Oncolytic virotherapy with helper-dependent adenoviral-based vectors expressing immunomodulatory molecules

Abstract

The present disclosure concerns combination therapy for cancer that utilizes (i) an oncolytic virus; (ii) a virus comprising nucleic acid encoding an immunomodulatory factor; and (iii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen. In particular embodiments, the virus comprises nucleic acid encoding an immunomodulatory factor comprises nucleic acid encoding IL-12 and/or antagonist anti-PD-L1 antibody.

Claims

1. A method of treating a cancer, comprising administering to a subject: (i) an oncolytic virus; (ii) a helper dependent adenovirus (HDAd) comprising a nucleic acid encoding IL-12 and an antagonist anti-PD-L1 antibody; and (iii) at least one cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain capable of specific binding to HER2; wherein the cancer comprises cells expressing HER2.

2. The method according to claim 1, wherein the oncolytic virus is an oncolytic adenovirus (OncAd).

3. The method according to claim 1, wherein the oncolytic virus is derived from adenovirus 5 (Ad5).

4. The method according to claim 1, wherein the oncolytic virus encodes an E1A protein which displays reduced binding to Rb protein as compared to E1A protein encoded by Ad5.

5. The method according to claim 1, wherein the oncolytic virus encodes an E1A protein lacking the amino acid sequence LTCHEACF (SEQ ID NO:52).

6. The method according to claim 1, wherein the oncolytic virus encodes an E1A protein comprising, or consisting of, the amino acid sequence SEQ ID NO:34.

7. The method according to claim 1, wherein the at least one cell comprising a CAR is a T cell.

8. The method according to claim 1, wherein the CAR comprises an antigen binding domain comprising: a VL domain comprising: LC-CDR1: SEQ ID NO:10; LC-CDR2: SEQ ID NO:11; LC-CDR3: SEQ ID NO:12; and a VH domain comprising: HC-CDR1: SEQ ID NO:13; HC-CDR2: SEQ ID NO:14; HC-CDR3: SEQ ID NO:15; or a VL domain comprising: LC-CDR1: SEQ ID NO:18; LC-CDR2: SEQ ID NO:19; LC-CDR3: SEQ ID NO:20; and a VH domain comprising: HC-CDR1: SEQ ID NO:21; HC-CDR2: SEQ ID NO:22; HC-CDR3: SEQ ID NO:23; or a VL domain comprising: LC-CDR1: SEQ ID NO:26; LC-CDR2: SEQ ID NO:27; LC-CDR3: SEQ ID NO:28; and a VH domain comprising: HC-CDR1: SEQ ID NO:29; HC-CDR2: SEQ ID NO:30; HC-CDR3: SEQ ID NO:31; or a VL domain comprising: LC-CDR1: SEQ ID NO:57; LC-CDR2: SEQ ID NO:58; LC-CDR3: SEQ ID NO:59; and a VH domain comprising: HC-CDR1: SEQ ID NO:60; HC-CDR2: SEQ ID NO:61; HC-CDR3: SEQ ID NO:62.

9. The method according to claim 1, wherein the CAR comprises an antigen binding domain comprising: a VL comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:16 and a VH comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:17, and further wherein the VL comprises LC-CDR1: SEQ ID NO:10; LC-CDR2: SEQ ID NO:11; and LC-CDR3: SEQ ID NO:12; and the VH comprises HC-CDR1: SEQ ID NO:13; HC-CDR2: SEQ ID NO:14; and HC-CDR3: SEQ ID NO:15; or a VL comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:24 and a VH comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:25, and further wherein the VL comprises LC-CDR1: SEQ ID NO:18; LC-CDR2: SEQ ID NO:19; and LC-CDR3: SEQ ID NO:20; and the VH comprises HC-CDR1: SEQ ID NO:21; HC-CDR2: SEQ ID NO:22; and HC-CDR3: SEQ ID NO:23; or a VL comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:32 and a VH comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:33, and further wherein the VL comprises LC-CDR1: SEQ ID NO:26; LC-CDR2: SEQ ID NO:27; and LC-CDR3: SEQ ID NO:28; and the VH comprises HC-CDR1: SEQ ID NO:29; HC-CDR2: SEQ ID NO:30; and HC-CDR3: SEQ ID NO:31; or a VL comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:63 and a VH comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:64, and further wherein the VL comprises LC-CDR: SEQ ID NO:57; LC-CDR2: SEQ ID NO:58; and LC-CDR3: SEQ ID NO:59; and the VH comprises HC-CDR1: SEQ ID NO:60; HC-CDR2: SEQ ID NO:61; and HC-CDR3: SEQ ID NO:62.

10. The method according to claim 1, wherein the helper dependent adenovirus comprises a nucleic acid encoding an enzyme capable of catalysing conversion of a non-toxic factor to a cytotoxic form.

11. The method according to claim 10, wherein the enzyme is selected from: thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase, horseradish peroxidase and carboxylesterase.

12. The method according to claim 1, wherein the method of treating a cancer comprises: (a) isolating at least one cell from a subject; (b) modifying the at least one cell to express or comprise a CAR specific for a cancer cell antigen, or a nucleic acid encoding a CAR specific for a cancer cell antigen, wherein the CAR comprises an antigen binding domain capable of specific binding to HER2; (c) optionally expanding the modified at least one cell, and; (d) administering the modified at least one cell to a subject.

13. The method according to claim 1, wherein the cancer is selected from head and neck cancer, nasopharyngeal carcinoma (NPC), cervical carcinoma (CC), oropharyngeal carcinoma (OPC), gastric carcinoma (GC), hepatocellular carcinoma (HCC) and lung cancer.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Embodiments and studies illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures.

(2) FIGS. 1A and 1B. FIG. 1A shows schematic representations of examples of HER2-specific CAR constructs. FIG. 1B shows a schematic of an example of a protocol for transducing T cells to produce HER2-specific CAR-T.

(3) FIG. 2. Graphs showing expression of the HER2-CARs, CCR7, CD45RO and PD-1 on T cells transduced with the indicated HER2-CAR constructs, as determined by flow cytometry.

(4) FIG. 3. Graphs showing expression of HER2-CAR, CCR7, CD45RO, PD-1, LAG-3 and TIM-3 on CD4 and CD8 T cells following transduction with anti-HER2 clone E4 CAR construct, as determined by flow cytometry.

(5) FIGS. 4A and 4B. FIG. 4A is a bar chart showing in vitro cell killing of MDA cells (which do not express HER2 at the cell surface; negative control), MDA-HER2 cells (which express HER2 at the cell surface; positive control), FaDu and SCC47 cells by anti-HER2 clone C5, E4 and F1 CAR-T cells (or non-transduced (NT) cells), as determined by .sup.51Cr release assay. FIG. 4B shows graphs indicating expression of HER2 on MDA-HER2 cells, FaDu and SCC47 cells but not on MDA cells, as determined by flow cytometry.

(6) FIG. 5. Bar chart showing in vitro cell killing of FaDu and SCC47 cells genetically modified to express firefly luciferase (ffLuc) by anti-HER2 clone C5, E4 and F1 CAR-T cells (or non-transduced (NT) cells), as determined by ffLuc activity assay. Data are presented as meanSD (n=4). *P<0.001.

(7) FIG. 6. FIG. 6 shows a schematic representation of the sequences of an example of an ICOSTAT oncolytic adenovirus construct.

(8) FIGS. 7A to 7F. Graphs showing the ability of ICOSTAT oncolytic adenovirus to kill A549 cells (FIGS. 7A and 7F), FaDu cells (FIG. 7B), SCC47 cells (FIG. 7C), WI-38 cells (FIG. 7D) and ARPE-19 cells (FIG. 7E) following infection with the indicated concentration of viral particles (Vp), as determined by MTS viability assay. Helper-dependent adenovirus (HDAd) is included as a control condition.

