG-605, NG-606 and EnAd to kill NHDF cells was assessed in co-culture with SKOV tumour cells and CD3+ T-cells using xCELLigence. NHDF cells and SKOV cells were seeded in a 48-well E-plate at 4?10.sup.3 and 1?10.sup.3 cells/well, respectively. Plates were incubated for 18 hrs, 37? C., 5% CO.sub.2, before cells were either infected with 100 ppc of EnAd, of NG-603, NG-604, NG-605 or NG-606 or were left uninfected. After 2 hour incubation, 37,500 CD3.sup.+ T-cells were added to each well. xCELLigence was used to measure target cell cytotoxicity every 15 minutes. The results (FIG. 25A) demonstrate that the FAP BiTE-expressing viruses NG-605 and NG606, but not EnAd or control BiTE-expressing viruses NG-603 and NG-604, were able to induce lysis of NHDF cells, with kinetics dependent on the promoter used for BiTE expression (faster with CMV promoter).

[0902] In a similar experiment, the ability of NG-603, NG-604, NG-605, NG-606 and EnAd to kill NHDF cells, was assessed in co-culture with SKOV and CD3+ T-cells using LDH cytotoxicity assay. NHDF cells and SKOV cells were seeded in a 96-well U-bottom plate at 8?10.sup.3 and 2?10.sup.3 cells/well, respectively, and either infected with 100 ppc of EnAd, of NG-603, NG-604, NG-605 or NG-606 or were left uninfected. After 2 hour incubation, 75,000 CD3+ T-cells were added to each well and plates were incubated at 37? C., 5% CO.sub.2. Supernatants were harvested at 0, 24, 48 and 96 hours post-treatment and cytotoxicity measured by LDH cytotoxicity assay. The results (FIG. 25B) demonstrate that the FAP BiTE-expressing viruses NG-605 and NG606, but not EnAd or control BiTE-expressing viruses NG-603 and NG-604, were able to induce lysis of NHDF cells, with kinetics dependent on the promoter used for BiTE expression.

[0903] As an extension of the LDH experiment above, the cells were also harvested at 0, 24, 48 and 96 hours post-treatment, stained with antibodies for CD45, CD69 and CD25 and analysed by flow cytometry. The results (FIG. 26) demonstrate that the FAP BiTE-expressing viruses NG-605 and NG-606, but not EnAd or control BiTE-expressing viruses NG-603 and NG-604, were able to induce T-cell activation, with kinetics dependent on the promoter used for BiTE expression.

[0904] In a similar experiment, the dependence on FAP to induce FAP BiTE-mediated T-cell activation was evaluated. In a 96-well U-bottom plate, SKOV cells were seeded at 2?10.sup.3 cells/well alone or in combination with NHDF cells at 8?10.sup.3 cells/well. Viral particles were added to each well at 100 ppc, and plates incubated at 37? C., 5% CO.sub.2. After two hours, 75,000 CD3+ T-cells were added and plates incubated further. At 96-hours post-infection, cells were harvested and stained for CD45 and CD25 and analysed by flow cytometry (FIG. 27A). The results demonstrate that the FAP BiTE-expressing viruses NG-605 and NG-606, only induced T-cell activation in the presence of FAP-positive NHDF cells.

[0905] In a similar experiment, the specificity of promoter (CMV or virus MLP/SA)-driven BiTE expression in NG-605 and NG-606 was investigated further. In a 96-well U-bottom plate, NHDF cells were seeded at 4?10.sup.3 cells/well. 100 viral particles per cell were added to each well, and plates incubated at 37? C., 5% CO.sub.2 After two hours, 40,000 CD3 cells were added and plates incubated further. At 72-hours post-infection, supernatants were harvested and cytotoxicity measured by LDH cytotoxicity assay. The results (FIG. 27B) demonstrate that the CMV-driven virus NG-605, but not SA-driven NG-606, was able to mediate killing of NHDF cells upon infection of NHDF cells alone.

[0906] The results indicate that NG-605 and NG-606 were both able to induce T cell activation and target cell lysis, although the kinetic profile was slightly different depending on the promoter used. Timelapse videos were obtained to observe viral or T cell-mediated lysis of target cells by recombinant FAP BiTE, EnAd, NG-603 or NG-605. NHDF cells were stained with CellTracker Orange CMTMR Dye (Life Tech, #C2927) and CD3+ T-cells were stained with CellTrace Violet Cell Proliferation Kit (Life Tech, #C34557) following manufacturer's protocols. Dyed NHDF were plated in a 24-well plate at 7.5?10.sup.3 cells/well in co-culture with 1.35?10.sup.4 DLD or SKOV tumour cells. Plates were incubated for 18 hrs, 37? C., 5% CO.sub.2. Cells were then treated with 300 ng/mL FAP BiTE or infected with 100 ppc of EnAd, NG-603, and NG-605 or left untreated. After two hours incubation, 100,000 dyed CD3+ T-cells were added to necessary wells, in addition to 1.5 ?M CellEvent Caspase 3-7 reagent (Life Tech, #C10423). Videos were obtained on a Nikon TE 2000-E Eclipse inverted microscope, with images captured every 15 minutes for 96 hours. Frames from the videos are shown in FIG. 28. The results show that the recombinant FAP BiTE and NG-605, but not EnAd or NG-603, were able to induce rapid lysis of NHDF cells.

[0907] In a similar experiment, NHDF cells were stained with CellTracker Green CMFDA Dye (Life Tech, #C2925) and CD3+ T-cells were stained with CellTrace Violet Cell Proliferation Kit (Life Tech, #C34557) following manufacturer's protocols. Dyed NHDF were plated in a 24-well plate at 7.5?10.sup.3 cells/well in co-culture with 1.35?10.sup.4 DLD or SKOV tumour cells. Plates were incubated for 18 hrs, 37? C., 5% CO.sub.2. Cells were then infected with 100 ppc of NG-607, NG-608, NG-609 or NG-610 or left uninfected. After two hours incubation, 100,000 dyed CD3+ T-cells were added to necessary wells. Videos were obtained on a Nikon TE 2000-E Eclipse inverted microscope, with images captured every 15 minutes for 96 hours. Frames from the videos are shown in FIG. 29. The results show that all viruses lead to tumour cell infection (RFP, red fluorescence, positive), but only NG-609 and NG-610 were able to induce rapid lysis of the co-cultured NHDF cells.

Example 15

[0908] In this series of experiments, the ability of EnAd and EpCAM or control BiTE viruses NG-601, NG-602, NG-603 and NG-604 to kill target cells, including tumour cells and fibroblasts, was evaluated.

Characterisation of Human T-Cell Activation and EpCAM-Positive Target Cell Lysis by EnAd, NG-601, NG-602, NG-603 and NG-604

[0909] The ability of EnAd and NG-601, NG-602, NG-603 and NG-604 to kill DLD tumour cells in the presence or absence of CD3+ T-cells was assessed using xCELLigence technology. DLD cells were plated in 48-well E-plate at 1.2?10.sup.4 cells/well. Plates were incubated for 18 hrs, 37? C., 5% CO.sub.2, before cells were either infected with EnAd at 100 ppc or were left uninfected. Two hours after infection, 75,000 CD3+ T-cells were added to the necessary wells. XCELLigence was used to measure target cell cytotoxicity every 15 minutes. The results (FIG. 30) demonstrate that NG-601 and NG-602 lead to significantly more rapid DLD cytotoxicity in a T cell-dependent manner.

[0910] In a similar experiment, the ability of EnAd and NG-601, NG-602, NG-603 and NG-604 to kill DLD tumour cells in the presence or absence of CD3+ T-cells was assessed using LDH cytotoxicity assay. DLD cells were plated in a 96-well U-bottom plate at 2?10.sup.4 cells/well and either infected with 100 ppc EnAd or were left uninfected. Two hours after infection, 150,000 CD3+ T-cells were added to the necessary wells. Plates were incubated at 37? C., 5% CO.sub.2 and supernatant harvested and analysed by LDH cytotoxicity assay at 0, 24, 48 and 72 hours post-infection. The results (FIG. 31) demonstrate that NG-601 and NG-602 lead to more rapid DLD cytotoxicity in a T cell-dependent manner.

[0911] As an extension of the LDH experiment above, the cells were also harvested at 0, 24, 48 and 96 hours post-treatment, stained with antibodies for CD45, CD69 and CD25 and analysed by flow cytometry to determine activation status of the CD3+ T-cells. The results (FIG. 32) demonstrate that the EpCAM BiTE-expressing viruses NG-601 and NG-602, but not EnAd or control BiTE-expressing viruses NG-603 and NG-604, were able to induce T-cell activation, with kinetics dependent on the promoter used for BiTE expression.

[0912] In another experiment, the ability of NG-601 to kill DLD tumour cells at varying multiplicity of infection (MOI) in the presence or absence of CD3+ T-cells was assessed using xCELLigence technology. DLD cells were plated in 48-well E-plate at 2?10.sup.4 cells/well. Plates were incubated for 18 hrs, 37? C., 5% CO.sub.2, before cells were either infected with NG-601 at MOI (ppc) varying from 0.001 to 10 or left uninfected. Two hours after infection, 150,000 CD3+ T-cells were added to the necessary wells. xCELLigence was used to measure target cell cytotoxicity every 15 minutes. The results (FIG. 33) demonstrate that NG-601 lead to more rapid DLD cytotoxicity in a T cell-dependent manner at MOI's as low as 0.001.

[0913] In a similar experiment, the ability of EnAd and NG-601, NG-602, NG-603 and NG-604 to kill SKOV tumour cells in the presence or absence of CD3+ T-cells was assessed using xCELLigence technology. SKOV cells were plated in 48-well E-plate at 1?10.sup.4 cells/well. Plates were incubated for 18 hrs, 37? C., 5% CO.sub.2, before cells were either infected with EnAd (100 ppc) or were left uninfected. Two hours after infection, 50,000 CD3+ T-cells were added to the necessary wells. xCELLigence was used to measure target cell cytotoxicity every 15 minutes. The results (FIG. 34) suggest that SKOV cells are resistant to EnAd-mediated cytotoxicity over the timeframe of this study, however NG-601 and NG-602 were able to induce rapid lysis of SKOV cells in the presence of CD3+ T-cells.

[0914] In a similar experiment, the ability of EnAd and NG-601, NG-602, NG-603 and NG-604 to kill SKOV cells in the presence or absence of CD3+ T-cells was assessed using LDH cytotoxicity assay. SKOV cells were plated in 96-well U-bottom plates at 2?10.sup.4 cells/well and either infected with EnAd (100 ppc) or were left uninfected. Two hours after infection, 150,000 CD3+ T-cells were added to the necessary wells. Plates were incubated at 37? C., 5% CO.sub.2 and supernatant harvested and analysed by LDH cytotoxicity assay at 0, 24, 48 and 72 hours post-infection. The results (FIG. 35) are consistent with previous data and suggest that SKOV cells are resistant to EnAd-mediated cytotoxicity over this time frame, however NG-601 and NG-602 are able to induce rapid lysis of SKOV cells in the presence of CD3+ T-cells.

[0915] As an extension of the LDH experiment above, the cells were also harvested at 0, 24, 48 and 96 hours post-treatment, stained with antibodies for CD45, CD69 and CD25 and analysed by flow cytometry to determine activation status of CD3+ T-cells (FIG. 36). The results demonstrate that the EpCAM BiTE-expressing viruses NG-601 and NG-602, but not EnAd or control BiTE-expressing viruses NG-603 and NG-604, were able to induce T-cell activation, with kinetics dependent on the promoter used for BiTE expression.

[0916] In a similar experiment, the ability of EnAd and NG-601, NG-602, NG-603 and NG-604 to activate cancer patient-derived CD3+ T-cells from a CD3.sup.+ EpCAM-negative primary ascites sample was assessed. EpCAM-positive DLD cells were plated at 1?10.sup.4 cells per well in a 96-well U-bottom plate and co-cultured with 100,000 ascites cells (unchanged from when received). Cells were infected with viral particles at 100 ppc or were left uninfected. After incubation at 37? C. for 48 hours, the total cell population was harvested and the expression level of CD25 on CD3+ T-cells determined by flow cytometry. The results (FIG. 37) demonstrate that the EpCAM BiTE-expressing viruses NG-601 and NG-602, but not EnAd or control BiTE-expressing viruses NG-603 and NG-604, were able to induce T-cell activation of patient-derived CD3+ T-cells.

[0917] The results indicate that both EpCAM BiTE viruses NG-601 and NG-602 were able to induce T cell activation and target cell lysis, although the kinetic profile was slightly different depending on the promoter used.

[0918] Timelapse videos were obtained to observe viral or T cell-mediated lysis of target cells by recombinant EpCAM BiTE, EnAd, NG-601 or NG-603. NHDF cells were stained with CellTracker Orange CMTMR Dye (Life Tech, #C2927) and CD3+ T-cells were stained with CellTrace Violet Cell Proliferation Kit (Life Tech, #C34557) following manufacturer's protocols. Dyed NHDF were plated in a 24-well plate at 7.5?10.sup.3 cells/well in co-culture with 1.35?10.sup.4 DLD or SKOV tumour cells. Plates were incubated for 18 hrs, 37? C., 5% CO.sub.2. Cells were then treated with 300 ng/mL EpCAM BiTE or infected with EnAd, NG-601 or NG-603 at 100 ppc or left untreated. After two hours incubation, 100,000 dyed CD3+ T-cells were added to necessary wells, in addition to 1.5 ?M CellEvent Caspase 3-7 reagent (Life Tech, #C10423). Videos were obtained on Nikon TE 2000-E Eclipse inverted, with images captured every 15 minutes for 96 hours. Frames from the videos are shown in FIG. 38. The results show that the recombinant EpCAM BiTE and NG-605 lead to rapid lysis of both DLD and SKOV target cells, but NHDF remained unaffected.

