ONCOLYTIC VIROTHERAPY AND IMMUNOTHERAPY

Abstract

Methods of treating a cancer, comprising administering to a subject: (i) a virus comprising nucleic acid encoding an antigen-binding molecule comprising: (a) an antigen-binding moiety specific for an immune cell surface molecule, and (b) an antigen-binding moiety specific for a cancer cell antigen; and (ii) an oncolytic virus, and/or (iii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen are disclosed. Also disclosed are articles and compositions for use in such methods.

Claims

1. A method of treating a cancer, comprising administering to a subject: (i) a virus comprising nucleic acid encoding an antigen-binding molecule comprising: (a) an antigen-binding moiety specific for an immune cell surface molecule, and (b) an antigen-binding moiety specific for a cancer cell antigen; and (ii) an oncolytic adeno virus (OncAd), and/or (iii) at least one T cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen.

2-3. (canceled)

4. The method according to claim 1, wherein the CAR and the antigen-binding moiety capable of binding to a cancer cell antigen are specific for non-identical cancer cell antigens.

5. The method according to claim 1, wherein the antigen-binding molecule comprises (a) a heavy chain variable region (VH) and a light chain variable region (VL) specific for an immune cell surface molecule associated via a linker sequence to (b) a VH and a VL specific for a cancer cell antigen.

6. The method according to claim 1, wherein the immune cell surface molecule is a CD3-TCR complex polypeptide, and/or wherein the cancer cell antigen is selected from CD44v6, HER2, CD19, PSCA, p53, CEA, GP100, EGFR, hTERT, NY-ES01, MAGE-A3, mesothelin and MUC-1.

7-8. (canceled)

9. The method according to claim 1, wherein the virus comprising nucleic acid encoding an antigen-binding molecule additionally comprises nucleic acid encoding IL-12 and/or an antagonist anti-PD-L1 antibody.

10. The method according to claim 1, wherein the virus comprising nucleic acid encoding an antigen-binding molecule is a helper-dependent adenovirus (HDAd).

11. The method according to claim 1, wherein the virus comprising nucleic acid encoding an antigen-binding molecule comprises nucleic acid encoding an enzyme capable of catalysing conversion of a non-toxic factor to a cytotoxic form, and wherein the enzyme is selected from: thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase, horseradish peroxidase and carboxylesterase.

12. (canceled)

13. The method according to claim 1, wherein the cell comprising a CAR is specific for the oncolytic virus.

14-15. (canceled)

16. The method according to claim 1, wherein the oncolytic virus is derived from adenovirus 5 (Ad5); wherein the oncolytic virus encodes an E1A protein which displays reduced binding to Rb protein as compared to E1A protein encoded by Ad5; wherein the oncolytic virus encodes an E1A protein lacking the amino acid sequence LTCHEACF (SEQ ID NO: 105); and/or wherein the oncolytic virus encodes an E1A protein comprising, or consisting of, the amino acid sequence SEQ ID NO:104.

17-20. (canceled)

21. The method according to claim 1, wherein the oncolytic virus comprises nucleic acid having one or more binding sites for STAT1.

22. 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 T 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 T cell, and; (d) administering the modified at least one T cell to a subject.

23. The method according to claim 1, wherein the method of treating a cancer comprises: (a) isolating immune cells from a subject; (b) generating or expanding a population of immune cells specific for an oncolytic virus by a method comprising: stimulating the immune cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of the oncolytic virus, and; (c) administering at least one immune cell specific for the oncolytic virus to a subject.

24. The method according to claim 1, wherein the cancer is selected from head and neck cancer, head and neck squamous cell carcinoma (HNSCC), nasopharyngeal carcinoma (NPC), oropharyngeal carcinoma (OPC), prostate carcinoma, pancreatic carcinoma, cervical carcinoma (CC), gastric carcinoma (GC), hepatocellular carcinoma (HCC), osteosarcoma (OS), ovarian cancer, colorectal cancer, breast cancer, HER2-positive breast cancer and lung cancer.

25. A combination, comprising: (i) a helper-dependent adenovirus (HDAd) comprising nucleic acid encoding an antigen-binding molecule comprising: (a) an antigen-binding moiety specific for an immune cell surface molecule, and (b) an antigen-binding moiety specific for a cancer cell antigen; and (ii) an oncolytic adenovirus (OncAd), and/or (iii) at least one T cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen.