(9) FIGS. 8A and 8B. Bar charts showing ability of ICOSTAT oncolytic adenovirus to replicate and act as helper for replication of helper-dependent adenovirus (HDAd), as determined by copy number analysis by quantitative real-time PCR. The virus designated Onc5/3AdicoSTAT is ICOSTAT. +HD indicates co-infection of ICOSTAT with HDAd.

(10) FIGS. 9A and 9B. Graphs showing the replication of ICOSTAT oncolytic adenovirus in FaDu cells (FIG. 9A) and SCC47 cells (FIG. 9B), in the presence or absence of 10 ng/ml IFN in the cell culture media.

(11) FIGS. 10A to 10D. FIG. 10A is a schematic representation of the HDAdIL-12_TK_PDL1 construct. FIG. 10B is a bar chart showing production of IL-12p70 by cells transfected with the indicated helper-dependent adenovirus (HDAd) constructs. FIG. 10C is a photograph of a western blot showing production of anti-PD-L1 minibody by cells transfected with the HDAd constructs. FIG. 10D is a photograph of a wells demonstrating HSV thymidine kinase production by cells transfected with the HDAd constructs.

(12) FIG. 11. Graph showing ELISA analysis of PD-L1 minibody avidity to recombinant human PD-L1, using serially diluted cell culture media of A549 cells which had been transfected with plasmid encoding GFP (pGFP; negative control), plasmid encoding the anti-PD-L1 minibody described in Tanoue et al. supra, (pPDL1 mini Tanoue) or plasmid encoding the anti-PD-L1 minibody encoded by HDAdIL-12_TK_PD-L1 (pPDL1 mini). Serially diluted anti-human PD-L1 antibody was used as a positive control (PDL1 IgG).

(13) FIGS. 12A and 12B. Schematic representations of the sequences of (FIG. 12A) an example of an Onc5/2E124 oncolytic adenovirus construct, and (FIG. 12B) a plasmid encoding an Onc5/2E124 oncolytic adenovirus construct.

(14) FIGS. 13A to 13D. Graphs showing the ability of Onc5/3Ad2E1A oncolytic adenovirus to kill FaDu cells (FIG. 13A), SCC47 cells (FIG. 13B), WI-38 cells (FIG. 13C) and ARPE-19 cells (FIG. 13D) following infection with the indicated concentration of viral particles (Vp), as determined by MTS viability assay. Helper-dependent adenovirus (HDAd) is included as a control condition.

(15) FIG. 14. Graph showing numbers of HER2-specific CAR T cells following the indicated number of days of in vitro cell culture after transuction with the indicated CAR constructs.

(16) FIGS. 15A to 15C. Images and graph showing the results of in vivo analysis of the anticancer activity of adoptively-transferred luciferase-expressing T cells in an orthotopic FaDu cell-derived model of squamous cell head and neck carcinoma. FIGS. 15A and 15B show the number and location of luciferase-expressing non-transduced T cells (NT), and cells expressing luciferase-expressing T cells expressing C5, F1 or A3 HER2-specific CARs within mice at the indicated number of days after infusion of the cells. FIG. 15C shows the percentage of surviving subjects in the different treatment groups at the inciated number of days after infusion of the cells. A negative control condition wherein mice were not administered with T cells is also shown (-).

(17) FIGS. 16A to 16C. Images and graphs showing the results of in vivo analysis of adoptively-transferred T cells in NSG mice. FIG. 16A shows the number and location of luciferase-expressing non-transduced T cells (NT), and cells expressing luciferase-expressing T cells expressing C5, F1 or A3 HER2-specific CARs within mice at the indicated number of days after infusion of the cells. FIG. 16B shows measurements for total flux (in photons per second; p/s) of ventral surface for mice of the different groups at the indicated number of days after infusion of the cells. FIG. 16C shows the weights of mice in the different treatment groups at the indicated number of days after infusion of the cells, expressed as a percentage of body weight at day 0.

(18) FIGS. 17A to 17C. Scatterplots and histograms showing the results of characterisation by flow cytometry of F1 HER2-specific CAR T cells used in experiments for in vivo analysis of the anti-cancer activity of the combination of CAdtrio and adoptively-transferred T cells. FIG. 17A shows the percentages of CD4+ T cells and CD8+ T cells within the F1.CAR-T population. FIG. 17B shows the percentage cells expressing HER2 CAR at the cell surface. FIG. 17C shows the percentages of cells within the F1.CAR-T population expressing CCR7 and/or CD45RO.

(19) FIGS. 18A to 18D. Images and graphs showing the results of in vivo analysis of the anti-cancer activity of the combination of CAdtrio and adoptively-transferred T cells, in an orthotopic FaDu cell-derived model of squamous cell head and neck carcinoma. FIG. 18A shows the number and location of luciferase-expressing non-transduced T cells (NT), and cells expressing luciferase-expressing T cells expressing F1 HER2-specific CAR within mice at the indicated number of days after infusion of the cells Top right figure (Y-axis is labelled as Total Flux) is Days post-injection of CAR T-cells. Bottom 2 figures are Days post-injection of CAdtrio. FIG. 18B shows measurements for total flux (in photons per second; p/s) of ventral surface for mice of the different groups at the indicated number of days after administration of CAdtrio. FIG. 18C shows the weights of mice in the different treatment groups at the indicated number of days after administration of CAdtrio, expressed as a percentage of body weight at day 0. FIG. 18D shows the percentage of surviving subjects in the different treatment groups at the inciated number of days after administration of CAdtrio. A negative control condition wherein mice were not administered with CAdtrio or T cells is also shown (-).

(20) FIGS. 19A to 19C. Images and graphs showing the results of in vivo analysis of the anti-cancer activity of the combination of different ratios of Onc5/3Ad2E124:HDAdIL-12_TK_PD-L1 and adoptively-transferred HER2-specific CAR T cells, in an orthotopic FaDu cell-derived model of squamous cell head and neck carcinoma. FIG. 19A shows the number and location of luciferase-expressing FaDu cells within mice at the indicated number of days after administration of CAdtrio. FIG. 19B shows measurements for total flux (in photons per second; p/s) of ventral surface for mice of the different groups at the indicated number of days after administration of CAdtrio. FIG. 19C shows the weights of mice in the different treatment groups at the indicated number of days after administration of CAdtrio, expressed as a percentage of body weight at day 0.

(21) FIGS. 20A to 20D. Bar charts and graphs showing the results of in vivo analysis of the combination of Onc5/3Ad2E124 and HDAdIL-12_TK_PD-L1 and ganciclovir (GCV), in an ectoptic FaDu cell-derived model of squamous cell head and neck carcinoma. FIGS. 20A and 20B show the GAPDH-normalised copy number of (FIG. 20A) Onc5/3Ad2E124 and (FIG. 20B) HDAdIL-12_TK_PD-L1 in tumors of mice administered with the combination of Onc5/3Ad2E124 and HDAdIL-12_TK_PD-L1 (CAdtrio) at 22 days post infection, with or without GCV treatment. FIG. 20C shows tumor volume in mm.sup.3 of mice administered with the combination of Onc5/3Ad2E124 and HDAdIL-12_TK_PD-L1 (CAdtrio) at the indicated number of days post-injection of CAdtrio, with or without GCV treatment. FIG. 20D shows IL-12 levels detected by ELISA analysis of blood samples obtained at the indicated number of days post-injection of CAdtrio, with or without GCV treatment.