Example 16

[0919] In this example, the activation of autologous tumour-associated lymphocytes from FAP.sup.+ primary malignant ascites from cancer patients by EnAd, NG-603, NG-604, NG-605 and NG-606 was evaluated. Patient samples considered suitable for further analysis were those containing CD3+ T-cells and FAP+ cells.

[0920] In the first experiment, unpurified (therefore unchanged from when received) ascites cells from a patient were seeded at 250,000 cells per well of a U-bottom 96-well plate in 100% ascites fluid. Cells were infected with viruses at 100 ppc, with untreated wells serving as negative controls. EnAd-CMV-GFP and EnAd-SA-GFP were also included in the experiment as a reporter to determine infection and late stage viral gene expression, respectively, with micrographs shown in FIG. 39. After incubation at 37? C. for 5 days, the total cell population was harvested and the expression level of CD25 on CD3+ T-cells (FIG. 40A) was determined. Total cell numbers per well were determined using precision counting beads. The results demonstrate that the FAP BiTE viruses NG-605 and NG-606 resulted in significant increases in T-cell activation of tumour-associated lymphocytes.

[0921] As an extension of the experiment above, replicate wells were harvested and the number of endogenous FAP+ cells determined by flow cytometry. Total cell numbers per well were determined using precision counting beads. The results (FIG. 40B) show that NG-605 and NG-606 resulted in a significant decrease in numbers of autologous FAP-expressing cells in the ascites samples, suggesting some FAP+ cells had been killed by the activated T-cells.

[0922] In a second experiment, unpurified (therefore unchanged from when received) ascites cells from a cancer patient were seeded at 250,000 cells per well of a U-bottom 96-well plate in either 100% ascites fluid or medium supplemented with 1% human serum. Cells were infected with viruses at 100 ppc, with untreated wells serving as negative controls. EnAd-CMV-GFP and EnAd-SA-GFP were also included as a reporter to determine infection and late stage viral gene expression, respectively, with micrographs shown in FIG. 41. After incubation at 37? C. for 5 days, the total cell population was harvested and the number of CD3+ T-cells (FIG. 42) and expression level of CD25 on CD3+ T-cells (FIG. 43) was determined. Total cell numbers per well were determined using precision counting beads. The results demonstrate that for this patient recombinant FAP BiTE and NG-605, but not NG-606, resulted in significant increase in T-cell activation of tumour-associated lymphocytes in media. Neither virus led to activation in ascites fluid.

[0923] As an extension of the experiment above, replicate wells were harvested and the number of FAP.sup.+ cells was determined by flow cytometry (FIG. 44). Total cell numbers per well were determined using precision counting beads. The results demonstrate that recombinant FAP BiTE and NG-605, but not NG-606, resulted in a significant decrease in numbers of autologous FAP-expressing cells in media. Neither virus led to a reduction in FAP+ cells in ascites fluid.

Example 17Materials and Methods

Cell Lines

[0924] HEK293A, DLD, SKOV3, MCF7, A431, A549 and PC3 cells (ATCC) were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich, UK) and CHO cells (ATCC) in Roswell Park Memorial Institute (RPMI-1640, Sigma-Aldrich, UK). Growth media was supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, UK) and 1% (v/v) penicillin/streptomycin (10 mg/mL, Sigma-Aldrich) and cells maintained in humidified atmosphere at 37? C. and 5% CO2. For virus infections and virus plasmid transfections cells were maintained in DMEM supplemented with 2% FBS. For recombinant BiTE plasmid transfections cells were maintained in DMEM without FBS. EpCAM expression of target cell lines was determined by flow cytometry.

Generation of EpCAM-Expressing Stable Cell Lines

[0925] The protein sequence of the EpCAM gene (ID: 4072) was obtained from NCBI database and DNA synthesised by Oxford Genetics Ltd (Oxford, UK). The EpCAM gene was cloned into pSF-Lenti vector by standard cloning techniques producing the pSF-Lenti-EpCAM vector. HEK293T cells were transfected using Lipofectamine 2000 with lentivirus EpCAM expression vector alongside pSF-C MVHIV-Gag-Pol, pSF-CMV-VSV-G, pSF-CMV-HIV-Rev (Oxford Genetics Ltd). Supernatants containing lentivirus were harvested 48 h later and mixed with polybrene (8 ?g/mL). Lentivirus/polybrene mixtures were added to CHO cells and incubated at 37? C. On day 4, the supernatant was exchanged for media containing 7.5 ?g/mL puromycin. Stable variants were then clonally selected and EpCAM expression of the parental cell lines or stable-transfected variant was determined by antibody staining with EpCAM or isotope control antibody and analysed by flow cytometry. Positive clones were expanded and used in further experiments.

Preparation of Peripheral Blood Mononuclear Cells (PBMC) and T Cell Isolation

[0926] PBMCs were isolated by density gradient centrifugation (Boyum, 1968) from whole blood leukocyte cones obtained from the NHS Blood and Transplant UK (Oxford, UK). Blood was diluted 1:2 with PBS and layered onto Ficoll (1,079 g/mL, Ficoll-Paque Plus, GE Healthcare) before centrifugation at 400 g for 30 min at 22? C. with low deceleration. After centrifugation, PBMCs were collected and washed twice with PBS (300 g for 10 min at room temperature) and resuspended in RPMI-1640 medium supplemented with 10% FBS. For extraction of CD3-positive T-cells from PBMCs, non-CD3 cells were depleted using Pan T Cell Isolation Kit (Miltenyi Biotec, #130-096-535), according to the manufacturer's protocol. For further isolation of CD4- and CD8-positive T-cells, CD3 T-cells underwent another round of purification using CD4+ Microbeads (Miltenyi Biotec, #130-045-101).

Processing Primary Ascites and Pleural Effusions

[0927] Primary human malignant ascites and pleural effusion samples were received from the Churchill Hospital, Oxford University Hospitals (Oxford, UK) following informed consent from patients with multiple indications of advanced carcinoma, including but not limited to ovarian, pancreatic, breast and lung. This work was approved by the research ethics committee of the Oxford Centre for Histopathology Research. Upon receipt, cellular and fluid fractions were separated and fluid used immediately or aliquots stored at ?20? C. for future analysis. The cellular fraction was treated with red blood cell lysis buffer (Roche, UK) following manufacturer's instructions. Cell number and viability was determined by trypan blue stain. Cell types present in each sample were determined by antibody staining for EpCAM, EGFR, FAP, CD45, CD11b, CD56, CD3, CD4, CD8, PD1 and CTLA4 and analysed by flow cytometry. For ex vivo T-cell activation and target cell lysis experiments fresh cells and fluid were used. In some cases, the adherent cells were passaged in DMEM supplemented with 10% FBS and expanded for later use.

BiTE Engineering and Production

[0928] BiTEs were generated by joining two scFvs of different specificities with a flexible GS linker. Each scFv is created by the joining of VH and VL domains from parental monoclonal antibodies by a linker. Each BiTE possessed an immunoglobulin light chain (Ig) N-terminal signal sequence for mammalian secretion and a C-terminal decahistidine affinity tag for detection and purification. BiTEs were engineered by standard DNA cloning techniques and inserted into a protein expression vector (pSFCMV-Amp) for cytomegalovirus (CMV) promoter-driven constitutive protein expression and secretion. pSF-CMV-EpCAMBiTE or pSF-CMV-ControlBiTE plasmid DNA were transfected into HEK293A cells using polyethylenimine (PEI, linear, MW 25000, Polysciences, USA) under the following conditions, 55 ?g of plasmid DNA: 110 ?g PEI (DNA:PEI ratio of 1:2 (w/w)) was added to cells, incubated at 37? C. for 4 h, then replaced with fresh serum-free DMEM and further incubated at 37? C., 5% CO2 for 48 h. Cells were transfected in parallel with pSF-CMV-GFP to ensure transfection efficiency. To harvest secreted protein, the supernatant of transfected cells was collected and centrifuged at 350 g, 4? C. for 5 min to remove cell components. Supernatants were transferred to 10,000 MWCO Amicon Ultra-15 Centrifugal Filter Units (Millipore). After centrifugation at 4750 g and 4? C., the volume of the retentate was adjusted with the flow through to obtain a 50-fold higher concentration. Aliquots of concentrated protein were stored at ?80? C.

Generation of BiTE-Expressing EnAdenotucirev

[0929] The plasmids pEnAd2.4-CMV-EpCAMBiTE, pEnAd2.4-SA-EpCAMBiTE, pEnAd2.4-CMV-ControlBiTE, pEnAd2.4-SA-ControlBiTE were generated by direct insertion of the transgene cassette encoding the EpCAM BiTE or control BiTE into the basic EnAd plasmid pEnAd2.4 using Gibson assembly technology. The transgene cassette contained a 5 short splice acceptor sequence or an exogenous CMV promoter, followed downstream by the EpCAM or control BiTE cDNA sequence and a 3 polyadenylation sequence. A schematic of the inserted transgene cassette is shown in FIG. 18. Correct construction of the plasmid was confirmed by DNA sequencing. The plasmids EnAd2.4-CMV-EpCAMBiTE, pEnAd2.4-SA-EpCAMBiTE, pEnAd2.4-CMV-ControlBiTE and pEnAd2.4-SA-ControlBiTE were linearised by restriction digest with the enzyme AscI prior to transfection in HEK293A cells. The production of virus was monitored by observation of cytopathic effect (CPE) in the cell monolayer. Once extensive CPE was observed the virus was harvested from HEK293A cells by three freeze-thaw cycles. Single virus clones were selected by serially diluting harvested lysate and re-infecting HEK293A cells, and harvesting wells containing single plaques. Serial infections of HEK293A cells were performed once an infection had reached full CPE in order to amplify the virus stocks. Once potent virus stocks were amplified the viruses were purified by double caesium chloride banding to produce EnAd-CMVEpCAMBiTE, EnAd-SA-EpCAMBiTE, EnAd-CMV-ControlBiTE, EnAd-SA-ControlBiTE virus stocks. These stocks were titred by TCID50 and picogreen assay (Life Technologies), following manufacturer's instructions.

Preparation of Supernatants

[0930] To evaluate BiTE-mediated cytokine release, DLD cells (20,000) were plated with 100,000 CD3+ T-cells in 96-well flat bottom plate alone or with 2 ng/?L EpCAM or control BiTE. After 48 h incubation at 37? C. and 5% CO2, supernatants were collected, cell components removed by centrifugation and aliquots stored at ?20? C. To assess BiTE transgene expression from recombinant viruses, HEK293A (1e6) or DLD cells (1.2e6) were infected with EnAd-CMV-EpCAMBiTE, EnAd-SA-EpCAMBiTE, EnAd-CMVControlBiTE, EnAd-SA-ControlBiTE or EnAd at 100 vp/cell. Cells were cultured for 72 h at which point the cytopathic effect (CPE) was advanced. Supernatants were collected and centrifuged for 5 min, 300 g to remove cell debris and stored at ?20? C. for future analysis.

Immunoblotting

[0931] Dot blot was used to measure the concentration of recombinant BiTE produced from plasmid transfections. Two-fold serial dilutions of each BiTE and of a protein standard (10?His-tagged (Cterminus) human Cathepsin D, Biolegend, #556704) were prepared. The molar concentration of the protein standard was adjusted to represent a BiTE concentration of 100 ?g/mL. Two ?L of each sample and protein standard was directly applied onto a nitrocellulose membrane. The membrane was air dried, blocked and probed with ?-6?His (C-terminus) antibody (1:5000, clone 3D5, Invitrogen, UK, #46-0693) for detection of C-terminally His-tagged proteins, followed by washing and incubation with antimouse secondary antibody (1:10000, Dako, #P0161) and detected by application of SuperSignal West Dura Extended Duration Substrate (Thermo Fisher, #34075) according to manufacturer's instructions. Supernatants of virus-infected HEK293A cells were analysed by Western blotting for BiTE expression. Supernatants were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane according to manufacturer's protocols (Bio-Rad). Membranes were further treated identically to that of dot blot protocol above.

Enzyme-Linked Immuno-Sorbent Assay (ELISA)

[0932] To assess EpCAM binding, ELISA plates were prepared by coating overnight at 4? C. with human EpCAM/TROP-1 protein (50 ng/well, Sino Biological Inc, #10694-H02H-50). Plates were blocked for 1 h at ambient temperature with 5% BSA, followed by incubation with diluted EpCAM BiTE-, Control BiTE- and empty pSF-CMV vector-transfected HEK293A supernatants (2 h, room temperature). Plates were washed three times with PBS-T and subsequently after every future binding step. Plates were incubated with anti-His (C-term) antibody (1:5000, clone 3D5, #46-0693, Invitrogen, UK) for 1 h, room temperature, followed by HRP conjugated anti-mouse-Fc (1:1000 in PBS/5% milk, Dako) for 1 h at room temperature. HRP detection was performed using 3.3.5.5-teramethylethylenediamine (TMB, Thermo-Fisher) and stop solution was used for terminating the reaction. Absorbance at 450 nm was measured on a Wallac 1420 plate reader (Perkin Elmer).