26. The combination according to claim 25, wherein the antigen-binding molecule comprises (a) a singlechain variable fragment (scFv) specific for an immune cell surface molecule associated via a linker to (b) a scFv specific for a cancer cell antigen.

27. The combination according to claim 25, wherein the immune cell surface molecule is a CD3-TCR complex polypeptide; and/or wherein the cancer cell antigen is selected from CD44v6, CD19, HER2, PSCA, p53, CEA, GP100, EGFR, hTERT, NY-ES01, MAGE-A3, mesothelin and MUC-1.

28-29. (canceled)

30. The combination according to claim 25, additionally comprising nucleic acid encoding IL-12 and/or an antagonist anti-PD-L1 antibody.

31. The combination according to claim 25, additionally comprising nucleic acid encoding an enzyme capable of catalysing conversion of a non-toxic factor to a cytotoxic form; wherein the enzyme is selected from: thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase, horseradish peroxidase and carboxylesterase.

32-39. (canceled)

40. A pharmaceutical composition comprising the components of the combination according to claim 25 and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

41-43. (canceled)

44. A method of treating cancer comprising administering to a subject the combination according to claim 25, wherein the cancer is selected from head and neck cancer, head and neck squamous cell carcinoma (HNSCC), nasopharyngeal carcinoma (NPC), prostate carcinoma, pancreatic carcinoma, cervical carcinoma (CC), oropharyngeal carcinoma (OPC), gastric carcinoma (GC), hepatocellular carcinoma (HCC), osteosarcoma (OS), ovarian cancer, colorectal cancer, breast cancer, HER2-positive breast cancer and lung cancer.

45. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0411] Embodiments and studies illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures.

[0412] FIG. 1. Histograms showing HER2 and CD44v6 expression by FaDu cells, FaDu.sup.HER2−/− and FaDu.sup.CD44−/− cells as determined by flow cytometry.

[0413] FIG. 2. Histograms showing HER2 and CD44v6 expression by FaDu cells analysed prior to administration to mice, and FaDu cells obtained from lymph nodes at 20 weeks post-administration.

[0414] FIG. 3. Bar chart showing in vitro cell killing of FaDu and FaDu.sup.CD44−/− cells by activated T cells (ATCs), either alone (ATC), or in the presence of cell culture supernatant containing anti-CD19 BiTE (ATC+CD19) or cell culture supernatant containing anti-CD44v6 BiTE (ATC+CD44v6). Data are presented as mean±SD (n=4). *P<0.001; **P<0.01.

[0415] FIGS. 4A to 4C. Histograms, scatterplots and bar chart showing the results of characterisation by flow cytometry of the immune cell population comprising activated T cells (ATCs) used in in vitro analysis of the anti-cancer activity of ATCs in the presence of cell culture supernatant containing anti-CD19 BiTE or cell culture supernatant containing anti-CD44v6 BiTE. 4A shows CD44v6 expression by different immune cell subsets. 4B and 4C show the numbers of the different immune cell subsets following incubation of the population comprising ATCs in the presence of FaDu cells (Control), and in the presence of cell culture supernatant containing anti-CD19 BiTE (CD19 BiTE), or cell culture supernatant containing anti-CD44v6 BiTE (CD44v6 BiTE). Data are presented as mean±SD (n=4). *P<0.001.

[0416] FIGS. 5A to 5F. Bar charts and images showing the results of in vitro analysis of cell killing and HDAd transgene expression. Firefly luciferase-labelled FaDu and FaDu.sup.HER2−/− cells were infected with HDAdCD44v6BiTE (BiTE), HDAdIL-12_PD-L1 (IL-12_PDL1) or HDAd Trio (Trio). 24 hours post-infection, HER2-specific CAR-T cells were added at an effector:target cell ratio of 1:10. Cell culture supernatant was collected and analysed for IL-12p70 by ELISA and anti-PD-L1 minibody by western blot, and cell viability was evaluated. Results obtained using FaDu cells are shown in 5A, 5C and 5E. Results obtained using FaDu.sup.HER2−/− cells are shown in 5B, 5D and 5F. 5A and 5B show IL-12p70 detected in the cell culture supernatant of uninfected cells (Control) or cells infected with the indicated HdAd. 5C and 5D show the cell viability of uninfected cells in the absence of co-culture with HER2-specific CAR-T cells (Control), or following co-culture with HER2-specific CAR-T cells (−), and cell viability of cells infected with the indicated HdAd following co-culture with HER2-specific CAR-T cells. 5E and 5F show anti-PD-L1 minibody detected in the cell culture supernatant of cells infected with the indicated HdAd. Data are presented as mean±SD (n=4) *P<0.001.