(22) FIGS. 21A to 21C. Bar chart and images showing the results of analysis of transgene expression in cancer cell lines infected with different HDAd viruses, cultured in the presence or absence of ganciclovir (GCV). FIG. 21A shows the level of IL-12 in cell culture supernatant as determined by ELISA. FIG. 21B shows anti-PD-L1 minibody detected in cell culture supernatant by western blot. FIG. 21C shows viable cells detected by Cystal Violet staining at the end of the experiment.

(23) FIGS. 22A and 22B. Scatterplots showing the results of characterisation by flow cytometry of Adenovirus-specific T cells (AdVSTs) used in experiments of Example 9. FIG. 22A shows the percentages of CD4+ T cells and CD8+ T cells within the AdVST population. FIG. 22B shows the percentages of cells within the AdVST population expressing CCR7 and/or CD45RO.

(24) FIGS. 23A to 23C. Scatterplots and histograms showing the results of characterisation by flow cytometry F1.CAR-transduced AdVSTs used in experiments of Example 9. FIG. 23A shows the percentages of CD4+ T cells and CD8+ T cells within the transduced population. FIG. 23B shows the percentage cells expressing HER2 CAR at the cell surface. FIG. 23C shows the percentages of cells within the F1.CAR-AdVST population expressing CCR7 and/or CD45RO.

(25) FIGS. 24A to 24D. Images and graphs showing the results of in vivo analysis of the anti-cancer activity of Adenovirus-specific T cells (AdVSTs), F1.CAR-transduced AdVSTs, the combination of F1.CAR-transduced AdVSTs with Onc5/3Ad2E124, and the combination of F1.CAR-transduced AdVSTs with Onc5/3Ad2E124+HDAdIL-12_TK_PD-L1 (CAdtrio). FIG. 24A shows the number and location of luciferase-expressing FaDu cells within mice at the indicated number of days after administration of CAdtrio. FIG. 24B shows measurements for total flux (in photons per second; p/s) of ventral surface for mice of the different groups at the indicated number of days after administration of CAdtrio. FIG. 24C shows the weights of mice in the different treatment groups at the indicated number of days after administration of CAdtrio, expressed as a percentage of body weight at day 0. FIG. 24D shows the percentage of surviving subjects in the different treatment groups at the indicated number of days after administration of CAdtrio. *P<0.04, **P<0.07, ***P<0.02 for FIG. 24B. *P<0.01, **P<0.04, ***P<0.02 for 24C. *P=0.03, **P=0.02 for FIG. 24D.

NUMBERED STATEMENTS OF DISCLOSURE

(26) Following numbered paragraphs (paras) describe particular aspects and embodiments of the present disclosure:

(27) 1. A method of treating a cancer, comprising administering to a subject: (i) an oncolytic virus; (ii) a virus comprising nucleic acid encoding an immunomodulatory factor; and (iii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen.

(28) 2. The method of para 1, wherein the oncolytic virus is an oncolytic adenovirus (OncAd).

(29) 3. The method of para 1 or para 2, wherein the oncolytic virus is derived from adenovirus 5 (Ad5).

(30) 4. The method of any one of paras 1 to 3, wherein the oncolytic virus encodes an E1A protein which displays reduced binding to Rb protein as compared to E1A protein encoded by Ad5.

(31) 5. The method of any one of paras 1 to 4, wherein the oncolytic virus encodes an E1A protein lacking the amino acid sequence LTCHEACF (SEQ ID NO:52).

(32) 6. The method of any one of paras 1 to 5, wherein the oncolytic virus encodes an E1A protein comprising, or consisting of, the amino acid sequence SEQ ID NO:34.

(33) 7. The method of any one of paras 1 to 6, wherein the oncolytic virus comprises nucleic acid having one or more binding sites for one or more transcription factors.

(34) 8. The method of any one of paras 1 to 7, wherein the oncolytic virus comprises nucleic acid having one or more binding sites for STAT1.

(35) 9. The method of any one of paras 1 to 8, wherein the virus comprising nucleic acid encoding an immunomodulatory factor is a helper-dependent adenovirus (HDAd).

(36) 10. The method of any one of paras 1 to 9, wherein the immunomodulatory factor is selected from: an agonist of an effector immune response or antagonist of an immunoregulatory response.

(37) 11. The method of any one of paras 1 to 10, wherein the virus comprising nucleic acid encoding an immunomodulatory factor comprises nucleic acid encoding IL-12 and/or antagonist anti-PD-L1 antibody.

(38) 12. The method of any one of paras 1 to 11, wherein the virus comprising nucleic acid encoding an immunomodulatory factor comprises nucleic acid encoding a thymidine kinase.

(39) 13. The method of any one of paras 1 to 12, wherein the at least one cell comprising a CAR specific for a cancer cell antigen is a T cell.

(40) 14. The method of any one of paras 1 to 13, wherein the CAR comprises an antigen binding domain capable of specific binding to HER2.

(41) 15. The method of any one of paras 1 to 14, wherein the CAR comprises an antigen binding domain comprising: a VL domain comprising: LC-CRD1: SEQ ID NO:10; LC-CRD2: SEQ ID NO:11; LC-CRD3: SEQ ID NO:12; and a VH domain comprising: HC-CRD1: SEQ ID NO:13; HC-CRD2: SEQ ID NO:14; HC-CRD3: SEQ ID NO:15;
or a VL domain comprising: LC-CRD1: SEQ ID NO:18; LC-CRD2: SEQ ID NO:19; LC-CRD3: SEQ ID NO:20; and a VH domain comprising: HC-CRD1: SEQ ID NO:21; HC-CRD2: SEQ ID NO:22; HC-CRD3: SEQ ID NO:23;
or a VL domain comprising: LC-CRD1: SEQ ID NO:26; LC-CRD2: SEQ ID NO:27; LC-CRD3: SEQ ID NO:28; and a VH domain comprising: HC-CRD1: SEQ ID NO:29; HC-CRD2: SEQ ID NO:30; HC-CRD3: SEQ ID NO:31.

(42) 16. The method of any one of paras 1 to 15, wherein the CAR comprises an antigen binding domain comprising: a VL comprising, or consisting of or consisting essentially of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:16 and a VH comprising, or consisting of or consisting essentially of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:17;
or a VL comprising, or consisting of or consisting essentially of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:24 and a VH comprising, or consisting of or consisting essentially of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:25;
or a VL comprising, or consisting of or consisting essentially of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:32 and a VH comprising, or consisting of or consisting essentially of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:33.

(43) 17. The method of any one of paras 1 to 16, wherein the method additionally comprises: (a) isolating at least one cell from a subject; (b) modifying the at least one cell to express or comprise a CAR specific for a cancer cell antigen, or a nucleic acid encoding a CAR specific for a cancer cell antigen, (c) optionally expanding the modified at least one cell, and; (d) administering the modified at least one cell to a subject.

(44) 18. The method of any one of paras 1 to 17, wherein the cancer is selected from head and neck cancer, nasopharyngeal carcinoma (NPC), cervical carcinoma (CC), oropharyngeal carcinoma (OPC), gastric carcinoma (GC), hepatocellular carcinoma (HCC) and lung cancer.

(45) 19. An oncolytic adenovirus (OncAd) encoding an E1A protein comprising, or consisting of, the amino acid sequence SEQ ID NO:34.

(46) 20. An oncolytic adenovirus (OncAd) comprising nucleic acid having one or more binding sites for STAT1.

(47) 21. The OncAd according to para 20, wherein the OncAd comprises a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO:51 or an equivalent sequence as a result of codon degeneracy.

(48) 22. A helper-dependent adenovirus (HDAd) comprising nucleic acid encoding IL-12 and/or antagonist anti-PD-L1 antibody.