Flow Cytometry

[0933] Flow cytometry analysis was performed on a FACSCalibur flow cytometer (BD Biosciences) and data processed with FlowJo v10.0.7r2 software (TreeStar Inc., USA). For classification of different cellular populations, antibodies specific for CD45 (HI30, Biolegend), CD11b (ICRF44, Biolegend), EpCAM (9C4, Biolegend) and FAP (427819, R&D Systems) were used. For analysis of T-cell populations, the following antibody clones coupled to different fluorophores were used: CD69 (FN50, Biolegend), CD25 (BC96, Biolegend), IFN? (4S.B3, Biolegend), ?CD107a antibody (H4A3, Biolegend), CD3 (HIT3a, Biolegend), CD4 (OKT4, Biolegend), CD8a (HIT8a, Biolegend), PD1 (H4A3, Biolegend). In each case, the appropriate isotype control antibody was used.

Characterisation of Human T-Cell Activation

CD69 and CD25 Expression Levels

[0934] The ability of the recombinant EpCAM BiTE or EpCAM BiTE viruses to induce T-cell activation was assessed by surface expression of CD69 and CD25. Human CD3 cells (75,000 cells/well in 96-well flatbottom plates) from PBMC or ascites samples were cultured alone or with DLD, SKOV, CHO, CHOEpCAM or ascites target cells (15,000) in the presence of media alone, EpCAM or control BiTE protein (2 ng/?L) or recombinant virus (100 vp/cell). In some cases, anti-PD1 (Invivogen, #hpd1ni-mab7) antibody was added at a final concentration of 2.5 ?g/mL. CD3 cells were incubated with CD3/CD28 Dynabeads (Thermo Fisher, #11131D) as positive control for T cell activation. Cells were cultured medium for 24 h at 37? C. unless stated otherwise and subsequently harvested with enzyme free cell dissociation buffer (Gibco, #13151014). Total cells were stained with antibodies for surface expression of CD69, CD25, CD3, CD4 or CD8 and analysed by flow cytometry. The effect of ascites fluid on T-cell activation (CD69, CD25) was investigated by polyclonally activating CD3-purified PBMC (100,000) by incubating with plate-immobilised CD3 antibody (7.5 ?g/mL, HIT3a, Biolegend, #300313) in RPMI-1640 or fluids isolated from the malignant ascites samples.

IFN? Expression

[0935] The ability of the EpCAM BiTE to induce T-cell activity was assessed by IFN? expression, by co-culture of T-cells for 6 h with DLD cells (200,000 CD3 cells/well, 40,000 DLD cells/well in a flat-bottom 96 well plate) and 2 ng/?L recombinant EpCAM or control BiTE. As a positive control, T cells were stimulated with soluble PMA/ionomycin cell activation cocktail (Biolegend, #423301). Brefeldin A (GolgiPlug, BD Biosciences) was added into the culture medium 5 h before harvest, at which point CD3+ T-cells were harvested and intracellularly stained for IFN? expression and analysed by flow cytometry.

T Cell Proliferation

[0936] To study T cell proliferation, 100,000 CFSE-labelled (CellTrace CFSE kit, Invitrogen, #C34554) CD3+ T cells were incubated with 20,000 DLD cells in 96 well plate format, with 2 ng/?L EpCAM or control BiTE. Five days after co-culture, cells were stained for CD3, CD4 or CD8 and CFSE fluorescence of viable CD3+ T-cells were measured by flow cytometry, with total cell number normalised using precision counting beads (5000/well, Biolegend, #424902). Fluorescence data was analysed and modelled using the proliferation function of FlowJo v7.6.5 software. Data is presented as the percentage of original cells that entered a proliferation cycle (% divided) or the average number of cell divisions that a cell in the original population has undergone (Division Index).

CD107a Degranulation

[0937] DLD cells (15,000 cells/well) were co-cultured with 75,000 CD3+ T-cells in a flat-bottom 96 well plate in the presence of media alone or 2 ng/?L of control or EpCAM BiTE. ?CD107a or isotype control antibodies were added directly to the culture medium. Monensin (GolgiStop, BD Biosciences) was added after 1 h of incubation at 37? C. and 5% CO2, followed by 5 h of further incubation. Cells were subsequently harvested, stained for CD3, CD4 or CD8 and analysed by flow cytometry.

Cytokine Release

[0938] Cytokines within supernatants harvested from cultures of DLD/PBMC or pleural effusion cells were quantified using the LEGENDplex Human T Helper Cytokine panel (Biolegend, #740001) and flow cytometry following the manufacturer's instructions. Cytokines included in the analysis are IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN? and TNF?.

In Vitro Target Cell Cytotoxicity Assay

[0939] Target cell cytotoxicity mediated by recombinant BiTE or viruses was assessed by LDH release or MTS assay. Target cells (DLD, SKOV, HT-29, A431, A549, PC3, CHO, CHO-EpCAM) were co-cultured with CD3, CD4 or CD8 T-cells (E:T 5:1) in a flat-bottom 96 well plate in the presence of media alone, diluted supernatants or virus (100 vp/cell). After 24 h of co-culture (unless stated otherwise), supernatants and cells were harvested and cytotoxicity determined by LDH assay (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, #G1780) or MTS viability assay (CellTiter 96 Cell Proliferation Assay, Promega, #G3580) as per manufacturer's instructions. Quantity of BiTE produced from virus-infected DLD cells was determined by comparing cytotoxicity induced by diluted viral supernatants to that of a standard curve generated using recombinant BiTE.

[0940] To evaluate oncolytic activity of the viruses, DLD cells were seeded in 96-well plate (25,000 cells/well) for 18 h at 37? C. and 5% CO2, before infection with increasing vp/cell (5-fold serial dilution, 100 to 5.12e-5 vp/cell) or left uninfected. DLD cytotoxicity was measured on day 5 by MTS viability assay. Dose response curves were fitted and IC50 determined using a four parameter non-linear fit model integrated into Prism 7 software (GraphPad Software). Cell viability was monitored in real-time using xCELLigence RTCA DP technology (Acea Biosciences). DLD, SKOV3 or MCF7 cells were plated in 48-well E-plate at 12,000 cells/well. Plates were incubated for 18 h, 37? C., 5% CO2, before cells were either treated with BiTE (2 ng/?L) or infected with virus (100 vp/cell) or left untreated. Two hours after infection, 75,000 CD3+ cells were added to the necessary wells. Cell impedance was measured every 15 min for a duration of up to 160 h. For ex vivo cytotoxicity assays, unpurified cells from ascites or pleural effusion samples were resuspended in ascites fluid and plated (1.5e5/well) in flat bottom 96-well plates. After incubation for the stated duration at 37? C., 5% CO2, supernatants were analysed by LDH assay or total cells were harvested by cell-dissociation buffer, stained for CD3, CD25 and EpCAM, and analysed by flow cytometry. For PD1 blocking experiments, anti-PD1 antibody (2.5 ?g/mL, Invivogen, #hpd1ni-mab7) antibody was included.

Viral Genome Replication and qPCR

[0941] The ability of EnAd-CMV-EpCAMBiTE, EnAd-SA-EpCAMBiTE, EnAd-CMV-ControlBiTE, EnAd-SAControlBiTE or EnAd to replicate their genomes was analysed by seeding DLD cells in 24-well plate (150,000 cells/well) for 18 h, 37? C., 5% CO2, before infection with 100 vp/cell. Wells were harvested 24 and 72 h post infection, and DNA purified using PureLink genomic DNA mini kit (Invitrogen, #K182001) according to the manufacturer's protocol. Total viral genomes were quantified by qPCR against EnAd hexon using specific primer-probe set (primers: TACATGCACATCGCCGGA/CGGGCGAACTGCACCA, probe: CCGGACTCAGGTACTCCGAAGCATCCT).

Microscopy

[0942] Brightfield and fluorescence images were captured on a Zeiss Axiovert 25 microscope. Time lapse videos were obtained to observe viral or T cell-mediated lysis of target cells by EnAd or EnAd-CMVEpCAMBiTE. Uninfected cells were used as a negative control. NHDF cells were stained with CellTracker Orange CMTMR Dye (Life Technologies, #C2927) and CD3+ cells were stained with CellTrace Violet Cell Proliferation Kit (Life Technologies, #C34557) following manufacturer's protocols. Dyed NHDF were plated in a 24-well plate at 7,500 cells/well in co-culture SKOV3 at 13,500 cell/well. Plates were incubated for 18 h, 37? C., 5% CO2. Cells were then treated with 300 ng/mL EpCAM BiTE or infected with 100 vp/cell of EnAd or EnAd2.4-CMV-EpCAMBiTE or left untreated. After 2 h incubation, 100,000 dyed CD3+ were added to necessary wells, in addition to 1.5 uM CellEvent Caspase 3-7 reagent (Life Technologies, #C10423). Images were captured on a Nikon TE 2000-E Eclipse inverted microscope (10? optical objective) at intervals of 15 min covering a period of 96 h. Time-lapse videos (12 frames/second) were generated using Image) software.

Statistics

[0943] In all cases of more than two experimental conditions being compared, statistical analysis was performed using a One-way ANOVA test with Tukey's Post Hoc analysis. All data is presented as mean?SD. The significant levels used were P=0.01-0.05 (*), 0.001-0.01 (**), 0.0001-0.001 (***). All in vitro experiments were performed in triplicate, unless stated otherwise.

Example 18Generation and Production of a BiTE Targeting EpCAM

[0944] A BiTE targeting EpCAM was engineered by joining two scFv specific for CD3E and EpCAM with a flexible glycine-serine (GS) linker. A control BiTE, recognising CD3E and an irrelevant antigen (the filamentous hemagglutinin adhesin (FHA) of Bordetella pertussis) was also produced. Both BiTEs were engineered to contain an N-terminal signal sequence for mammalian secretion and a C-terminal decahistidine affinity tag for detection and purification (FIG. 45A). To characterise the functionality of the recombinant BiTEs, they were cloned into expression vectors under transcriptional control of the CMV immediate early promoter (pSF-CMV-EpCAMBiTE and pSF-CMV-ControlBiTE, respectively).

[0945] Adherent HEK293 cells (HEK293A) were transfected with the expression vectors and supernatants harvested and concentrated 50-fold for further analysis. To estimate the amount of BiTE produced, samples were serially diluted and evaluated, using anti-His, in a dot blot using decahistidine-tagged cathepsin D as a standard. In this way it was possible to estimate the level of BiTEs produced into the supernatant to be approximately 20 ?g/mL at 48 h post transfection (of 1.8e7 HEK293A cells) (FIG. 46A). Specific binding of the EpCAM BiTE and not the control BiTE to recombinant EpCAM protein was demonstrated by ELISA (FIG. 46B).

Example 19Characterisation of Human T-Cell Activation by Recombinant EpCAM BiTE

[0946] The ability of recombinant EpCAM BiTE protein to activate PBMC-derived T cells was evaluated by adding unstimulated human primary CD3+ cells to a culture of human DLD colorectal carcinoma cells, which are known to express EpCAM on their surface (Karlsson et al, 2008). Addition of 2.5 ng/ml EpCAM BiTE (as supernatant from transduced HEK293A cells) led to a significant increase in T cell activation markers CD69 and CD25 (FIGS. 45B & C), whereas the control BiTE had no effect. Exposure of CD3 cells to the EpCAM BiTE in the absence of tumour cells gave a very modest increase in CD69 and CD25, and this indicates that antibody-mediated clustering of CD3 is essential for full activation by this anti-CD3 binding. T cells stimulated by the EpCAM BiTE in the presence of tumour cells also showed a significant increase in the production of gamma interferon (FIG. 45D) and cell proliferation (FIG. 45E) whereas the control BiTE had no effect. The aim of T cell activation is to cause degranulation-mediated cytotoxicity, and expression of surface CD107a/LAMP1 (indicating degranulation, Aktas et al.) was strongly upregulated by the EpCAM BiTE but not by control (FIG. 45F)

[0947] The release of cytokines following EpCAM BiTE-mediated activation of PBMC-derived T cells in the presence of DLD cells was characterised by flow cytometry using a cytokine bead array. As before the control BiTE showed little activity, although the EpCAM BiTE triggered release of several cytokines, including high levels of IL-2, IL-6, IL-10, IL-13, gamma interferon and TNF (FIG. 45G). Production of IL-2, gamma interferon and TNF are generally associated with a Th1 response, whereas IL-6 and IL-10 are more often linked to a Th2 response (Mosmann & Sad, 1996).

Example 20Specificity of Recombinant EpCAM BiTE

[0948] Most human epithelial cells express EpCAM, so to assess whether the effect of the EpCAM BiTE was antigen-specific, Chinese Hamster Ovary cells (CHO cells) were engineered using a lentiviral vector to express human EpCAM on their surface. In the presence of EpCAM BiTE and CHO-EpCAM cells, exogenously added PBMC-derived T cells showed strong activation (assessed by CD25 expression see FIG. 47A) and associated cytotoxicity (FIG. 47B) that was not seen with parental CHO control cells or control BiTEs. This indicates that the cytotoxicity of the EpCAM BiTE is antigen-specific.

[0949] We then assessed whether the EpCAM BiTE would kill a range of tumour cells, and whether the level of EpCAM BiTE-mediated cytotoxicity observed was dependent on the density of EpCAM expression. Cytotoxicity of T cells in the presence of the EpCAM BiTE was measured in six different carcinoma cell lines, with greatest cytotoxicity observed in DLD and A431, and least in A549 and PC3 (FIG. 47C). This showed a loose association with the surface levels of EpCAM (determined by flow cytometry), where A549 and PC3 cells showed the lowest levels and DLD the highest (FIG. 47D). This suggests that the presence and level of EpCAM expression do influence the degree of cytotoxicity, although other factors (perhaps the intrinsic resistance of cells to granzyme-mediated apoptosis) also play a role in determining the overall level of cell killing.