[0417] FIGS. 6A to 6D. Graphs and bar charts showing the results of in vitro analysis of cell killing and HDAd transgene expression. FaDu and FaDu.sup.HER2−/− cells were infected with Onc.Ad alone, HDAd Trio alone or Onc5/3Ad2E1Δ24+HDAd Trio (CAdTrio), at various different viral particle/cell concentrations. Cell culture supernatant was collected was analysed for IL-12p70 by ELISA, and cell viability was evaluated. Results obtained using FaDu cells are shown in 6A and 6C, and results obtained using FaDu.sup.HER2−/− cells are shown in 6B and 6D. 6A and 6B show the cell viability of cells infected with the indicated virus/combination of viruses, at the indicated viral particle/cell concentrations. 6C and 6D show IL-12p70 detected in the cell culture supernatant of cells infected with the indicated virus/combination of viruses. *P<0.001.

[0418] FIGS. 7A to 7C. Graphs and images showing the results of in vivo analysis of the anti-cancer activity of the combination of HER2-specific CAR-T cell therapy with different OncAd+HDAd combinations in an ectopic FaDu cell-derived model of squamous cell head and neck carcinoma. FaDu cells were transplanted subcutaneously into the right flank of NSG mice, and mice were untreated (Control), or administered with firefly luciferase-labelled HER2-specific CAR-T cells alone (CART), or in combination with Onc5/3Ad2E1Δ24 and HDAdCD44v6BiTE (BiTE+CART), Onc5/3Ad2E1Δ24 and HDAdIL-12 PD-L1 (12_PD+CART) or Onc5/3Ad2E1Δ24 and HDAd Trio (Trio+CART). 7A shows tumor volumes at the indicated number of days after administration of the OncAd+HDAd combinations. 7B and 7C show the total flux (in photons per second; p/s) of ventral surface for mice of the different treatment groups at the indicated number of days after infusion of the CAR-T cells.

[0419] FIGS. 8A and 8B. Images and graphs showing the results of in vivo analysis of the anti-cancer activity of the combination of HER2-specific CAR-T cell therapy with different OncAd+HDAd combinations in an orthotopic FaDu cell-derived model of squamous cell head and neck carcinoma. Firefly luciferase-labelled FaDu cells were transplanted orthotopically into NSG mice, and mice were untreated, or administered with HER2-specific CAR-T cells alone (CART), or in combination with Onc5/3Ad2E1Δ24 and HDAdIL-12_PD-L1 (12_PDL1+CART) or Onc5/3Ad2E1Δ24 and HDAd Trio (Trio+CART). 8A and 8B show the total flux (in photons per second; p/s) of ventral surface for mice of the different treatment groups at the indicated number of days after infusion of the CAR-T cells.

[0420] FIGS. 9A to 9C. Images and graphs showing the results of in vivo analysis of the anti-cancer activity of the combination of HER2-specific CAR-T cell therapy with different OncAd+HDAd combinations in an orthotopic FaDu cell-derived model of squamous cell head and neck carcinoma. FaDu cells were transplanted orthotopically into NSG mice, and mice were untreated, or administered with firefly luciferase-labelled HER2-specific CAR-T cells alone (CART), or in combination with Onc5/3Ad2E1Δ24 and HDAdIL-12_PD-L1 (12_PDL1+CART) or Onc5/3Ad2E1Δ24 and HDAd Trio (Trio+CART). 9A and 9B show the total flux (in photons per second; p/s) of ventral surface for mice of the different treatment groups at the indicated number of days after infusion of the CAR-T cells. 9C shows percentage of surviving subjects in the different treatment groups at the indicated number of days after infusion of the CAR-T cells.

[0421] FIG. 10. Scatterplots and histograms showing the results of characterisation by flow cytometry of firefly luciferase-labelled HER2-specific CAR-T cells prior to infusion, and HER2-specific CAR-T cells obtained from the tongue and lymph nodes of mice at 120 days post-infusion, from mice treated with Onc5/3Ad2E1Δ24 and HDAdIL-12_PD-L1 (12_PDL1) or Onc5/3Ad2E1Δ24 and HDAd Trio (Trio). The CAR-T cells were analysed for CD4, CD8 and CAR expression.