(49) 23. The HDAd according to para 22, wherein the HDAd additionally comprises nucleic acid encoding a thymidine kinase.

(50) 24. A chimeric antigen receptor (CAR) comprising an antigen binding domain comprising: a VL domain comprising: LC-CRD1: SEQ ID NO:10; LC-CRD2: SEQ ID NO:11; LC-CRD3: SEQ ID NO:12; and a VH domain comprising: HC-CRD1: SEQ ID NO:13; HC-CRD2: SEQ ID NO:14; HC-CRD3: SEQ ID NO:15;
or a VL domain comprising: LC-CRD1: SEQ ID NO:18; LC-CRD2: SEQ ID NO:19; LC-CRD3: SEQ ID NO:20; and a VH domain comprising: HC-CRD1: SEQ ID NO:21; HC-CRD2: SEQ ID NO:22; HC-CRD3: SEQ ID NO:23;
or a VL domain comprising: LC-CRD1: SEQ ID NO:26; LC-CRD2: SEQ ID NO:27; LC-CRD3: SEQ ID NO:28; and a VH domain comprising: HC-CRD1: SEQ ID NO:29; HC-CRD2: SEQ ID NO:30; HC-CRD3: SEQ ID NO:31.

(51) 25. The CAR according to para 24, wherein the CAR comprises an antigen binding domain comprising: a VL comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:16 and a VH comprising, or consisting of or consisting essentially of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:17;
or a VL comprising, or consisting of or consisting essentially of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:24 and a VH comprising, or consisting of or consisting essentially of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:25;
or a VL comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:32 and a VH comprising, or consisting of or consisting essentially of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO:33.

(52) 26. A nucleic acid, optionally isolated or man-made, encoding the oncolytic adenovirus (OncAd) according to any one of paras 19 to 21, the helper-dependent adenovirus (HDAd) according to para 22 or para 23, or the chimeric antigen receptor (CAR) according to para 24 or para 25.

(53) 27. A cell comprising the oncolytic adenovirus (OncAd) according to any one of paras 19 to 21, the helper-dependent adenovirus (HDAd) according to para 22 or para 23, the chimeric antigen receptor (CAR) according to para 24 or para 25, or the nucleic acid according to para 26, optionally wherein the cell is man-made and not found in nature.

(54) 28. A pharmaceutical composition comprising the oncolytic adenovirus (OncAd) according to any one of paras 19 to 21, the helper-dependent adenovirus (HDAd) according to para 22 or para 23, the chimeric antigen receptor (CAR) according to para 24 or para 25, the nucleic acid according to para 26 or the cell according to para 27 and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

(55) 29. A method of treating cancer comprising administering to a subject the oncolytic adenovirus (OncAd) according to any one of paras 19 to 21, the helper-dependent adenovirus (HDAd) according to para 22 or para 23, the chimeric antigen receptor (CAR) according to para 24 or para 25, the nucleic acid according to para 26, the cell according to para 27 or the pharmaceutical composition according to para 28.

(56) 30. The oncolytic adenovirus (OncAd) according to any one of paras 19 to 21, the helper-dependent adenovirus (HDAd) according to para 22 or para 23, the chimeric antigen receptor (CAR) according to para 24 or para 25, the nucleic acid according to para 26, the cell according to para 27 or the pharmaceutical composition according to para 28 for use in a method of treating a cancer.

(57) 31. Use of the oncolytic adenovirus (OncAd) according to any one of paras 19 to 21, the helper-dependent adenovirus (HDAd) according to para 22 or para 23, the chimeric antigen receptor (CAR) according to para 24 or para 25, the nucleic acid according to para 26, the cell according to para 27 or the pharmaceutical composition according to para 28 in the manufacture of a medicament for treating a cancer.

(58) 32. The method, the use or the use according to any one of paras 29 to 31, wherein the cancer is selected from head and neck cancer, nasopharyngeal carcinoma (NPC), cervical carcinoma (CC), oropharyngeal carcinoma (OPC), gastric carcinoma (GC), hepatocellular carcinoma (HCC) and lung cancer.

(59) 33. A kit of parts comprising a predetermined quantity of the oncolytic adenovirus (OncAd) according to any one of paras 19 to 21, the helper-dependent adenovirus (HDAd) according to para 22 or para 23, the chimeric antigen receptor (CAR) according to para 24 or para 25, the nucleic acid according to para 26, the cell according to para 27 or the pharmaceutical composition according to para 28.

EXAMPLES

(60) In the following Examples, the inventors describe the generation functional characterisation of novel HER-2 specific CARs and CAR-T cells, oncolytic adenoviruses and helper-dependent adenovirus.

Example 1: HER2-Specific CAR-T Cells

(61) 1.1 Generation of HER2-Specific CAR Constructs and CAR-T Cells

(62) HER2-binding CAR constructs were prepared. Briefly, DNA encoding scFv (i.e. VL domain and VH domain joined by a linker sequence) for the anti-HER2 antibody clone C5, E4, F1 or A3 was cloned into a CAR construct backbone comprising a 5 signal peptide (SP), and CD28 transmembrane (TM) and intracellular domain sequence, with a 3 CD3 intracellular domain sequence. The three HER2-binding CAR constructs are represented schematically in FIG. 1A.

(63) HER2 specific CAR-T cells were generated as represented graphically in FIG. 1B. Briefly, human PBMCs were isolated from blood samples by with Ficoll density gradient centrifugation. Cells were treated by stimulation with anti-CD3(OKT3)/anti-CD28 in the presence of IL-2 to promote T cell activation and proliferation, and the cells were transduced with retrovirus encoding the HER2 CAR constructs. T-cells were expanded by culture in the presence of 100 IU/mL recombinant human IL-2, and were frozen at 6 days post-transduction. The HER2-specific CAR construct-transduced T cells were readily expanded by culture in vitro (see e.g. FIG. 14). T-cells were thawed and expanded in the presence of 100 IU/mL of recombinant human IL-2 for 5 days and used for in vitro/in vivo experiments and phenotypic analysis.

(64) 1.2 Characterisation of the HER2-Specific CAR-T Cells

(65) 1.2.1 Expression of Surface Markers and HER2 CARs

(66) T cells transduced with HER2 CAR construct encoding scFv for anti-HER2 antibody clone E4 were characterised by flow cytometry for expression of different cell surface molecules. Expanded HER2 specific CAR T-cells were stained with fluorescently-labelled monoclonal antibodies for 30 minutes at 4 C. Discrimination of live/dead cells was achieved by including 7AAD in stainings (BD Pharmingen). Stained cells were analyzed using a Gallios flow cytometer and Kaluza software (BD Bioscience), according to manufacturer's instructions.

(67) The results are shown in FIGS. 2 and 3. Strong surface expression of the HER2-CARs was detected on the transduced cells (FIG. 2).

(68) FIG. 3 shows the results of characterisation of T cells transduced with HER2(E4)-CAR. CD3+ cells, CD4+ cells and CD8+ cells expressing HER2(E4)-CAR were shown to have increased expression of PD-1, LAG-3 and TIM-3, and to have reduced level of expression of CCR7 as compared to non-transduced cells (FIG. 3).

(69) 1.2.2 Cell Killing Activity

(70) The HER2-CAR-T cells were analysed for their ability to kill HER2 expressing cancer cells in vitro in cell killing assays.

(71) In a first experiment, cells of the HER2 negative MDA cell line (negative control), MDA cells stably expressing HER2 (MDA-HER2; positive control), pharynx squamous cell carcinoma cell line FaDu or the head and neck squamous carcinoma cell line SCC47 cells were labelled with Chromium-51 (.sup.51Cr) and co-cultured with non-transduced T-cells (NT) or the HER2-CAR-T cells expressing the indicated CARs at an effector:target cell ratio of 20:1 for 4 hours. After centrifugation, .sup.51Cr levels in the cell culture media were counted using a liquid scintillation counter. The results are shown in FIG. 4A; the HER2-CAR-T cells were shown to kill HER2-expressing cancer cells. Similar results were obtained when the experiments were performed using an effector:target cell ratio of 10:1.