Example 21BiTE Mediated Activation of CD4+ and CD8+ T Cell Subsets

[0950] To determine which T cell types are activated by the EpCAM BiTE, PBMC-derived T cells were incubated with DLD cells and activated using the BiTE prior to flow analysis. Both CD4+ and CD8+ cells showed high levels of expression of CD69 and CD25 (FIG. 49A), although the percentage of activated CD4 cells was generally slightly greater. EpCAM BiTE-mediated T cell proliferation was assessed using CFSE stain (FIG. 49B), and degranulation by expression of CD197a/LAMP1 (FIG. 49C) and again similar levels of activation were seen for both CD4+ and CD8+ cells. Finally, levels of tumour cell cytotoxicity achieved were compared using EpCAM BiTE to activate purified CD4+ and CD8+ subsets. All T cell preparations showed similar cytotoxicity (FIG. 49D), indicating that both CD4+ and CD8+ cells can contribute to the BiTE-mediated cytotoxicity observed.

Example 22Expression of the EpCAM BiTE from Oncolytic Adenovirus, EnAdenotucirev

[0951] EnAdenotucirev (EnAd) is an oncolytic adenovirus, a chimera of group B type 11 and type 3 adenovirus with a mosaic E2B region, a nearly complete E3 deletion and a smaller E4 deletion mapped to E4orf4 (Kuhn 2008). Currently undergoing several early phase clinical trials for treatment of cancer, the virus combines good systemic pharmacokinetics and promising clinical activity with the possibility to encode and express transgenes (Calvo 2014, Boni 2014). The EpCAM BiTE was encoded within EnAd immediately downstream of the fibre gene, using a shuttle vector inserted into the virus backbone by Gibson assembly (FIG. 18). The BiTE was placed either under transcriptional control of a CMV immediate early promoter (EnAd-CMV-EpCAM BITE), or was placed downstream of a splice acceptor site for the adenovirus major late promoter (MLP; EnAd-SA-EpCAM BiTE). In the former configuration the BiTE should be expressed whenever the virus successfully infects a cell, whereas expression from the MLP splice acceptor site will only occur when the MLP is activated in cells that are permissive to virus replication. A control BiTE (recognising CD3 and FHA) was also introduced to create two corresponding control viruses.

[0952] The viruses were cloned, rescued in HEK293A cells, and a large batch of each was prepared in a hyperflask and purified twice by caesium chloride banding. Infection of DLD with parental EnAd and the recombinant BiTE viruses yielded similar amounts of viral genomes (measured by qPCR) at all timepoints tested, indicating the BiTE transgene does not interfere with the viral replication kinetics (FIG. 51A). Next we investigated the replication and oncolytic properties of the viruses in the absence of human T-cells. DLD cells were infected with virus batches at increasing virus particles (vp)/cell, and the cytotoxicity measured by MTS assay on day 5. All of the recombinant viruses, including those with EpCAM and control BiTEs, regulated by the CMV promoter or splice acceptor, showed cytotoxic activity indistinguishable from the parental virus, showing that the genetic modification had not changed the intrinsic oncolytic activity of the virus (FIG. 51B).

[0953] To assess BiTE expression and secretion, the BiTE-expressing EnAd viruses were used to infect HEK293A cells, and 72 h supernatants were examined by western blotting using an anti-His antibody. As shown in FIG. 51C, all four viruses (two expressing the control BiTE and two expressing the EpCAM BiTE) showed similar levels of BiTE secreted into the supernatant.

Example 23Selective Killing of EpCAM Positive Cells by Virally Produced EpCAM BiTE

[0954] The supernatants from EnAd-EpCAM BiTE-infected HEK293A cells were added to cultures of CHO and CHO-EpCAM cells, either with or without PBMC-derived T cells; T cell activation and cytotoxicity to the CHO/CHO-EpCAM cells was measured after 24 h. In the case of CHO cells, there was no increase in T cell expression of CD25 (FIG. 51D) nor any cytotoxicity observed with any treatment (FIG. 51E). However, T cells I incubated with the CHO-EpCAM cells showed substantial increases in CD25 expression using supernatants from HEK293A cells that had been infected with either EnAd-CMV-EpCAM BiTE or EnAd-SA-EpCAM BiTE viruses (FIG. 51D). As expected this translated into selective cytotoxicity to CHO-EpCAM cells only when T cells were added in the presence of supernatant from 293A cells that had been infected with either EnAd-CMV-EpCAM BiTE or EnAd-SA-EpCAM BiTE viruses (FIG. 51E). Crucially there was no cytotoxicity in the absence of T cells, or when using supernatants from HEK293A that cells had been infected with EnAd expressing the control BiTE.

Example 24Superior Cytotoxicity of EnAd Expressing EpCAM BiTE

[0955] EnAd kills most carcinoma cells quickly by direct oncolysis (Kuhn 2008), although some cellsnotably SKOV3 ovarian carcinoma cellsare partially resistant and killed more slowly. We therefore reasoned that the consequences of arming End to secrete EpCAM BiTE, leading to cytotoxic activation of T cells might be particularly evident in SKOV3 cells. Cells were therefore exposed to virus (100 vp/cell) 24 h after seeding and cell death monitored by xCELLigence system. PBMC-derived T cells were added (or not) to the SKOV3 cell culture 2 h later. In the absence of T cells, the tumour cells grew for approximately 72 h (manifest by the increasing Cell Index signal in FIG. 53A) but cell growth then reached a plateau and remained stable, independent of virus infection, up until at least 160 h). All tested viruses, including parental EnAd, induced no observable target cell cytotoxicity during the time measured. However, when co-cultured with PBMC-derived T cells, both the CMV- and SA-EpCAM BiTE-armed viruses induced rapid SKOV3 lysis, with CMV-driven induced lysis within 16 h, and SA within 44 h following addition of T cells (FIG. 53B). Importantly, parental EnAd or the non-specific BiTE control viruses demonstrated no target cell lysis in this time frame even with the addition of Tcells. This result was confirmed by LDH assay, in which co-cultures identical to above were set up, with cytotoxicity measured at 24, 48 and 96 h post-infection (FIG. 48). These results are further supported by similar findings in DLD cells in which EpCAM BiTE expressing viruses induced cytotoxicity at a significantly quicker rate than the control BiTE viruses (FIG. 50A+B).

[0956] To confirm that target cell cytotoxicity is mediated via T cell activation, CD3 cells were harvested at each timepoint and activation status determined by CD69 and CD25 expression, demonstrating similar kinetics of expression as observed for cytotoxicity (FIGS. 53C & D, FIGS. 50C & D). The approximate quantity of EpCAM BiTE produced from infected DLD cells was determined by comparing cytotoxicity (Abs490) induced by infected DLD supernatants to the cytotoxicity induced by known quantities of recombinant BiTE (i.e. creation of a standard curve (Abs490)). DLD in co-culture with CD3-purified PBMC (1:5) were incubated with recombinant BiTE (FIG. 50E) or infected DLD supernatant (FIG. 50F) and LDH release was measured at 24 h, This allowed us to determine that EpCAM BiTE was produced at 165 ?g and 50 ?g per million DLD for EnAd-CMV-EpCAMBiTE and EnAd-SAEpCAMBiTE, respectively. The EC50 for the EpCAM BiTE is 7.4 ng/ml (FIGS. 50E & F), and therefore EpCAM BiTE is produced by the recombinant virus at levels that are likely to reach therapeutic doses.

[0957] Cytotoxicity of EpCAM BiTE-expressing EnAd was visualised by time lapse video microscopy. SKOV3 tumour cells (unlabelled) were co-incubated with normal human fibroblasts (EpCAM-negative, labelled red, serving as non-target control cells) and PBMC-derived T cells (labelled blue) in the presence of a caspase stain (CellEvent Caspase 3-7 reagent produces a green stain when caspases are activated). Again the combination of EpCAM BITE-expressing EnAd, combined with exogenous T cells, gave dramatic cytotoxicity to the SKOV3 tumour cells, which showed strong induction of apoptosis when infected with EnAd-CMV-EpCAMBiTE, but not parental EnAd. Importantly, the EpCAM-negative NHDF in co-culture remained viable throughout. Representative fluorescent images at different time points from the SKOV3 videos are shown in FIG. 53E. Equivalent time lapse videos showing DLD cells (which are intrinsically more sensitive to the virus) cocultured with NHDF are also shown.

Example 25EpCAM BiTE can Overcome Immune Suppression, Activate Endogenous T Cells and Kill Endogenous Tumour Cells within Malignant Peritoneal Ascites

[0958] Three clinical samples of malignant peritoneal ascites samples containing EpCAM-positive tumour cells and primary fibroblasts (as control, non EpCAM-expressing cells) were expanded ex vivo and the mixed primary cell populations were incubated with PBMC-derived T-cells and treated with free BiTE or 100 vp/cell EnAd-EpCAMBiTE in culture medium. After 72 h, the level of EpCAM-positive target cells (FIG. 55A) or non-target fibroblast activation protein (FAP)-positive fibroblasts (FIG. 55B) were measured by flow cytometry. Activation of T cells was analysed by measuring CD25 expression (FIG. 55C). The free EpCAM BiTE and the EpCAM BiTE-expressing viruses induced T-cell activation, leading to a depletion of EpCAM-positive tumour cells, with primary FAP-positive (EpCAM-negative) fibroblasts showing no change in numbers. This was observed in all the patients' samples, and none of the other treatments showed any significant effects. This demonstrates that the EpCAM BiTE (or oncolytic virus encoding it) can mediate activation and selective cytotoxicity by PBMC-derived T cells to human ovarian ascites tumour cells.

[0959] Malignant exudates likely represent an environment of potential immune tolerance with suppressed immune responses commonly observed in patients with late-stage metastatic cancer. To test this hypothesis we polyclonally stimulated PBMC-derived T cells with anti-CD3 antibodies in culture media or the presence of 100% ascites fluid from five patients with peritoneal malignancies. Whereas in RPMI medium the anti-CD3 antibody gave approximately 50% of T cells positive for both CD25 and CD69, the presence of ascites fluid appeared to attenuate the activation of T-cells as determined by decreased antibody-mediated elevation of CD69/CD25 expression, and this was particularly noticeable for patient fluid #2 (FIG. 56A). This supports our notion that components of ascites fluid may exert an immune suppressive or tolerising effect. However, this attenuation in the increase of activation markers did not correlate with a suppression of T-cell degranulation, with CD107a externalisation in ascites fluid similar to that in culture medium (FIG. 56B). It follows that BiTEs may be able to bypass tumour microenvironment-associated mechanisms of T-cell immunosuppression (Nakamura & Smyth, 2016).

[0960] We therefore investigated the ability of PBMC-derived T cells and EpCAM BiTE to mediate target cell cytotoxicity in the presence of immunosuppressive ascites fluid. T-cells incubated with ascites fluid 1 and 2 induced similar lysis of the human breast adenocarcinoma MCF7 cell line as when in RPMI culture medium (measured using xCELLigence), although the cytotoxicity showed a delay of about 8 h in the presence of patient ascites fluid #2 (FIG. 56C). In addition to the immune suppressive fluid and tumour cells present, ascites contain tumour-associated lymphocytes and supporting cells of the tumour stroma, providing a unique tumour-like model system to test BiTE-mediated activation of endogenous patient-derived T-cells. Following a 24 h incubation of total endogenous cells and the ascites fluid with the free recombinant BiTE, activation of patient T cells was assessed (FIG. 56D). In this highly clinically-relevant setting the EpCAM BiTE (but not the control counterpart) induced CD69 and CD25 expression, albeit CD25 at lower levels when the experiment was performed in 100% ascites fluid than in simple medium. These data suggest that the EpCAM BiTE can overcome at least some of the immune suppressive effects of peritoneal ascites fluid to activate endogenous T cells. Cytotoxicity was assessed by measuring release of LDH, and the BiTE caused a significant rise both when the experiment was performed in medium and also in 100% ascites fluid. This indicates that some of the ascites cells had been killed by BiTE-mediated cytotoxicity, although given the multiple cell types present in primary ascites it is not possible to define what proportion of tumour cells are killed.

Example 26EnAd Expressing EpCAM BiTE can Activate Endogenous T Cells to Kill Endogenous Tumour Cells within Malignant Pleural Exudates

[0961] To study the effects of the EpCAM BiTE-expressing viruses in another clinically relevant setting, we obtained several samples of pleural exudates from patients with a range of malignancies. At initial screening (an example is shown in FIG. 52), samples considered suitable for further analysis were those containing CD3 and EpCAM-positive cells. We also assessed the expression of PD1 by endogenous T cells following their initial isolation, and whereas only 10% of PBMC-derived T cells expresses PD1, all the malignant effusion samples T cells were at least 40% positive for PD1 and reached sometimes as high as 100% (FIG. 54). Unpurified total cells (isolated by centrifugation and resuspended) were incubated at fixed concentrations in 100% pleural effusion fluid in the presence of 500 ng/mL free EpCAM BiTE or 100 vp/cell virus encoding BiTE. After 5 days, the total cell population was harvested, and the total number of CD3+ cells (FIG. 57A) was measured. Compared to untreated controls, only samples receiving the free EpCAM BiTE or EnAd encoding EpCAM BiTE showed T cell proliferation. This confirms that the EpCAM BiTE was binding to the EpCAM target and crosslinking CD3 to stimulate endogenous T cells. The expression level of CD25 on CD3 cells was also determined (FIG. 57B). The free EpCAM BiTE induced significant T-cell activation of tumour associated lymphocytes (assessed by CD25 expression) in all patients' samples, even within the likely immune-tolerising environment of the pleural effusion fluid. The addition of an anti-PD1 blocking antibody had no effect on EpCAM BiTE mediated activation of T cells in this setting (FIGS. 54B & C). There was noticeable variation between patients (although little between samples from the same patient), with activation ranging from 50% to 90% dependent on the donor. Similarly, samples treated with EnAd expressing the EpCAM BiTE showed high activation in some patients (ranging from 10-20% up to 80%, for both EnAd-CMV-EpCAM BITE and EnAd-SA-EpCAM BiTE).