[0422] FIGS. 11A to 11C. Graphs and images showing the results of in vivo analysis of the anti-cancer activity of HER2-specific CAR-T cell therapy and OncAd+HDAd in an ectopic PC-3 cell-derived model of prostate adenocarcinoma. PC-3 cells were transplanted subcutaneously into the right flank of NSG mice, and mice were untreated (Control), administered with firefly luciferase-labelled HER2-specific CAR-T cells alone (CART), the combination of Onc5/3Ad2E1Δ24 and HDAd Trio (CAdVEC), or firefly luciferase-labelled HER2-specific CAR-T cells in combination with Onc5/3Ad2E1Δ24 and HDAd Trio (CAd+CART; Trio+CART). 11A shows tumor volumes at the indicated number of days after administration of the OncAd+HDAd combinations. 11B and 11C show the total flux (in photons per second; p/s) of ventral surface for mice of the different treatment groups at the indicated number of days after infusion of the CAR-T cells.

[0423] FIGS. 12A to 12C. Graphs and images showing the results of in vivo analysis of the anti-cancer activity of HER2-specific CAR-T cell therapy and OncAd+HDAd in an ectopic CAPAN-1 cell-derived model of pancreatic adenocarcinoma. CAPAN-1 cells were transplanted subcutaneously into the right flank of NSG mice, and mice were untreated (Control), administered with firefly luciferase-labelled HER2-specific CAR-T cells alone (CART), the combination of Onc5/3Ad2E1Δ24 and HDAd Trio (CAdVEC), or firefly luciferase-labelled HER2-specific CAR-T cells in combination with Onc5/3Ad2E1Δ24 and HDAd Trio (CAd+CART; Trio+CART). 12A shows tumor volumes at the indicated number of days after administration of the OncAd+HDAd combinations. 12B and 12C show the total flux (in photons per second; p/s) of ventral surface for mice of the different treatment groups at the indicated number of days after infusion of the CAR-T cells.

[0424] FIG. 13. Bar chart showing the results of in vitro analysis of cell killing of FaDu, FaDu.sup.HER2−/− and FaDu.sup.CD44v6−/− cells in the presence of AdVSTs alone (AdVST), or in the presence of cell culture supernatant containing anti-CD19 BiTE (AdVST+CD19), cell culture supernatant containing anti-HER2 BiTE (AdVST+HER2), cell culture supernatant containing anti-CD44v6 BiTE (AdVST+CD44v6) or cell culture supernatant containing anti-HER2 BiTE and anti-CD44v6 BiTE (AdVST+2×BiTEs). Data are presented as mean±SD (n=5). *P<0.001; **P<0.01.

EXAMPLES

Example 1: Materials and Methods

1.1 Generation of Antigen-Specific CAR-T Cells

[0425] 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 FRP5 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 encoded CAR is shown in SEQ ID NO:131.

[0426] HER2 specific CAR-T cells were subsequently generated. Briefly, human PBMCs were isolated from blood samples by Ficoll density gradient centrifugation. Cells were stimulated 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 construct. 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.

[0427] PSCA-specific CAR-T cells were generated in the same way, using the PCSA-specific CAR construct “2G.CAR.PSCA” described in Watanabe et al., Oncoimmunology (2016) 5(12): e1253656, which is hereby incorporated by reference in its entirety (represented schematically in FIG. 1A of Watanabe et al., Oncoimmunology (2016) 5(12): e1253656).

[0428] 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.

1.2 Helper-Dependent Ad (HDAd) Constructs

[0429] Novel constructs encoding a helper-dependent adenovirus were prepared using recombinant DNA techniques.

[0430] HDAdCD19BiTE contains sequence encoding an anti-CD19 bispecific T cell engager (BiTE), which comprises scFv specific for CD19 joined via a linker to scFv specific for CD3 (clone OKT3). The CD19 scFv comprises the VH and VL of clone FMC63. The nucleotide sequence for HDAdCD19BiTE is shown in SEQ ID NO:123, and the encoded BiTE is shown in SEQ ID NO:93.

[0431] HDAdCD44v6BiTE contains sequence encoding an anti-CD44v6 BiTE, which comprises scFv specific for CD44v6 joined via a linker to scFv specific for CD3 (clone OKT3). The CD44v6 scFv comprises the VH and VL of clone BIWA8 described e.g. in US 2005/0214212 A1. The nucleotide sequence for HDAdCD44v6BiTE is shown in SEQ ID NO:121, and the encoded BiTE is shown in SEQ ID NO:64.