(72) Expression of HER2 on MDA-HER2, FaDu and SCC47 was confirmed by flow cytometry. Briefly, the cells were were stained with fluorescently-labelled monoclonal anti-HER2 antibody or isotype control antibody for 30 minutes at 4 C. Discrimination of live/dead cells was achieved by including 7AAD in stainings (BD Pharmingen). Stained cells were analyzed using a Gallios flow cytometer and Kaluza software (BD Bioscience), according to manufacturer's instructions. The results are shown in FIG. 4B; MDA cells were confirmed not to express HER2, whilst MDA-HER2, FaDu and SCC47 express HER2.

(73) In a separate experiment, FaDu and SCC47 cells genetically modified to express firefly luciferase (ffLuc) were seeded in wells of 24-well plates, and co-cultured with HER2(C5)-CAR-T cells, HER2(E4)-CAR-T cells, or HER2(F1)-CAR-T cells at an effector:target cell ratio of 1:5 for 3 days, and ffLuc activity was measured using a plate reader (Life Technologies). The results are shown in FIG. 5; the HER2-CAR-T cells were shown to kill HER2-expressing cancer cells, as evidenced by a reduction in ffLuc activity (relative light units, RLU). Similar results were obtained when the experiment was performed using an effector:target cell ratio of 1:20.

Example 2: OncAd Constructs

(74) 2.1 Generation of OncAd Constructs

(75) Novel constructs encoding oncolytic adenovirus are prepared using recombinant DNA techniques. In particular embodiments, an OncAd is produced upon modification of a known virus. For example, a region encoding E1A protein from adenovirus 5, such as one lacking the sequence LTCHEACF (SEQ ID NO:52) involved in binding the Rb protein, is replaced with sequence encoding E1A protein from adenovirus 2, similarly lacking the sequence LTCHEACF (SEQ ID NO:52).

(76) ICOSTAT shown in FIG. 6 was produced from ICOVIR15 disclosed e.g. in Rojas et al. 2010 Mol Ther 18 1960-1971. Briefly, the region of ICOVIR15 encoding eight copies of a binding site for the transcription factor E2F was replaced with a region encoding eight tandem copies of a binding site for the transcription factor STAT1. The sequence of ICOSTAT is shown in SEQ ID NO:51.

(77) Onc5/3Ad2E124 (also referred to herein as Onc5/2E124) shown in SEQ ID NO:55 and represented schematically in FIG. 12 was also prepared by using recombinant DNA techniques. Onc5/3Ad2E124 has a similar structure as Onc524 disclosed e.g. in Fueyo et al. 2000 Oncogene 19:2-12 (hereby incorporated by reference in its entirety; Onc524 is also referred to in Fueyo et al. as 24), but differs in that Onc5/3Ad2E124 encodes E1A protein from adenovirus type 2 (Ad2) lacking the sequence LTCHEACF (SEQ ID NO:52), rather than E1A protein from adenovirus type 5 (Ad5) lacking the sequence LTCHEACF (SEQ ID NO:52).

(78) 2.2 Cell Killing Activity

(79) The ability of an oncolytic adenovirus of choice or ICOSTAT as generated in Example 2.1 to kill cancer cells may be analysed for example by MTS assay. Briefly, cells of the human alveolar basal epithelial adenocarcinoma cell line A549 cells, FaDu cells, SCC47 cells, or non-cancerous WI-38 human lung fibroblasts or ARPE-19 human retinal pigmented epithelial cells were seeded in wells of 96-well plates and infected with different amounts of a helper-dependent, non-replicating adenovirus (HDAd; as a negative control), an oncolytic adenovirus of choice (e.g. Onc5/3Ad2E124 described in Example 2.1), or ICOSTAT described in Example 2.1 above.

(80) Cells may be cultured for 4 days, for example, and then MTS reagents (Promega) may be added to each well, with cells being incubated at 37 C. for 2 hours. Live cells may be analyzed by measuring the absorbance at 490 nm with a plate reader. Readings may be normalized using the readings for untreated cells of each type (i.e. untreated cells=100% cell viability), and wells lacking cells would be considered 0%.

(81) In particular embodiments, the oncolytic virus of choice is able to kill cancer cells in a dose-dependent manner. The oncolytic virus of choice also exhibits a lower level of cell killing of non-cancerous cells, such as WI-38 and ARPE-19 cells as compared to the level of killing by the virus of cancerous cells, in specific embodiments.

(82) FIGS. 7A to 7F show that ICOSTAT is able to kill cancer cells (i.e. A549, FaDu and SCC47 cells) in a dose-dependent manner (FIGS. 7A to 7C and 7F), and exhibits a lower level of cell killing of non-cancerous cells WI-38 and ARPE-19 cells as compared to the level of killing of the cancerous cells (FIGS. 7D and 7E).

(83) FIGS. 13A to 13D show that Onc5/3Ad2E124 is able to kill cancer cells (i.e. FaDu and SCC47 cells) in a dose-dependent manner (FIGS. 13A and 13B), and exhibits a lower level of cell killing of non-cancerous WI-38 and ARPE-19 cells as compared to the level of killing of the cancerous cells (FIGS. 13C and 13D).

(84) 2.3 Ability to Help Helper-Dependent Adenovirus (HDAd)

(85) The ability of an oncolytic adenovirus of choice or ICOSTAT as generated in Example 2.1 to assist replication of a helper-dependent adenovirus (HDAd) may be analysed by co-infecting cancer cells with the OncoAd and HDAd, and determining virus copy number. Briefly, FaDu or SCC47 cells are plated in 24-well plates and infected with 10 viral particles per cell of HDAd alone, or OncAd+HDAd (at an OncAd:HDAd ratio of 1:10). Cells are harvested at 48 hours post-infection, DNA is extracted and both HDAd and Onc.Ad vector copies are analyzed by quantitative real-time PCR (10 min at 95 C. and then 45 cycles of 10 s at 95 C., 15 s at 60 C., and 30 s at 72 C.) using a Bio-Rad iQ5 real-time PCR detection system (Bio-Rad), and Applied Biosystems SYBR green PCR master mix (Life Technologies). Copy number is normalized using copy number detected for GAPDH.

(86) In particular embodiments, the oncolytic virus of choice is able to replicate itself and the HDAd sufficiently.

(87) FIGS. 8A and 8B show that ICOSTAT (designated Onc5/3AdicoSTAT in the figures) was found to be able to replicate itself (FIG. 8A) and the HDAd (FIG. 8B).

(88) 2.4 Effect of IFN on Replication of ICOSTAT in Cancer Cells

(89) The effect of IFN treatment on replication of ICOSTAT OncAd was analysed. Briefly, FaDu and SCC47 cells are plated in 24-well plates, and the cells are infected with 10 vp/cell of the oncolytic virus of choice or icoSTAT 3 hours post-infection cell culture medium is replaced with medium containing, or not containing, 10 ng/mL recombinant IFN at 3 hours post-infection, and cell culture media are replaced with fresh media with/without 10 ng/mL recombinant IFN again at 24 and 48 hours post-infection. Cells are harvested at 3, 24, 48 and 72 hours post-infection, DNA is extracted from the cells, viral copy numbers are analysed by quantitative real-time PCR and normalized using copy number detected for GAPDH.