[0962] Interestingly, the patient showing the lowest BiTE-mediated activation also showed the lowest level of background T cell activation. Parental EnAd, or EnAd expressing control BiTEs, or free control BiTEs caused no stimulation above background.

[0963] We assessed the ability of the BiTE-expressing viruses to mediate EpCAM-targeted cytotoxicity by measuring residual levels of EpCAM positive cells by flow cytometry at the end of the five day incubation (FIG. 57C). The free EpCAM BiTE, and the two viruses encoding EpCAM BiTE, caused a marked depletion of autologous EpCAM-expressing cells in every case, whereas the other treatments had little or no effect on the level of EpCAM-positive cells. In the case of Sample #1 there is a slightly decreased viability with all EnAd based viruses compared to the untreated control, and this is likely to represent the effects of direct viral oncolysis. In conjunction with the lack of influence of the PD1 blocking antibody on T cell activation, it had no effect on EpCAM BiTE mediated killing of target cells, with near complete cytotoxity of EpCAM+ cells (patients 2, 3 & 4) in the absence of the PD1 blocker (FIG. 54D).

[0964] The different effects of parental EnAd and EnAd-CMV-EpCAM BiTE are shown by microscopy in FIG. 57D, where expression of the BiTE decreases the presence of tumour cells and expands the T cell population. The associated flow cytometry plots confirm the substantial expansion and activation of T cells following treatment with the EpCAM BiTE-expressing virus.

[0965] Finally the effects of the various treatments were characterised by measuring the levels of key cytokines produced using a LEGENDplex protein array (FIG. 57E). By far the greatest fold increases were in gamma interferon, which rose nearly 1000-fold following treatment with the free EpCAM BiTE or EnAd encoding EpCAM BiTE. These two treatments also caused approximately 10-fold increases in expression of IL-5, IL-13, tumour necrosis factor (TNF), IL17A and IL17F, characteristic of activated T cells. EnAd alone (or expressing the control BiTE) also caused a 10-fold rise in gamma interferon, but otherwise no treatments caused any appreciable changes in cytokine expression.

Example 27Discussion

[0966] Oncolytic viruses offer an intriguing new strategy to combine several therapeutic modalities within a single targeted, self-amplifying, agent (Keller & Bell, 2016; Seymour & Fisher, 2016). As they replicate selectively within cancer cells and spread from cell to cell, some oncolytic viruses are thought to mediate cell death by non-apoptotic death pathways (Ingemarsdotter et al, 2010; Li et al, 2013), as part of the process allowing virus particles to escape from dying cells. EnAd, in particular, kills cells by a pro-inflammatory process known as oncosis or ischemic cell death (Dyer, 2017). This non-apoptotic death mechanism causes release of several pro-inflammatory cellular components, such as ATP, HMGB1 and exposure of calreticulin (known as damage-associated molecular patterns, DAMPs) (Weerasinghe & Buja, 2012), and is likely pivotal to the ability of the virus to promote an effective anticancer immune response. In addition to the consequences of direct lysis, however, viruses offer the potential to encode and express other anticancer biologics, obviating delivery challenges and ensuring the biologic achieves its highest concentration within the tumour microenvironment. Imlygic encodes GM-CSF, however the potential for arming viruses is virtually limitless and provides many exciting opportunities to design multimodal therapeutic strategies with additive or synergistic anticancer effects (de Gruijl et al, 2015; Hermiston & Kuhn, 2002).

[0967] Encoding BiTEs within oncolytic viruses provides a powerful means to activate tumour infiltrating lymphocytes to become cytotoxic and lyse antigen-positive target cells, providing a completely separate therapeutic modality from the effects of direct viral lysis. In this study we have shown that BiTE-targeted cytotoxicity is fully antigen-specific, can be mediated by both CD4 and CD8 T cells (Brischwein et al, 2006) and can be incorporated into an oncolytic adenovirus and expressed only in cells that allow virus replication. In addition the current study shows, for the first time, that endogenous T cells within liquid cancer biopsies can be activated by BiTEs and virus-encoded BiTEs and can kill endogenous tumour cells without any additional stimulation or reversal of immune suppression. Importantly, this can happen even in the primary fluids that comprise the microenvironment of peritoneal ascites or pleural effusions, as surrogates for the immune suppressive microenvironment of solid tumours.

[0968] Arming oncolytic viruses to express BiTEs combines two quite distinct therapeutic mechanisms, with the former providing lytic death of tumour cells that are permissive for virus infection, and the latter targeting T cell cytotoxicity via a specific, chosen, antigen. This provides considerable flexibility in the design of a therapeutic approach, perhaps using the BiTEs to deliver cytotoxicity to tumour-associated cells that are relatively resistant to kill by the virus directly. For example, while we have exemplified the technology here using a BiTE that recognises a carcinoma-associated antigen (EpCAM), it is also possible to use the BiTE approach to target cytotoxicity to tumour-associated fibroblasts or other stromal cells. Indeed, even when the targets for BiTE-recognition are not restricted to expression in the tumour microenvironment, by linking BiTE production to virus replication allows expression of the BiTE to be spatially restricted to the tumour, minimising systemic toxicities. This is important, as BiTEs administered intravenously show relatively short circulation kinetics (Klinger et al, 2012) and are often associated with considerable on-target off-tumour toxicities (Teachey et al, 2013).

[0969] The possibility to encode BiTEs within oncolytic viruses has been previously explored using an oncolytic vaccinia virus with an Ephrin A2-targeting BiTE. This agent showed that the Ephrin BiTE could mediate activation of PBMCs and antigen-targeted killing of tumour cells both in vitro and in vivo. Intriguingly, although the BiTE could activate T cells it did not lead to T cell proliferation without the addition of exogenous IL-2, whereas the BiTE used in the current study led to extensive proliferation both of PBMC in vitro and of tumour-associated lymphocytes using the clinical biopsy samples ex vivo.

[0970] We believe that the differences observed may reflect the different BiTE design, the different oncolytic virus used or perhaps depend on the antigen density giving sufficient crosslinking of CD3 on the T cells.

[0971] One central aim of oncolytic virus therapy is to create an anticancer T cell response that recognises patient specific neoantigens as well as public tumour associated antigens. Lytic viruses may do this by stimulating improved antigen presentation by lysing tumour cells in the context of DAMPs alongside virus-related pathogen-associated molecular patterns (PAMPs). Immunohistochemical staining of resected colon tumours, following intravenous delivery of EnAd, suggest the virus promotes a strong influx of CD8+ T cells into tumour tissue (Garcia-Carbonero, 2017). However, while this is potentially a very powerful approach, adaptive T cell responses are ultimately dependent on the expression of MHC class I antigens by tumour cells, to allow targeted killing. Loss of MHC expression is a well documented immune evasion strategy for tumours (Garrido et al, 2016). It is noteworthy that both cytotoxic strategies that are immediately engaged by BiTE-armed oncolytic viruses operate independently of MHC class I by the tumour cells, and therefore can be employed to kill cancer cells even when tumour cells have lost MHC expression.

[0972] The present study thus demonstrates that encoding BiTEs within EnAd provides a particularly promising strategy to achieve targeted expression in disseminated tumours, exploiting the known blood-stability and systemic bioavailability of the virus, which has now been studied in several early phase clinical trials. Notably, in a study where the virus is given intravenously a few days prior to resection of primary colon cancer, subsequent immunohistological assessment of tumour sections showed that the virus had reached to regions through the tumours and gave strong intranuclear hexon signals, indicating successful infection and virus replication selectively in tumour cells. This confirms preclinical data (Di et al, 2014; Illingworth, 2017) indicating that this virus is stable in 100% human blood and should be capable of tumour targeted infection of disseminated and metastatic malignancies in human patients.

[0973] BiTEs could be encoded by EnAd without any loss of oncolytic virulence (FIG. 51B), reflecting the considerable transgene packaging capacity of the virus. The presence of the transgene will not affect the physicochemical properties of the virus particles, hence the modified viruses should exhibit exactly the same clinical pharmacokinetics as the parental agent, and should be capable of expressing the encoded BiTE selectively within tumours throughout the body. This provides an exciting and potentially very effective new approach to systemically targeted cancer immunotherapy that should now be prioritised for clinical assessment.

Example 28

Immunosuppression of Human T-Cell Activation and Target Cell Cytotoxicity by Patient Malignant Exudate Fluids

[0974] Malignant exudates represent an environment of potential immune tolerance with suppressed immune responses commonly observed in patients with late-stage metastatic cancer. The quantity of IL-10, considered to be an anti-inflammatory cytokine, was measured in normal serum or patient malignant exudate fluids (A, peritoneal ascites; P, pleural effusion) using Human IL-10 ELISA MAX kit (Biolegend, 430604). IL-10 levels in the exudates (88.1-633.4 pg/mL) were far in excess of those measured in normal serum (7.2-10 pg/mL). See FIG. 58.

[0975] The ability of CD3/CD28 beads (Gibco, 11161D) to activate PBMC T-cells in the presence of normal serum, ascites or pleural fluid was investigated. Human PBMC T-cells (100,000 cells per well in 96 well plate) were treated with CD3/CD28 beads (following manufacturers instructions) in normal serum or patient exudate fluid (50%). T-cells were left untreated in each fluid as negative control. After 24 hours of culture, cells were harvested and the expression levels of CD69 and CD25 on CD3+ T-cells were then analysed by antibody staining and flow cytometry represented as percentage of dual positive (CD69+CD25+ cells) (FIG. 59). In normal serum the anti-CD3/CD28 beads gave approximately 60% of T cells dual positive for both CD25 and CD69, whereas the presence of ascites fluid attenuated T cell activation in 6/12 fluids.

[0976] In a similar experiment, 100,000 T-cells were treated with CD3/CD28 beads in the presence of normal serum, ascites or pleural fluid (50%). Anti-CD107a or isotype control antibody were added directly to culture medium. After 1 hour, monensin was added (BD Golgistop, BD Biosciences) according to manufacturers instructions. After 5 further hours, cells were harvested and analysed by flow cytometry to determine degranulation (FIG. 60). In normal serum the anti-CD3/CD28 beads gave approximately 22.5% of T cells degranulated, whereas the presence of ascites fluid attenuated T cell activation in 10/12 fluids. The level of degranulation was significantly correlative (Pearson co-efficient, r=?0.7645; p=0.0038) with quantity of IL-10 in each fluid (FIG. 61).

[0977] In a similar experiment, 75,000 T-cells were co-cultured with 15,000 SKOV3 and EpCAM in the presence of normal serum, ascites or pleural fluid (50%). T-cells were treated with control BiTE in each fluid as negative control. After 24 hours of culture, cells were harvested and the expression levels of CD69 and CD25 on CD3+ T-cells were then analysed by antibody staining and flow cytometry represented as percentage of dual positive (CD69+CD25+ cells) (FIG. 62). In normal serum the EpCAM BiTE gave approximately 67.6% of T cells dual positive for both CD25 and CD69, whereas the presence of ascites fluid attenuated T cell activation in 0/12 fluids, and slightly induced activation in 4/10 fluids.

[0978] In a similar experiment, 75,000 T-cells were co-cultured with 15,000 SKOV3 and EpCAM in the presence of normal serum, ascites or pleural fluid (50%). T-cells were treated with control BiTE in each fluid as negative control. Anti-CD107a or isotype control antibody were added directly to culture medium. After 1 hour, monensin was added (BD Golgistop, BD Biosciences) according to manufacturers instructions. After 5 further hours, cells were harvested and analysed by flow cytometry to determine degranulation (FIG. 63). In normal serum the EpCAM BiTE beads gave approximately 41.4% of T cells degranulated, whereas the presence of ascites fluid attenuated T cell activation in 2/12 fluids.

[0979] The ability of EnAd-SA-EpCAMBiTE and EnAd-SA-ControlBiTE to induce T cell-mediated target cell lysis in malignant exudate fluids was assessed using xCELLigence technology. SKOV cells were plated in 48-well E-plate at 1e4 cells/well respectively. Plates were incubated for 18 hrs, 37? C., 5% CO2, before cells were either infected with 100 virus particles per cell (ppc) or were left uninfected. After two hours, PBMC T-cells (5:1) in normal serum or patient exudate fluid (final, 50%) were added. xCELLigence was used to measure target cell cytotoxicity every 10 minutes (FIG. 64). The results suggest that BiTE-mediated SKOV3 lysis by T-cells is independent of fluid used.