[0432] HDAdHER2BiTE contains sequence encoding an anti-HER2 BiTE, which comprises scFv specific for HER2 joined via a linker to scFv specific for CD3 (clone OKT3). The HER2 scFv comprises the VH and VL of clone FRP5. The nucleotide sequence for HDAdCD44v6BiTE is shown in SEQ ID NO:122, and the encoded BiTE is shown in SEQ ID NO:83.

[0433] HDAd Trio contains sequence encoding expression cassettes for (i) an anti-CD44v6 BiTE, which comprises scFv specific for CD44v6 joined via a linker to scFv specific for CD3 (clone OKT3), (ii) human IL-12p70 (sequence encoding alpha and beta chains), and (iii) an anti-PD-L1 minibody derived from YW243.55.570 (atezolizumab). The three coding sequences each have their own polyA signal sequences. The nucleotide sequence for HDAd Trio is shown in SEQ ID NO:125.

[0434] HD2xBiTEs contains sequence encoding (i) an anti-HER2 BiTE, which comprises scFv specific for HER2 joined via a linker to scFv specific for CD3, and (ii) an anti-CD44v6 BiTE, which comprises scFv specific for CD44v6 joined via a linker to scFv specific for CD3. The anti-HER2 BiTE and anti-CD44v6 BiTE are encoded by the same expression cassette, joined by a T2A autocleavage linker sequence. The nucleotide sequence for HD2xBiTEs is shown in SEQ ID NO:122, and the encoded BiTE is shown in SEQ ID NO:103.

[0435] 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). The three coding sequences each have their own polyA signal sequences. The nucleotide sequence for HDAdIL-12_TK_PD-L1 is shown in SEQ ID NO:120.

[0436] HDAdIL-12_PD-L1 contains sequence encoding human IL-12p70 protein and anti-PD-L1 minibody derived from YW243.55.570 (atezolizumab). The anti-PD-L1 minibody of this construct consists of scFv for YW243.55.570 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).

1.3 OncAd Construct

[0437] Constructs encoding oncolytic adenovirus were prepared using recombinant DNA techniques.

[0438] Onc5/3Ad2E1Δ24 (also referred to herein as “Onc5/2E1Δ24”) shown in SEQ ID NO:126 was prepared by using recombinant DNA techniques. Onc5/3Ad2E1Δ24 has a similar structure as Onc5Δ24 disclosed e.g. in Fueyo et al. 2000 Oncogene 19:2-12 (hereby incorporated by reference in its entirety; Onc5Δ24 is also referred to in Fueyo et al. as “Δ24”), but differs in that Onc5/3Ad2E1Δ24 encodes E1A protein from adenovirus type 2 (Ad2) lacking the sequence LTCHEACF (SEQ ID NO:105), rather than E1A protein from adenovirus type 5 (Ad5) lacking the sequence LTCHEACF (SEQ ID NO:105).

1.4 CAdTrio and CAdIL-12 PD-L1

[0439] “CAd Trio” as used in the present Examples refers to the combination of Onc5/3Ad2E1Δ24 (described in Example 1.3) and HDAd Trio described in Example 1.2.

[0440] “CAdIL-12_PD-L1” as used in the present Examples refers to the combination of Onc5/3Ad2E1Δ24 (described in Example 1.3) and HDAdIL-12_PD-L1 described in Example 1.2.

1.5 Cell Lines

[0441] The following cell lines are used in the experiments described in the present Examples:

[0442] FaDu—cell line derived from human pharynx squamous cell carcinoma.

[0443] FaDu.sup.CD44−/−—cell line obtained by CRISPR/Cas9-KO modification of FaDu cells to specifically knockout the gene encoding CD44.

[0444] FaDu.sup.HER2−/−—cell line obtained by CRISPR/Cas9-KO modification of FaDu cells to specifically knockout the gene encoding HER2.

[0445] PC-3—cell line derived from metastatic human prostate adenocarcinoma.

[0446] CAPAN-1—cell line derived from metastatic human pancreatic adenocarcinoma.

1.6 Generation of Activated T Cells (ATCs)

[0447] Activated T cells (ATCs) were prepared as follows.

[0448] 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.

[0449] PBMCs were isolated from blood samples obtained from healthy donors according to the standard Ficoll-Paque method.

[0450] 1×10.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% CO2 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.

[0451] ATCs were maintained in culture, and subsequently harvested and used in experiments or cryopreserved between days 5-7.