(90) FIGS. 9A and 9B show that ICOSTAT was able to replicate in FaDu cells and SCC47 cells, in the presence or absence of IFN.

Example 3: Helper-Dependent Ad (HDAd) Constructs

(91) 3.1 HDAd Constructs and Production

(92) A novel construct encoding a helper-dependent adenovirus was prepared using recombinant DNA techniques. The coding sequence of the resulting construct designated HDAdIL-12_TK_PD-L1 is represented schematically in FIG. 10A. HDAdIL-12_TK_PD-L1 contains sequence encoding expression cassettes for (i) human IL-12p70 (sequence encoding alpha and beta chains), (ii) HSV-1 thymidine kinase, and (iii) an anti-PD-L1 minibody (comprising the CDRs of anti-PD-L1 clone H12_gl described e.g. in WO 2016111645 A1) including a HA tag. The three coding sequences each have their own polyA signal sequences.

(93) The HDAd HD28E4EGFP construct containing an EGFP transgene driven by the CMV promoter (HDAdeGFP) was produced as described in Farzad et al. Oncolytics 2014 1: 14008.

(94) The HDAd HDIL12_PDL1 contains sequence encoding human IL-12p70 protein and anti-PD-L1 minibody derived from YW243.55.S70 (atezolizumab). The anti-PD-L1 minibody of this construct consists of scFv for YW243.55.S70 fused with a hinge, CH2 and CH3 regions of human IgG1 and a C-terminal HA tag (as described e.g. in Tanoue et al. Cancer Res. (2017) 77(8):2040-2051).

(95) 3.2 Expression of Encoded Proteins

(96) Cancer cells were transfected with plasmid HDAd vectors, and medium samples were collected to analyze IL-12p70 and anti-PD-L1 minibody levels in the cell culture media of the transfected cells at 48 hours post-transfection.

(97) IL-12p70 levels in media were measured using the BD cytokine multiplex bead array system (BD Biosciences), according to manufacturer's instructions. The results are shown in FIG. 10B. Cells transfected with the HDAdIL-12_TK_PD-L1 construct were found to produce higher levels of IL-12p70 than cells transfected with the HDIL-12_PD-L1 construct.

(98) Secretion of anti-PD-L1 minibodies into the cell culture medium was detected by western blot analysis, using an anti-HA antibody (to detect the HA-tagged minibodies). FIG. 10C shows that cells transfected with the HDAdIL-12_TK_PD-L1 construct secreted the anti-PD-L1 minibody into the cell culture medium.

(99) In another experiment, cells were transfected with the different constructs and at 8 hours post-transfection the cell culture media was replaced with medium containing 10 ng/ml Ganciclovir (GCV). Cell culture medium was then replaced with medium containing 10 ng/ml every 24 hours, and after 7 days, the wells were stained with Crystal Violet solution to reveal viable cells.

(100) The results are shown in FIG. 10D, and confirm that cells transfected with the HDAdIL-12_TK_PD-L1 construct express thymidine kinase.

(101) In further experiments A549, FaDu or SCC47 cells (n=4 wells per condition) were infected in vitro with HDAdIL-12_TK_PD-L1, HDAd_PD-L1 (see e.g. Tanoue et al., supra), or a control HDAd encoding eGFP (see Farzad et al., supra). The cells were either cultured for 48 hours in the absence of ganciclovir, or medium was changed at 8 hours post-infection and every 24 hours thereafter with medium containing 10 ng/ml ganciclovir.

(102) Secretion of IL-12 into the cell culture supernatant was analysed by ELISA, and secretion of anti-PD-L1 minibody was analysed by western blot using an anti-HA antibody (the anti-PD-L1 minibody comprises a C-terminal HA-tag). At the end of the experiment wells were stained with Crystal Violet solution to reveal viable cells.

(103) The results are shown in FIGS. 21A to 21C, and confirmed expression of the transgenes encoded by the HDAds in the different cancer cell lines analysed.

(104) 3.3 Confirmation of Anti-PD-L1 Minibody Binding to PD-L1

(105) The ability of the anti-PD-L1 minibody encoded by HDAdIL-12_TK_PD-L1 to bind to PD-L1 was analysed by ELISA.

(106) Briefly, Immulon 2 high binding 96-well plates (VWR) were coated with 500 ng/well of recombinant human PD-L1 (BioVision). After blocking plate with PBS-T containing 3% BSA, serially diluted cell culture media of A549 cells which had been transfected with plasmid encoding GFP (pGFP; negative control), plasmid encoding the anti-PD-L1 minibody described in Tanoue et al. supra, (pPDL1 mini Tanoue) or plasmid encoding the anti-PD-L1 minibody encoded by HDAdIL-12_TK_PD-L1 (pPDL1 mini) were added and incubated at 4 C. for 24 hours. Serially diluted anti-human PD-L1 antibody starting from 10 g/well (BioLegend) was used as a positive control (PDL1 IgG). After washing plate with PBS-T, HRP-labeled anti-human IgG (for PD-L1 mini and PDL1 mini Tanoue) or HRP-labeled anti-mouse IgG (BioRad; for PD-L1 IgG and Iso IgG) were added for detection, and incubated at room temperature for 1 hour. The plate was then developed, and absorbance at 450 nm was measured using Tecan reader (TECAN).

(107) The results are shown in FIG. 11. The anti-PD-L1 minibody comprising the CDRs of anti-PD-L1 antibody clone H12 was found to bind to human PD-L1 in a dose-dependent fashion, with comparable (or greater) avidity as compared to the avidity of binding by anti-PD-L1 minibody described in Tanoue et al. supra.

Example 4: Analysis of Treatment of Cancer In Vivo

(108) The anticancer effect of treatment with the combination of (1) an oncolytic virus of choice+HDAdIL-12_TK_PD-L1+HER2-CAR-T and (2) ICOSTAT+HDAdIL-12_TK_PD-L1+HER2-CAR-T is demonstrated in vivo in mouse xenograft tumour models.

(109) In a first experiment, 110.sup.6 FaDu cells are injected subcutaneously in PBS into NSG male mice. After 12 days, 110.sup.8 viral particles (1) oncolytic virus and HDAdIL-12_TK_PD-L1 or (2) ICOSTAT+HDAdIL-12_TK_PD-L1 are injected intratumorally at an OncAd:HDAd ratio of 1:20.

(110) In a second experiment, 0.510.sup.6 FaDu cells are injected orthotopically into NSG male mice. After 6 days, 110.sup.8 viral particles (1) oncolytic virus and HDAdIL-12_TK_PD-L1 or (2) ICOSTAT+HDAdIL-12_TK_PD-L1 are injected intratumorally at an OncAd:HDAd ratio of 1:20.

(111) In both experiments, 3 days after administration of the viral particles, 110.sup.6 HER2-CAR T cells are administered intravenously.

(112) In both experiments, control conditions are included as follows:

(113) TABLE-US-00002 Condition OncAd HDAd CAR T 1 (test Of choice HDAdIL- HER2 CAR-T condition) 12_TK_PD-L1 2 (test ICOSTAT HDAdIL- HER2 CAR-T condition) 12_TK_PD-L1 3 HDAdIL- HER2 CAR-T 12_TK_PD-L1 4 Of choice HER2 CAR-T 5 ICOSTAT HER2 CAR-T 6 Of choice HDAdIL- 12_TK_PD-L1 7 ICOSTAT HDAdIL- 12_TK_PD-L1 8 Of choice 9 ICOSTAT 10 HDAdIL- 12_TK_PD-L1 11 HER2 CAR-T

(114) Tumor size is monitored and tumour volumes are calculated using the formula: Width.sup.2Length0.5.