[0980] Unpurified ascites cells (therefore unchanged from when received) are seeded at 100,000 cells per well of a flat-bottom 96-well plate in RPMI media or ascites fluid. Cells were treated with EpCAM or control BiTE, with untreated wells serving as a negative control. After incubation at 37 C for 24 hours, cells were harvested, and the expression level of CD25 and CD69 on CD3 cells determined (FIG. 65). The results demonstrate that EpCAM BiTE resulted in significant increase in T-cell activation (CD69/CD25 dual positive) of tumour-associated lymphocytes, slightly increased by ascites fluid. In a similar experiment, unpurified ascites cells (therefore unchanged from when received) are seeded at 100,000 cells per well of a flat-bottom 96-well plate in RPMI media or ascites fluid. Cells were treated with EpCAM, control BiTE or recombinant BiTE viruses (100 vp/cell), with untreated wells serving as a negative control (FIG. 66). After incubation at 37 C for 5 days, the total cell population was harvested, and the number of CD3+ cells (FIG. 66A) and expression level of CD25 on CD3 cells determined (FIG. 66B) and the number of endogenous EpCaM+ cells determined by flow cytometry (FIG. 66C). Total cell numbers per well were determined using precision counting beads. The results demonstrate that EpCAM BiTE and EnAd expressing EpCAM BiTE resulted in significant increase in T-cell activation (CD3 number, CD25) of tumour-associated lymphocytes and cytotoxicity of EpCAM+ cells in both RPMI media and ascites fluid.

[0981] As an extension of the experiment above, six more patient exudate samples (for a total of 7) were treated identically in ascites fluid (FIG. 67) and number of CD3+(FIG. 67A), CD25 expression of T-cells (FIG. 67B) and number of EpCAM+ cells (FIG. 67C) determined by flow cytometry. The results show that EpCAM BiTE and EnAd expressing EpCAM BiTE resulted in significant increase in T-cell activation (CD3 number, CD25) of tumour-associated lymphocytes and cytotoxicity of EpCAM+ cells reproducibly in a range of exudate biopsy samples.

Example 29

FAP BiTE Mediate Activation of T-Cells and Killing of FAP+ Cells by Different Donor T-Cells

[0982] In other experiments, methods described in Example 2 were used to further evaluate the T-cell activating properties of recombinant FAP BiTE protein tested in co-cultures of NHDF and T-cells, comparing to control BiTE and polyclonal T-cell activation using anti-CD3/CD28 Dynabeads. Supernatants taken after 24 hours of culture were tested by ELISA for IFN? (FIG. 68A) and by cytokine bead array (LEGENDplex human T helper cytokine panel, BioLegend #74001) for a panel of cytokines (FIG. 68B). The control BiTE induced no significant change in any cytokine, however the FAP-BiTE led to strong increases in gamma interferon, IL-2, TNF?, IL-17 and IL-10, consistent with different subsets of T-cells being stimulated, and production of IFN? was far greater than that triggered by anti-CD3/CD28.

[0983] Stimulation with the FAP BiTE, but not control BiTE, in the presence of NHDF cells also induced rapid degranulation (within 6 hr) of T-cells, both CD4+ and CD8+ subsets, as determined by the externalisation of CD107a/LAMP1 on the T-cell surface (as assessed by flow cytometry), which is strongly correlative with their ability to kill target cells (FIGS. 69A&B). This induction of degranulation by the FAP BiTE translated to potent fibroblast lysis (FIG. 69C), as measured by LDH release after 24 h co-culture with PBMC T-cells (EC.sub.50 of ?2.5 ng/mL) with induced T-cell activation and cytotoxicity observed using 6/6 donor T-cells (FIG. 69D). No cytotoxicity was induced by the control BiTE, consistent with T-cells remaining in an inactivated state.

Example 30

[0984] Effect of FAP BiTE and EnAd-FAP BiTE Viruses on Cells in Primary Malignant Ascites Samples from Different Cancer Patients

[0985] As a follow-on to studies described in Example 16, fresh primary malignant peritoneal ascites from further cancer patients were obtained for study of EnAd FAP BiTE virus activities. Three patient samples containing both EpCAM.sup.+ tumour cells and FAP fibroblasts were expanded ex vivo, and the mixed (adherent) cell populations were cultured with PBMC-derived T-cells and unmodified or BiTE expressing EnAd viruses. After 72 h, total cells were harvested and the number of FAP+(FIG. 70A) and EpCAM+ cells (FIG. 70B) determined by flow cytometry. Additionally, the activation status of T-cells (by CD25 expression) was measured (FIG. 70C). Infection with both EnAd-CMV-FAPBiTE and EnAd-SA-FAPBiTE induced T-cell activation and FAP+ cell depletion in all patient samples, with no significant change in levels of EpCAM+ tumour cells. Parental EnAd or the control viruses induced no observable T cell activation, with FAP+ cell numbers remaining similar to the uninfected control. Importantly, this depletion in FAP+ fibroblasts consistently led to a strong reduction in levels of the immunosuppressive cytokine TGF? detected in supernatants (FIG. 70D).

[0986] In a second series of experiments, total (and unpurified) cells from five patient biopsy samples were evaluated to assess the activity of endogenous tumour-associated T-cells in the samples. Cells were plated in 50% ascites fluid and treated with recombinant control or FAP BiTE proteins, or 100 vp/cell of EnAd or EnAd-BiTE viruses. After 5 days incubation, T-cell activation (by CD25 expression) and residual number of FAP+ cells was measured by flow cytometry (FIGS. 71A&B). In all 3 patient samples, recombinant FAP-BiTE and EnAd-CMV-FAP BiTE induced strong T-cell activation, with up to ?80% of patient-derived T-cells activated, which caused a marked depletion FAP.sup.+ fibroblasts. Interestingly, EnAd-SA-FAP-BiTE induced CD25 expression in 2/3 samples, with no observable activation or FAP+ cell depletion in patient 1. This is probably due to insufficient tumour cells being present for infection by the virus and production of BiTE protein (no EpCAM.sup.+ tumour cells were detected in this sample by flow cytometry), consistent with the requirement for tumour cells for MLP (SA)-driven transgene expression (this likely also explains the lack of T-cell activation and FAP+ cell depletion by EnAd-SA-FAP-BiTE virus with the patient ascites sample illustrated in FIGS. 42-44). Collectively, the data shows that EnAd expressing FAP-BiTE can, following infection of tumor cells, reproducibly lead to activation of tumour-associated T-cells to kill endogenous fibroblasts.

[0987] Another experiment investigated whether FAP-BiTE activity could be improved by blocking the PD-1 checkpoint, using a patient biopsy sample in which T-cells were 73.6% PD-1 positive and FAP.sup.+ cells were 62.9% PDL1-positive (FIG. 72A). Co-cultures similar to those described above were set up in the presence or absence of a purified blocking mouse IgG2b antibody to human PDL1 (BioLegend, clone 29E.2A3) at a final concentration of 2.5 ?g/mL. After 2 days of culture, total cells were harvested and residual FAP+ cells and T-cell activation was measured. The inclusion of the blocking anti-PDL1 antibody led to a modest increase in CD25 induction (FIG. 72B) and a two-fold higher IFN? production (FIG. 72C), without altering the depletion of FAP+ cells (FIG. 72D) with near complete lysis by day 2 in either setting.

[0988] Tumour-associated lymphocytes (TALs) isolated from ovarian cancer patient ascites are reported to have enriched expression of PD-1 and impaired effector functionsincluding cytotoxicity and IFNg production. Consistent with this, PD-1 expression was 2-fold higher on CD3+ cells from six cancer patient ascites biopsies than on those in peripheral blood mononuclear cells (PBMCs) from three healthy donors (FIG. 73A). To evaluate the functionality of the T-cells within these cancer biopsy samples, NHDF cells and unpurified PBMC or ascites cells (the % CD3+ cells for each of the samples is shown in FIG. 73B) were co-cultured with control or FAP BiTE-containing supernatants, and supernatants were harvested 5 days later and tested for IFN? by ELISA (FIG. 73C). No IFN? was induced by the control BiTE. Three of the ascites cell samples produced IFN? at a similar level to that of the PBMC samples, while the other three had an attenuated response to the FAP BiTE. We next investigate the ability of these T-cells to induce BiTE-mediated lysis of the NHDF cells. NHDF were plated, and PBMC or ascites cells added along with BiTE-containing supernatants and the viability of cells in the culture monitored in real-time using the xCELLigence cytotoxicity assay system. Despite the variability in IFN? production, all ascites samples induced full cytotoxicity of NHDF cells when added with the FAP BiTE, with an overall similar rate of BiTE-mediated NHDF lysis to that seen with when effected by PBMCs (FIG. 73D).

[0989] To investigate whether the FAP BiTE can mediate T-cell activation in the presence patient malignant exudate samples (all at 50%), PBMC T-cells were activated with control or FAP BiTEs in the presence of NHDF cells, or activated with anti-CD3/CD28 Dynabeads, either in 50% normal human serum (NS) or different (cell-free) malignant exudate samples. Whereas in normal serum 74% of T-cells were activated (dual-positive for both CD25 and CD69) at 24 h following stimulation with the anti-CD3/CD28 beads, 3/5 tested ascites fluid significantly attenuated T-cell activation compared to the response in NS (FIG. 74A). However, when PBMCs were cultured with NHDF and stimulated with the FAP BiTE, there was no observable suppression of T-cell activation in the presence of any of the exudate fluids (FIG. 74B), demonstrating that the FAP BiTE can overcome immunosuppressive mechanisms to activate T-cells.

Example 31

EnAd-FAPBiTE-Mediated Oncolysis and T Cell Stimulation Polarise CD11b+ TAMs in Patient Ascites to a More Activated Phenotype

[0990] To investigate whether the production of Th1 cytokines, including IFN?, TNF? and IL-2, by FAP BiTE-mediated activation of T-cells, and the subsequent elimination of FAP.sup.+ fibroblasts (and associated reduction in TGF?1 was associated other shifts in the tumour microenvironment from immunosuppressive and pro-oncogenic towards anti-tumour activity, the effect on tumour-associated macrophages (TAMs) in an unseparated ascites cell sample was evaluated. Total unpurified patient ascites cells were plated in 50% ascites fluid and treated with free control or FAP BiTE or infected with EnAd-SA-control BiTE or EnAd-SA-FAPBiTE virus (at 100 vp/cell). In parallel, some cells were treated in with IFN? to induce an activated CD11b myeloid cell phenotype. After 3 days incubation, the activation status of T-cells was first measured; CD25+ cells measured by flow cytometry and IFN? secretion by ELISA.

[0991] Treatment with FAP BiTE and EnAd-SA-FAPBiTE led to approximately 60% of CD3+ T-cells becoming CD25+(FIG. 75A) and large quantities of IFN? in culture supernatants (FIG. 75B). No increase above background by the control BiTE or control virus was observed for CD25 expression or IFN?. To evaluate TAM polarisation, the expression levels of CD64 and CD86 (M1 or activated macrophage markers) and CD206 and CD163 (M2 or TAM markers) were measured on CD11b+ cells by flow cytometry (FIG. 75C). Treatment with free FAP BiTE or EnAd expressing FAP BiTE induce a more activated phenotype, manifested by significant increases in CD64 expression, and strong decreases CD206 and CD163similar to that observed when IFN? was spiked into the cultures. While treatment with free FAP BiTE or control virus induced no clear change in CD86 above background in this experiment, the EnAd expressing FAP BiTE induced a large increase in CD86 expression, indicating that EnAd virus infection and FAP BiTE activity may synergize to activate primary myeloid cells within a suppressive tumour microenvironment such as the malignant ascetic fluid samples tested here. In this study, IFN? treatment induced a modest decrease in CD86, indicating that the strong increase in CD86 observed by EnAd-SA-FAPBiTE may be via an IFN?-independent mechanism.

Example 32

EnAd-FAPBiTE Activates Tumour-Infiltrating Lymphocytes and Induces Cytotoxicity in Solid Prostate Tumour Biopsies Ex Vivo

[0992] Tissue slice cultures provide one of the most realistic preclinical models of diverse tissues, organs and tumours. To evaluate the activity of the FAP BiTE expressing viruses in this highly clinically-relevant setting, several paired punch biopsies of malignant and benign prostate tissue from resected human prostates were studies. At initial screening, prostate tissue was reproducibly shown to have circular rings of EpCAM+ tumour cells (FIG. 76A) interspersed between large regions of stroma containing scattered CD8 T-cells (FIG. 76B). FAP staining was found on fibroblasts adjacent to tumour regions (FIG. 76C).

[0993] Cores were sliced by a vibratome to 300 ?m thickness and slice cultures established in the presence of virus (1.5e9 vp/slice), or left uninfected. After 7 days, slices were fixed, paraffin-embedded, sectioned and T-cell activation status was assessed by immunohistochemistry (IHC) by staining for CD25 expression (FIG. 76D). Only samples receiving EnAd-CMV-FAPBiTE or EnAd-SA-FAPBiTE showed activation of tumour-infiltrating T-cells, manifest by strong CD25 staining. Neither untreated or control virus-treated had detectable CD25-positive cells. Supernatants from these slice cultures taken at 4 and 7 days post-infection were tested for IFN? and IL-2 by ELISA, with increases in IFN? detected from malignant, but not benign, prostate slice cultures infected with either FAP BiTE virus (FIG. 76E) and IL-2 detected in cultures with EnAd-SA-FAPBiTE virus (FIG. 76F). The EnAd-SA-FAPBiTE induced higher quantities of IFN?, which were detectable earlier, than the CMV-driven FAPBiTE virus.

Example 33EnAd Viruses Expressing EpCAM or FAP BiTEs

[0994] Five viruses (NG-611, NG-612, NG-613, NG-614, NG-617) were generated that encode a single BiTE (Table 8).