(115) The use of the combination of oncolytic virus, HDAdIL-12_TK_PD-L1 and HER2 CAR-T (test condition 1) is found to have an improved antitumour effect as compared to the use of any of the agents alone (conditions 8, 10 or 11), or compared to the use of two of the three agents (conditions 3, 4 and 6).

(116) Similarly, the use of the combination of ICOSTAT, HDAdIL-12_TK_PD-L1 and HER2 CAR-T (test condition 2) is found to have an improved antitumour effect as compared to the use of any of the agents alone (conditions 9, 10 or 11), or compared to the use of two of the three agents (conditions 3, 5 and 7).

(117) Similar results are observed when xenograft tumours are established using SCC47 cells and A549 cells.

Example 5: Analysis of the Anti-Cancer Activity of the HER2-Specific CAR-T Cells In Vivo

(118) The anti-cancer activity of the HER2-specific CAR-T cells (see Example 1 above) was investigated in vivo in a FaDu cell-derived xenograft model of squamous cell head and neck cancer.

(119) Briefly, 0.510.sup.6 FaDu cells were injected orthotopically into NSG male mice. After 9 days, mice were injected via the tail vein with 110.sup.6 T cells genetically modified to express firefly luciferase, which had not been transduced with a HER2-CAR construct, or with 110.sup.6 firefly luciferase-expressing T cells which had been transduced with the C5, F1 or A3 CAR constructs. A control condition was included in the experiment in which mice were not injected with T cells at day 9.

(120) Luciferase activity (and thus number and distribution of the administered T cells), body weight, survival of the mice was monitored over time. Luciferase activity was monitored by intraperitoneal injection of D-Luciferin (1.5 mg per mouse), and imaging of the mice 10 min later using an IVIS imager (Xenogen).

(121) FIGS. 15A and 15B show the images acquired on days 0, 4, 7, 14, 28, 42, 56 and 70 following injection of the luciferase-expressing T cells (i.e. the non-transduced T cells or HER2-specific CAR-T cells) (days refer to days after ffLuc T cell injection). The systemically infused T cells were shown to migrate to the site of the orthotopic tumors. The T cells which had not been modified to express HER2-specific CARs were undetectable after 7 days. By contrast, the HER2-specific CAR-T cells persisted and remained detectable throughout the experiment.

(122) FIG. 15C shows percentage survival of mice subjected to the different treatments over the course of the experiment. Administration of HER2-specific CAR-T cells was found to increase survival.

(123) In a separate experiment NOD scid gamma (NSG) mice were injected via the tail vein with 110.sup.6 firefly luciferase-expressing T cells which had not been transduced with a HER2-CAR construct, or with 110.sup.6 firefly luciferase-expressing T cells which had been transduced with the C5, F1 or A3 CAR construct. Luciferase activity was monitored as described above, and body weight of the mice was also monitored over time.

(124) The results of the experiment are shown in FIGS. 16A to 16C. The C5 CAR-T cells were found to expand non-specifically in NSG mice (FIG. 16A). No significant weight loss was observed in NSG mice administered with the HER2-specific CAR-T cells (FIG. 16C).

Example 6: Analysis of of the Anti-Cancer Activity of the Combination of Oncolytic Virus, HDAd Virus and HER2-Specific CAR-T Cells In Vivo

(125) The anti-cancer activity of a combination of oncolytic virus, HdAd and HER-specific CAR-T cell therapy was investigated in vivo in a FaDu cell-derived xenograft model of squamous cell head and neck cancer.

(126) Briefly, 0.510.sup.6 FaDu cells were injected orthotopically into NSG male mice. After 6 days, one group of mice was then injected intratumorally with a combination of Onc5/3Ad2E124 (described in Example 2.1) and HDAdIL-12_TK_PD-L1 described in Example 3.1 (this combination of OncAd and HdAd is referred to herein as CAdtrio). A total of 110.sup.7 viral particles were administered, at a 1:10 ratio of Onc5/3Ad2E124:HDAdIL-12_TK_PD-L1.

(127) Three days later, mice were injected via the tail vein with 110.sup.6 T cells engineered to express firefly luciferase, which had been transduced with the HER2-specific CAR construct corresponding to clone F1. A control group of mice which had not been administered with CAdtrio was injected via the tail vein with 110.sup.6 firefly luciferase-expressing T cells which had not been transduced with a HER2-CAR construct, and a further control group of mice was not administered with CAdtrio nor injected with T cells. Luciferase activity, body weight and survival of the mice was monitored over time.

(128) Prior to their use in the experiment the F1.CART cells were characterised flow cytometry, and the results are shown in FIGS. 17A to 17C. The cells were found to comprise 72.5% CD4+ cells and CD8+ cells. 87% of the cells were determined to express HER2 CAR at the cell surface. 39% of the cells were CCDR7+CD45RO+, and 59.2% of the cells were CCR7-CD45RO+.

(129) The results of the experiments analysing the therapeutic efficacy of the combination of oncolytic virus, HDAd virus and HER2-specific CAR-T cells to treat cancer in vivo are shown in FIGS. 18A to 18D. The combination of Onc5/3Ad2E124, HDAdIL-12_TK_PD-L1 and F1.CART was found to improve survival over treatment with F1.CART cells alone.

(130) In further experiments two different ratios of Onc5/3Ad2E124 to HDAdIL-12_TK_PD-L1 were investigated.

(131) Briefly, 0.510.sup.6 FaDu cells modified to express firefly luciferase were injected orthotopically into NSG male mice. After 6 days, mice were injected intratumorally with: (i) 110.sup.7 viral particles of CAdtrio, at a ratio of Onc5/3Ad2E124:HDAdIL-12_TK_PD-L1 of 1:10; (ii) 110.sup.7 viral particles of CAdtrio, at a ratio of Onc5/3Ad2E124:HDAdIL-12_TK_PD-L1 of 1:20; (iii) 110.sup.8 viral particles of CAdtrio, at a ratio of Onc5/3Ad2E124:HDAdIL-12_TK_PD-L1 of 1:10; or (iv) 110.sup.8 viral particles of CAdtrio, at a ratio of Onc5/3Ad2E124:HDAdIL-12_TK_PD-L1 of 1:20.

(132) Three days later, mice were injected via the tail vein with 110.sup.6 T cells which had been transduced with the F1 CAR construct (not expressing firefly luciferase). The cancer was monitored over time by analysis of luciferase activity as described above, and the body weight of the mice was also monitored.

(133) The results of the experiments are shown in FIGS. 19A to 19C. Mice administered with a 1:10 ratio of Onc5/3Ad2E124:HDAd IL-12_TK_PD-L1 generally had fewer luciferase-expressing FaDu cells than those administered with a 1:20 ratio of Onc5/3Ad2E124:HDAdIL-12_TK_PD-L1, and mice administered with 110.sup.8 viral particles of CAdtrio generally had fewer luciferase-expressing FaDu cells than those administered with 110.sup.7 viral particles of CAdtrio (FIG. 19B).

Example 7: Analysis of the Anti-Cancer Activity of the Combination of Oncolytic Virus, HDAd Virus and Ganciclovir (GCV) In Vivo

(134) The anti-cancer activity of a combination of oncolytic virus and HdAd (encoding thymidine kinase) (l,e, CAdtrio) in conjunction with ganciclovir (GCV) was investigated in vivo in a FaDu cell-derived xenograft model of squamous cell head and neck cancer.

(135) Ectopic FaDu tumors were established by subcutaneous injection of FaDu cells into the flanks of mice. The mice were subsequently injected intratumorally with 110.sup.8 viral particles of CAdtrio, at a ratio of Onc5/3Ad2E124:HDAdIL-12_TK_PD-L1 of 1:10. One group of mice (n=5) was then injected intraperitoneally on days 2, 3, 4, 5, 7, 10, 14, 17 and 21 days after CAdtrio injection with 10 mg/kg of ganciclovir.