TABLE-US-00008 TABLE 8 Virus ID Transgene Cassette NG-611 (SEQ ID NO: 96) SSA.sup.1-EpCamBiTE.sup.2-His.sup.3-PA.sup.4 NG-612 (SEQ ID NO: 97) SSA.sup.1-FAPBiTE.sup.5-His.sup.3-PA.sup.4 NG-613 (SEQ ID NO: 98) SA.sup.6-FAPBiTE.sup.5-His.sup.3-PA.sup.4 NG-614 (SEQ ID NO: 99) SA.sup.6-FAPBiTE.sup.7-His.sup.3-PA.sup.4 NG-617 (SEQ ID NO: 100) SSA.sup.1-FAPBiTE.sup.5-PA.sup.4 .sup.1SEQ ID NO. 55; .sup.2SEQ ID NO. 83; .sup.3SEQ ID NO. 84; .sup.4SEQ ID NO. 65; .sup.5SEQ ID NO. 85; .sup.6SEQ ID NO. 86; .sup.7SEQ ID NO. 87;

[0995] In each transgene cassette, the cDNA encoding the BiTE was flanked at the 5 end with either a short splice acceptor sequence (SSA, SEQUENCE ID NO: 55) or a longer splice acceptor sequence (SA, SEQUENCE ID NO: 86). At the 3 end of the BiTE, a SV40 late poly(A) sequence (PA, SEQUENCE ID NO: 65) was encoded preceded by either a Histidine tag (HIS, SEQ ID NO. 41) or no tag. In viruses NG-611, NG-612, NG-613 and NG-617 the anti-CD3 portion of the BiTE molecule used a single chain variant of the mouse anti-human CD3E monoclonal antibody OKT3.

Virus Production

[0996] The plasmid pEnAd2.4 was used to generate the plasmids pNG-611, pNG-612, pNG-613, pNG-614 and pNG-617 by direct insertion of synthesised transgene cassettes (SEQ ID NOs: 88-92, respectively). The pNG-611 transgene cassette encodes for an EpCam targeting BiTE (SEQ ID NO. 93), the pNG-612, pNG-613 and pNG-617 transgene cassettes encode a FAP targeting BiTE of SEQ ID NO. 94 and the pNG-614 transgene cassette encodes a FAP targeting BiTE of SEQ ID NO. 95. Schematics of the transgene cassettes are shown in FIG. 77A to C. Construction of plasmid DNA was confirmed by restriction analysis and DNA sequencing.

[0997] The plasmids, pNG-611, pNG-612, pNG-613, pNG-614 and pNG-617, were linearised by restriction digest with the enzyme AscI to produce the virus genomes. The viruses were amplified and purified according to methods given below.

[0998] Digested DNA was purified by phenol/chloroform extraction and precipitated for 16 hrs, ?20? C. in 300 ?l>95% molecular biology grade ethanol and 10 ?l 3M Sodium Acetate. The precipitated DNA was pelleted by centrifuging at 14000 rpm, 5 mins and was washed in 500 ?l 70% ethanol, before centrifuging again, 14000 rpm, 5 mins. The clean DNA pellet was air dried, resuspended in 500 ?l OptiMEM containing 15 ?l lipofectamine transfection reagent and incubated for 30 mins, RT. The transfection mixture was then added drop wise to a T-25 flask containing 293 cells grown to 70% confluency. After incubation of the cells with the transfection mix for 2 hrs at 37? C., 5% CO.sub.2 4 mls of cell media (DMEM high glucose with glutamine supplemented with 2% FBS) was added to the cells and the flasks was incubated 37? C., 5% CO.sub.2.

[0999] The transfected 293 cells were monitored every 24 hrs and were supplemented with additional media every 48-72 hrs. The production of virus was monitored by observation of a significant cytopathic effect (CPE) in the cell monolayer. Once extensive CPE was observed the virus was harvested from 293 cells by three freeze-thaw cycles. The harvested viruses were used to re-infect 293 cells in order to amplify the virus stocks. Viable virus production during amplification was confirmed by observation of significant CPE in the cell monolayer. Once CPE was observed the virus was harvested from 293 cells by three freeze-thaw cycles. The amplified stocks of viruses were used for further amplification before the viruses were purified by double caesium chloride banding to produce purified virus stocks.

Virus Activity Assessed by qPCR

[1000] A549 cells, either infected for 72 hrs with 1ppc NG-611, NG-612, NG-617, enadenotucirev or left uninfected, were used for quantification of viral DNA by qPCR. Cell supernatants were collected and clarified by centrifuging for 5 mins, 1200 rpm. DNA was extracted from 45 ?L of supernatant using the Qiagen DNeasy kit, according to the manufacturer's protocol. A standard curve using enadenotucirev virus particles (2.5e10-2.5e5vp) was also prepared and extracted using the DNeasy kit. Each extracted sample or standard was analysed by qPCR using a virus gene specific primer-probe set to the early gene E3.

[1001] Quantification of the number of detected virus genomes per cell demonstrated that NG-611, NG-612, and NG-617 showed significant genome replication in A549 cell lines (FIG. 77D). This was similar for all viruses tested including the parental virus enadenotucirev, indicating that inclusion of the BiTE transgene does not impact virus replicative activity. No virus genomes could be detected in uninfected cells (data not shown).

T Cell Activation and Degranulation Mediated by BiTE Expressing Viruses.

Carcinoma Cell Infection

[1002] A549 cells were seeded into 24 well plates at a density of 2.5e5 cells/well. Plates were incubated for 4 hrs, 37? C., 5% CO.sub.2, before cells were either infected with 1ppc of NG-611, NG-612, enadenotucirev or were left uninfected. At 24, 48 or 72 hrs post-infection supernatants were harvested from the cells, clarified by centrifuging for 5 mins, 1200 rpm and snap frozen.

T Cell Assay

[1003] FAP expressing lung fibroblast cell lines MRC-5, or EpCam expressing ovarian carcinoma cells, SKOV3 were seeded into 48 well plates at densities of 5.7e4 cells/well and 1.2e5 cells/well, respectively. Plates were incubated for 4 hrs, 37? C., 5% CO.sub.2, before media was replaced with 150 ?L/well of thawed supernatant harvested from the A549 plates. Purified CD3 T cells isolated form human PBMC donors were then also added to the plates to give a ratio of T cells to MRC-5 or SKOV3 of 2 to 1. The co-cultures were incubated for 16 hrs, 37? C., 5% CO.sub.2 before cellular supernatants were collected for ELISA analysis and T cells harvested for flow cytometry analysis. Culture media containing non-adherent cells was removed from co-culture wells and centrifuged (300?g). The supernatant was carefully removed, diluted 1 in 2 with PBS 5% BSA and stored for ELISA analysis. The adherent cell monolayers were washed once with PBS and then detached using trypsin. The trypsin was inactivated using 10% FBS RPMI media and the cells were added to the cell pellets that had been collected from the culture supernatants. The cells were centrifuged (300?g), the supernatant discarded and the cell pellet washed in 2004 of PBS. The cells were centrifuged again then resuspended in 504 of PBS containing Live/Dead Aqua (Life tech) for 15 minutes at RT. The cells were washed once in FACs buffer before staining with panels of directly conjugated antibodies: anti-CD3 conjugated to AF700; anti-CD25 conjugated to BV421; anti-HLA-DR conjugated to PE/CY5; anti-CD40L conjugated to BV605; anti-CD69 conjugated to PE and anti-CD107a conjugated to FITC. A sample of cells from each co-culture condition was also stained with relevant isotype control antibodies. All staining was carried out in FACs buffer in a total volume of 504/well for 15 minutes, 4? C. Cells were then washed twice with FACs buffer (2004) before resuspension in 2004 of FACs buffer and analysis by Flow cytometry (Attune).

Upregulation of T Cell Activation Markers

[1004] Flow cytometry analysis of T cell activation was assessed by expression of the T cell activation markers CD25, CD69, HLA-DR and CD40L or the T cell degranulation marker, CD107a on live, single cells. These data showed that when co-cultured with EpCam.sup.+ SKOV3 cells the number of T cells expressing CD25, CD69, HLA-DR, CD40L or cell surface CD107a was significantly increased when NG-611 supernatants were added to the cells compared to NG-612, enadenotucirev or untreated control supernatants (FIG. 78). For all these markers little T cell activation was stimulated by supernatants from A549 cells infected for 24 hrs however, by 48 hrs post-infection, supernatants stimulated significant T cell activation across all markers. This was also the case at 72 hrs post-infection.

[1005] When co-cultured with FAP.sup.+ MRC-5 cells the number of T cells expressing CD25, CD69, HLA-DR, CD40L or cell surface CD107a was significantly increased when NG-612 supernatants were added to the cells compared to NG-611, enadenotucirev or untreated control supernatants (FIG. 79). Some T cell activation could also be observed with the NG-611 virus, which was likely due to low but detectable expression of EpCam (?5%) on the MRC-5 cell lines engaging the EpCam BiTE expressed by the NG-611 virus (FIG. 80). For all these markers, little T cell activation was stimulated by supernatants from A549 cells infected for 24 hrs however, by 48 hrs post-infection, supernatants stimulated significant T cell activation across all markers. CD25 and CD69 markers were also upregulated following incubation with supernatants harvested 72 hrs post-infection, however, activation markers, HLA-DR, CD40L and CD107a were detected at lower levels with supernatants harvested 72 hrs post-infection than 48 hrs post-infection. This could be due to high levels of BiTE present at this later stage of infection leading to rapid and potent T cell activation that means the effector functions need to measured at timepoints earlier than 16 hrs post-incubation with the supernatants.

[1006] For detection of IFN? expression, co-culture supernatants were diluted into 5% BSA/PBS assay buffer (in a range of 1:10 to 1:1000) and ELISA was carried out using the Human IFN gamma Quantikine ELISA kit (R&D systems) according to the manufacturer's protocol. The concentration of secreted IFN? was determined by interpolating from the standard curve. Expression of IFN? could only be detected in the supernatants of co-cultures using NG-611 on SKOV3 cells FIG. 81A) or NG-611, NG-612 on MRC-5 cells (FIG. 81B).

Example 34: Immune Activation and Anti-Tumour Efficacy of BiTE Expressing Viruses In Vivo

[1007] NSG mice humanised CD34+ haematopoietic stem cells (from Jackson Labs) were implanted with HCT116 tumour cells subcutaneously on both flanks at 18 weeks post engraftment. Once tumours reached 80-400 mm.sup.3 mice were grouped such that each treatment arm had an equivalent distribution of tumour volumes, 7 mice per group. Mice were injected intratumorally with either saline, enadenotucirev or NG-611 at 5?10.sup.9 particles per injection, 2 injections per tumour. Tumours on both flanks were treated. Tumour volume was measured 3-4 times per week and demonstrated that NG-611 treatment resulted in a significant anti-tumour response out to 20 days post-dosing compared to enadenotucirev or untreated controls (FIG. 82a). After the 20 days post-dosing one tumour from 4 mice in each group was processed for flow cytometry while remaining tumours were frozen on dry ice.

Flow Cytometry

[1008] Tumour samples were mechanically disaggregated immediately following resection in a small volume of RPMI media. Disaggregated tumours were then passed through a 70 ?m cell strainer and centrifuged at 300 g for 10 minutes. Cell pellets were resuspended in 1004 of PBS containing Live/Dead Aqua (Life tech) for 15 minutes on ice. The cells were washed once in FACs buffer (5% BSA PBS) before staining with a panel of directly conjugated antibodies: anti-CD8 (RPA-T8, AF700); anti-CD4 (RPA-T4, PE); anti-CD45 (2D1, APC-Fire 750); anti-CD3 (OKT3, PerCP-Cy5.5); anti-CD25 (M-A251, PE-Dazzle 594); anti-CD69 (FN50, APC); anti-HLA-DR (L243, BV605); anti-CD107a (H4A3, FITC). A pool of tumour cell suspensions was also stained with relevant isotype control antibodies. All staining was carried out in FACs buffer in a total volume of 504/well for 20 minutes at 4? C. Cells were washed three times with FACs buffer (2004) before resuspension in 2004 of FACs buffer and analysis by Flow cytometry (Attune). FACs analysis demonstrated that the ratio of CD8 to CD4 T cells in the tumour was significantly increased in NG-611 treated tumours compared to enadenotucirev treated or untreated controls (FIG. 82b).

Example 35EnAd Viruses Co-Expressing FAP BiTEs and Immune-Modulatory Cytokines and Chemokines

[1009] Three viruses (NG-615, NG-640 and NG-641) were generated that encoded a FAP BiTE and immunomodulatory proteins (Table 9).

TABLE-US-00009 TABLE 9 Virus ID Transgene Cassette NG-615 SSA.sup.1-FAPBiTE.sup.2-E2A.sup.3-Flt3L.sup.4-P2A.sup.5-MIP1?.sup.6-T2A.sup.7- (SEQ ID NO: 101) IFN?.sup.8-PA.sup.9 NG-640 SSA.sup.1-FAPBiTE.sup.2-P2A.sup.5-CXCL10.sup.10-T2A.sup.7-CXCL9.sup.11- (SEQ ID NO: 102) PA.sup.6 NG-641 SSA.sup.1-FAPBiTE.sup.5-P2A.sup.5-CXCL10.sup.10-T2A.sup.7-CXCL9.sup.11- (SEQ ID NO: 103) E2A.sup.3-IFN?.sup.8-PA.sup.6 NG-615 SA.sup.12-FAPBiTE.sup.2-E2A.sup.3-Flt3L.sup.4-P2A.sup.5-MIP1?.sup.6-T2A.sup.7- (SEQ ID NO: 298) IFN?.sup.8-PA.sup.9 .sup.1SEQ ID NO. 55; .sup.2SEQ ID NO. 87; .sup.3SEQ ID NO. 63; .sup.4SEQ ID NO. 105; .sup.5SEQ ID NO. 61; .sup.6SEQ ID NO. 107; .sup.7SEQ ID NO. 64; .sup.8SEQ ID NO. 109; .sup.9SEQ ID NO. 65; .sup.10SEQ ID NO. 110; .sup.11SEQ ID NO. 111; .sup.12SEQ ID NO. 86

Virus Production

[1010] The plasmid pEnAd2.4 was used to generate the plasmids pNG-615, pNG-616, pNG-640 and pNG-641 by direct insertion of synthesised transgene cassettes (SEQ ID NOs: 112-114, respectively). NG-615 and NG-616 contain four transgenes encoding for a FAP-targeting BiTE (SEQ ID NO: 94), Flt3L (SEQ ID NO. 115), MIP1? SEQ ID NO. 116) and IFN? (SEQ ID NO. 117). NG-640 and NG-641 encode for a FAP targeting BiTe (SEQ ID NO. 94), CXCL9 (SEQ ID NO. 118) and CXCL10 (SEQ ID NO. 119), NG-641 also contains a fourth transgene encoding IFN? (SEQ ID NO. 117) Schematics of the transgene cassettes are shown in FIG. 83A to C. Construction of plasmid DNA was confirmed by restriction analysis and DNA sequencing.