(136) Blood samples were collected from the mice on days 2, 7, 14 and 21 and analysed by ELISA for IL-12 expression. Tumor volumes were monitored throughout the experiment. At day 22 Onc.Ad and HDAd vector copy numbers were determined in DNA extracted from the tumors by quantitative real-time PCR analysis, and normalised using the copy number detected for GAPDH.

(137) The results of the experiments are shown in FIGS. 20A to 20D. Ganciclovir (GCV) treatment did not significantly influence Onc.Ad vector copy number at day 22 (FIG. 20A), but significantly decreased HDAd vector copy number (FIG. 20B). GCV treatment was also found to improve tumor control (FIG. 20C), but did not significantly influence the levels of IL-12 in the blood (FIG. 20D).

Example 8: Generation of Oncolytic Virus-Specific T Cells and HER-Specific CAR-Expressing Oncolytic Virus-Specific T Cells

(138) 8.1 Generation and Characterisation of Oncolytic Virus-Specific T Cells

(139) Adenovirus-specific T cells (AdVSTs) and activated T cells (ATCs) were prepared as follows.

(140) Anti-CD3 (clone OKT3) and anti-CD28 agonist antibodies were coated onto wells of tissue culture plates by addition of 0.5 ml of 1:1000 dilution of 1 mg/ml antibodies, and incubation for 2-4 hr at 37 C., or at 4 C. overnight.

(141) PBMCs were isolated from blood samples obtained from healthy donors according to the standard Ficoll-Paque method.

(142) ATCs:

(143) 110.sup.6 PBMCs (in 2 ml of cell culture medium) were stimulated by culture on the anti-CD3/CD28 agonist antibody-coated plates in CTL cell culture medium (containing 50% Advanced RPMI, 50% Click's medium, 10% FBS, 1% GlutaMax, 1% Pen/Strep) supplemented with 10 ng/ml IL-7 and 5 ng/ml IL-15. The cells were maintained at 37 C. in a 5% CO.sub.2 atmosphere. The next day, 1 ml of the cell culture medium was replaced with fresh CTL medium containing 20 ng/ml IL-7 and 10 ng/ml IL-15.

(144) ATCs were maintained in culture, and subsequently harvested and used in experiments or cryopreserved between days 5-7.

(145) AdVSTs:

(146) 110.sup.6 PBMCs (in 2 ml of cell culture medium) were stimulated by culture on the anti-CD3/CD28 agonist antibody-coated plates in CTL cell culture medium supplemented with 10 ng/ml IL-7 and 100 ng/ml IL-15.

(147) 20 l of a 200-fold dilution of Adenovirus-specific Hexon Pepmix (JPT Cat #PM-HAdV3) or Penton PepMix (JPT Cat #PM-HAdV5) was added to the wells. The cells were maintained at 37 C. in a 5% CO.sub.2 atmosphere. After 48 hours cells were fed with CTL medium, with added IL-7 and IL-15 to a final concentration of 10 ng/ml IL-7 and 100 ng/ml IL-15.

(148) 8.2 Generation of CAR-Expressing, Oncolytic Virus-Specific T Cells

(149) On day 3, AdVSTs were resuspended at a concentration of 0.12510.sup.6 cells/ml in CTL cell culture medium containing 10 ng/ml IL-7 and 100 ng/ml IL-15.

(150) Retronectin coated plates were prepared by incubation of RetroNectin (Clontech) diluted 1:100 in PBS for 2-4 hr at 37 C., or at 4 C. overnight. The wells were washed with CTL medium, 1 ml of retroviral supernatant of HER2-specific CAR retrovirus was added to wells, and plates were centrifuged at 2000 g for 1.5 hr. At the end of the centrifugation step retroviral supernatant was aspirated, and 2 ml of AdVST suspension (i.e. 0.2510.sup.6 cells) was added to wells of the plate. Plates were centrifuged at 400 g for 5 min, and incubated at 37 C. in a 5% CO.sub.2 atmosphere.

(151) After 48 hrs (i.e. on day 6) the cell culture medium was aspirated and replaced with CTL cell culture medium containing 10 ng/ml IL-7 and 100 ng/ml IL-15.

(152) On day 9 cells were harvested and used in experiments or cryopreserved, or subjected to a second stimulation to expand CAR-expressing AdVSTs (see Example 8.3).

(153) 8.3 Expansion of AdVSTs and CAR-AdVSTs

(154) AdVSTs and CAR-expressing AdVSTs were expanded by further stimulations as desired, as follows.

(155) Pepmix-pulsed autologous ATCs were used as APCs, and K562cs cells (see e.g. Ngo et al., J Immunother. (2014) 37(4):193-203) were used as costimulatory cells. The final ratio of AdVSTs or CAR-AdVSTs:ATCs:K562cs cells in the stimulation cultures was 1:1:3-5.

(156) AdVSTs or CAR-AdVSTs were resuspended to a concentration of 0.210.sup.6 cells/ml in CTL medium.

(157) 110.sup.6 ATCs were incubated with 10 l of 200-fold dilution of Adenovirus-specific Hexon Pepmix (JPT Cat #PM-HAdV3) or Penton PepMix (JPT Cat #PM-HAdV5) at 37 C. for 30 min. The ATCs were subsequently irradiated at 30Gy and harvested. 3-510.sup.6 K562cs cells were irradiated at 100Gy.

(158) The ATCs and K562cs cells were then mixed in a total volume of 5 ml CTL medium, and 20 ng/ml IL-7 and 200 ng/ml IL-15 was added, 1 ml of this mixture was added to wells of a 24 well plate, and 1 ml of AdVST suspension or CAR-AdVST suspension was added to the wells.

(159) Cells were maintained at 37 C. in a 5% CO.sub.2 atmosphere. After 3-4 days cell culture medium was added as necessary, and after 6-7 days cells the expanded AdVSTs or CAR-AdVSTs were harvested for use in experiments.

Example 9: Analysis of the Anti-Cancer Activity of Combinations of Oncolytic Virus, HDAd, Oncolytic Virus-Specific T Cells and CAR-Expressing Oncolytic Virus-Specific T Cells In Vivo

(160) The anti-cancer activity of different combinations of oncolytic virus, HDAd, oncolytic virus-specific T cells and CAR-expressing oncolytic virus-specific T cells was investigated in vivo in a FaDu cell-derived xenograft model of squamous cell head and neck cancer.

(161) Briefly, 0.510.sup.6 FaDu cells engineered to express firefly luciferase were injected orthotopically into NSG male mice. After 6 days groups of mice were injected intratumorally with: (i) 110.sup.7 viral particles of CAdtrio, at a ratio of Onc5/3Ad2E124:HDAdIL-12_TK_PD-L1 of 1:10; or (ii) 110.sup.7 viral particles of Onc5/3Ad2E124.

(162) Three days later, mice were injected via the tail vein with: (a) 110.sup.6 AdVSTs, or (b) 110.sup.6 AdVSTs transduced with anti-HER2 CAR clone F1 (prepared as described in Example 8).

(163) Prior to their use in the experiment the AdVSTs and F1.CAR-AdVSTs were characterised by flow cytometry, and the results of the analysis are shown in FIGS. 22A and 22B, and FIGS. 23A to 23C.

(164) The cancer was monitored over time by analysis of luciferase activity as described above, and the body weight of the mice was also monitored.

(165) The results of the experiments are shown in FIGS. 24A to 24D. The greatest level of tumor control was observed in mice treated with a combination of CAdtrio+HER2-specific CAR-expressing AdVSTs (i.e. treatment group (i)(b)).