[1011] The plasmids, pNG-615, pNG-616, pNG-640 and pNG-641, were linearised by restriction digest with the enzyme AscI to produce the virus genomes. The viruses were amplified and purified according to methods detailed in Example 33.

Virus Activity Assessed by qPCR and Transgene ELISA

Carcinoma Cell Infection

[1012] A549 cells either infected for 72 hrs with 1ppc NG-615, enadenotucirev or left uninfected were used for quantification of viral DNA by qPCR and analysis of transgene expression by ELISA. Cell supernatants were collected and clarified by centrifuging for 5 mins, 1200 rpm. 454 of supernatant was used for DNA analysis and the remaining supernatant was used for ELISA.

qPCR

[1013] DNA was extracted from the supernatant sample using the Qiagen DNeasy kit, according to the manufacturer's protocol. A standard curve using enadenotucirev virus particles (2.5e10-2.5e5vp) was also prepared and extracted using the DNeasy kit. Each extracted sample or standard was analysed by qPCR using a virus gene specific primer-probe set to the early gene E3. Quantification of the number of detected virus genomes per cell demonstrated that NG-615 showed significant genome replication in A549 cell lines at a level similar to that of the parental virus enadenotucirev (FIG. 84). These data indicated that inclusion of the BiTE and three immunomodulatory transgenes does not significantly impact virus replicative activity. No virus genomes could be detected in uninfected cells.

ELISA

[1014] IFN? ELISA was carried out using the Verikine Human IFN alpha Kit (Pbl assay science), MIP1? ELISA was carried out using the Human CCL3 Quantikine ELISA kit (R & D systems) and Flt3L ELISA was carried out using the Flt3L human ELISA kit (Abcam). All assays were carried out according to the manufacturers' protocol.

[1015] The concentrations of secreted IFN?, MIP? or FLt3L were determined by interpolating from the standard curves. IFN?, MIP1? and Flt3 L expression could be detected in the cellular supernatant of NG-615 but not enadenotucirev or untreated control cells (FIG. 85).

T Cell Activation and Degranulation Mediated by BiTE Expressing Viruses.

Carcinoma Cell Infection

[1016] A549 cells were seeded into 24 well plates at a density of 2.5e5 cells/well. Plates were incubated for 4 hrs, 37? C., 5% CO.sub.2, before cells were either infected with 1ppc of NG-612, NG-615, enadenotucirev or were left uninfected. At 24, 48 or 72 hrs post-infection supernatants were harvested from the cells, clarified by centrifuging for 5 mins, 1200 rpm and snap frozen.

T Cell Assay

[1017] FAP expressing lung fibroblast cell lines MRC-5 were seeded into 48 well plates at a density of 5.7e4 cells/well. Plates were incubated for 4 hrs, 37? C., 5% CO.sub.2, before media was replaced with 150 ?L/well of thawed supernatant harvested from the A549 plates. Purified CD3 T cells isolated form human PBMC donors were then also added to the plates to give a ratio of T cells to MRC-5 of 2 to 1. The co-cultures were incubated for 16 hrs, 37? C., 5% CO.sub.2 before cellular supernatants were collected for ELISA analysis and T cells harvested for flow cytometry analysis according to the methods detailed in Example 29.

Upregulation of T Cell Activation Markers

[1018] Flow cytometry analysis of T cell activation was assessed by expression of the T cell activation markers CD25, CD69, HLA-DR and CD40L or the T cell degranulation marker, CD107a on live, CD3+, single cells. These data showed that when co-cultured with FAP.sup.+ MRC-5 cells the number of T cells expressing CD25, CD69, HLA-DR, CD40L or CD107a was significantly increased when NG-615 or 612 supernatants were added to the cells compared to enadenotucirev or untreated control supernatants (FIG. 86).

Secretion of the Stimulatory Cytokine IFN?

[1019] For detection of IFN? expression, co-culture supernatants were diluted into 5% BSA/PBS assay buffer (in a range of 1:10 to 1:1000) and ELISA was carried out using the Human IFN gamma Quantikine kit (RandD Systems) according to the manufacturer's protocol. The concentration of secreted IFN? was determined by interpolating from the standard curve. Expression of IFN? could only be detected in the supernatants of co-cultures using NG-612 or NG-615 infected A549 supernatants (FIG. 87).

Example 36EnAd Virus Co-Expressing a BiTE Targeting FAP and a BiTE Targeting EpCam

[1020] The virus NG-618 was generated that encoded two BiTE molecules, one targeting EpCam (EpCam BiTE) and one targeting FAP (FAP BiTE) (Table 10).

TABLE-US-00010 TABLE 10 Virus ID Transgene Cassette NG-618 SSA.sup.1-EpCAMBiTE.sup.2-P2A.sup.3-FAPBiTE.sup.4-PA.sup.5 (SEQ ID NO: 120) .sup.1SEQ ID NO. 55; .sup.2SEQ ID NO. 121; .sup.3SEQ ID NO. 106; .sup.4SEQ ID NO. 122; .sup.5SEQ ID NO. 65;

Virus Production

[1021] The plasmid pEnAd2.4 was used to generate the plasmid pNG-618 by direct insertion of a synthesised transgene cassettes (SEQ ID NO. 123). The NG-618 virus contains two transgenes encoding an EpCam targeting BiTE (SEQ ID NO. 93) and a FAP targeting BiTE (SEQ ID NO. 95). A schematic of the transgene cassette is shown in FIG. 88. Construction of plasmid DNA was confirmed by restriction analysis and DNA sequencing.

[1022] The plasmid pNG-618, was linearised by restriction digest with the enzyme AscI to produce the virus genomes. The viruses were amplified and purified according to methods detailed in Example 33

T Cell Activation and Degranulation Mediated by BiTE Expressing Viruses.

Carcinoma Cell Infection

[1023] A549 cells were seeded into 6 well plates at a density of 1.2e6 cells/well. Plates were incubated for 4 hrs, 37? C., 5% CO.sub.2, before cells were either infected with NG-611, NG-612, NG-618, enadenotucirev or were left uninfected. At 72 hrs post-infection supernatants were harvested from the cells and clarified by centrifuging for 5 mins, 1200 rpm.

T Cell Assay

[1024] FAP expressing lung fibroblast cell lines MRC-5 and EpCam expressing A549 cells, were seeded into 24 well plates at a density of 1.5e5 cells/well. MRC-5 and A549 cells were also mixed at a 1 to 1 ratio and seeded in to 24 plates at a total cell density of 1.5e5 cells/well. Plates were incubated for 4 hrs, 37? C., 5% CO.sub.2, before media was replaced with 300 ?L/well of thawed supernatant harvested from the A549 plates. Purified CD3 T cells isolated form human PBMC donors were then also added to the plates to give a ratio of T cells to MRC-5 or SKOV3 cells of 2 to 1. The co-cultures were incubated for 16 hrs, 37? C., 5% CO.sub.2 before cellular supernatants were collected for ELISA analysis and T, MRC-5 and A549 cells harvested for flow cytometry analysis.

Detection of FAP and EpCam on MRC-5 or SKOV Cells

[1025] Flow cytometry analysis of detectable FAP or EpCam on the surface of MRC-5 or SKOV cells, respectively was assessed by washing the cells once in FACs buffer before staining with panels of directly conjugated antibodies: anti-FAP conjugated to AF647; anti-EpCam conjugated to PE. Analysis showed that FAP expression was no longer detectable on the MRC-5 cells that had been incubated with supernatant from cells infected with FAP-BiTE expressing virus, NG-618 but was detected on >80% of cells incubated with supernatants from cells treated with EnAd, or the untreated cells (FIG. 89A). These data indicate that FAP-BiTE produced by the NG-618 viruses binds to its FAP target on the MRC-5 cells occluding binding of the anti-FAP antibody. Live, large, single cells SKOV cells were assessed for detectable expression of EpCam. EpCam expression was only detectable at low levels on the SKOV cells that had been incubated with supernatants from cells infected with EpCam-BiTE expressing virus, NG-618 (17% of cells), but was detected on >40% of cells incubated with supernatants from cells treated with EnAd or the untreated cells (FIG. 89B). Collectively these data indicate that NG-618 produces BITE molecules that bind to EpCam and FAP target proteins.

Upregulation of T Cell Activation Markers

[1026] Flow cytometry analysis of T cell activation was assessed by expression of the T cell activation markers CD25, CD69, HLA-DR and CD40L or the T cell degranulation marker, CD107a on live, CD3+, single cells. These data showed that when co-cultured with FAP.sup.+ MRC-5 cells the number of T cells expressing CD25, CD40L or CD107a was significantly increased when NG-618 supernatants were added to the cells compared enadenotucirev or untreated control supernatants (FIG. 90). The number of T cells expressing CD25, CD40L or CD107a was also significantly increased when NG-618 supernatants were added to the EpCam.sup.+ SKOV3 cells compared to enadenotucirev or untreated control supernatants (FIG. 91). These data demonstrate that both BiTE molecules expressed by the NG-618 virus are functional in terms of inducing T cell activation.

Analysis of T Cell Mediated Target (MRC-5 and SKOV) Cell Killing

[1027] Flow cytometry analysis of MRC-5 and SKOV cell viability was assessed by staining the cells in 504 of PBS containing Live/Dead Aqua (Life tech) for 15 minutes at RT. The cells were washed once in FACs buffer before staining with panels of directly conjugated antibodies: anti-FAP conjugated to AF647; anti-EpCam conjugated to PE. MRC-5 and SKOV cell viability was significantly reduced following incubation with NG-618 supernatant samples, whereas no significant cell death was detectable in the enadenotucirev or untreated control supernatants FIG. 92. These data demonstrate the functional ability of NG-618 coexpressed FAP and EpCam targeting BiTes to induce T cell mediated cell killing of target cells.

TABLE-US-00011 SEQUENCES SEQIDNO:25:FAPBiTE-P2A-RFP(ITALICS= leader, BOLD= furincleavagesite,UNDERLINE= P2A sequence,lowercase= RFP) MGWSCIILFLVATATGVHSDIVMTQSPDSLAVSLGERATINCKSSQSLLYS RNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISS LQAEDVAVYYCQQYFSYPLTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVQ SGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGI PNYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEG HAMDYWGQGTLVTVSSGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFT RYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYM ELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGG SGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRW IYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTF GGGTKVEIKHHHHHHHHHHRRKRGSGATNFSLLKQAGDVEENPGPmselik enmhmklymegtvnnhhfkctsegegkpyegtqtmkikvveggplpfafdi latsfmygskafinhtqgipdffkqsfpegftwerittyedggvltatqdt sfqngciiynvkingvnfpsngpvmqkktrgweantemlypadgglrghsq malklvgggylhcsfkttyrskkpaknlkmpgfhfvdhrlerikeadkety veqhemavakycdlpsklghr SEQIDNO:26:Control(Anti-FHA)BiTE-P2A-RFP (ITALICS= leader,BOLD= furincleavagesite, UNDERLINE= P2Asequence,lowercase= RFP) MGWSCIILFLVATATGVHSELDIVMTQAPASLAVSLGQRATISCRASKSVS SSGYNYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHP VEEEDAATYYCQHSREFPLTFGAGTKLEIKSSGGGGSGGGGGGSSRSSLEV QLQQSGPELVKPGASVKISCKTSGYTFTGYTMHWVRQSHGKSLEWIGGINP KNGGIIYNQKFQGKATLTVDKSSSTASMELRSLTSDDSAVYYCARRVYDDY PYYYAMDYWGQGTSVTVSSAKTTPPSVTSGGGGSDVQLVQSGAEVKKPGAS VKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRF TITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSS GEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMN WYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAAT YYCQQWSSNPLTFGGGTKVEIKHHHHHHHHHHRRKRGSGATNFSLLKQAGD VEENPGPmselikenmhmklymegtvnnhhfkctsegegkpyegtqtmkik vveggplpfafdilatsfmygskafinhtqgipdffkqsfpegftweritt yedggvltatqdtsfqngciiynvkingvnfpsngpvmqkktrgweantem lypadgglrghsqmalklvgggylhcsfkttyrskkpaknlkmpgfhfvdh rlerikeadketyveqhemavakycdlpsklghr SEQIDNO:33:Spliceacceptorsequence CAGG SEQIDNO:55shortspliceacceptor(SSA)DNA sequence(nullsequence) CAGG SEQIDNO:58Kozaksequence(nullsequence) CCACC