1.7 Generation of Oncolytic Virus-Specific T Cells

[0452] Adenovirus-specific T cells (AdVSTs) were prepared as follows.

[0453] 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.

[0454] PBMCs were isolated from blood samples obtained from healthy donors according to the standard Ficoll-Paque method.

[0455] 1×10.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.

[0456] 20 μl 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% CO2 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.

1.8 Generation of CAR-Expressing Oncolytic Virus-Specific T Cells

[0457] On day 3, AdVSTs were resuspended at a concentration of 0.125×10.sup.6 cells/ml in CTL cell culture medium containing 10 ng/ml IL-7 and 100 ng/ml IL-15.

[0458] 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. 0.25×10.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% CO2 atmosphere.

[0459] 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.

[0460] On day 9 cells were harvested and used in experiments or cryopreserved, or subjected to a second stimulation to expand CAR-expressing AdVSTs.

1.9 Expansion of AdVSTs and CAR-AdVSTs

[0461] AdVSTs and CAR-expressing AdVSTs were expanded by further stimulations as desired, as follows.

[0462] 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.

[0463] AdVSTs or CAR-AdVSTs were resuspended to a concentration of 0.2×10.sup.6 cells/ml in CTL medium.

[0464] 1×10.sup.6 ATCs were incubated with 10 μl 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 30 Gy and harvested. 3-5×10.sup.6 K562cs cells were irradiated at 100 Gy.

[0465] 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.

[0466] Cells were maintained at 37° C. in a 5% CO2 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 2: Analysis of CD44v6 and HER2 Expression in Head and Neck Cancer

[0467] FaDu cells, FaDu.sup.CD44−/− cells and FaDu.sup.HER2−/− cells were analysed for expression of HER2 and CD44v6 by flow cytometry using antibodies specific for the respective targets.

[0468] The results are shown in FIG. 1. FaDu cells were found to express both of HER2 and CD44v6. FaDu.sup.CD44−/− cells were found to express HER2, but did not express CD44v6. FaDu.sup.HER2−/− cells were found to express CD44v6, but did not express HER2.

Example 3: Analysis of FaDu Cell Phenotype Following Treatment with HER2-Specific CAR-T Cells and CAd12_PD-L1

[0469] The phenotype of cells of a FaDu cell-derived xenograft model of squamous cell head and neck cancer was investigated following treatment with CAd12_PD-L1 and HER2-specific CAR-T cells.

[0470] Briefly, 0.5×10.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 1×10.sup.8 viral particles of CAd12_PD-L1, at a ratio of Onc5/3Ad2E1Δ24:HDAdIL-12_PD-L1 of 1:20; and three days later, mice were injected via the tail vein with HER2-specific CAR-T cells (see Example 1.1).

[0471] 20 weeks post injection FaDu cells were obtained from the lymph node by FACS sorting of luciferase-expressing cells, and analysed by flow cytometry for expression of HER2 and CD44v6. Expression of HER2 and CD44v6 was also analysed in FaDu cells engineered to express firefly luciferase prior to administration.

[0472] The results are shown in FIG. 2. The FaDu cells that persisted following treatment with CAd12_PD-L1 and HER2-specific CAR-T cells did not express HER2, but did express CD44v6.

Example 4: T Cell Mediated Cell Killing of Cancer Cells Facilitated by BiTEs Specific for Cancer Cell Antigens

[0473] Constructs encoding CD19-specific and CD44v6-specific BiTEs were analysed for their ability to promote cell killing of FaDu cells by activated T cells (ATCs).

[0474] Briefly, FaDu cells were infected with 200 viral particles/cell of HDAdCD19BiTE or HDAdCD44v6BiTE (see Example 1.2) by addition of viral particles to cell culture medium of the cells in culture. Cell culture supernatant was collected at 48 hours post-infection.

[0475] ATCs (see Example 1.6) were co-cultured with firefly Luciferase (ffLuc)-labelled FaDu cells or FaDu.sup.CD44−/− cells (see Example 1.5) at an effector:target cell ratio of 1:10, in the presence of cell culture supernatant containing CD19-specific BiTE or CD44v6-specific BiTE.

[0476] After 72 hours, cell killing was analysed by Luciferase assay. Briefly, cells were washed with PBS, and lysis buffer was added. Cell lysates were collected, and the residual cancer cells were determined by measuring ffLuc activity using plate reader. Readings were normalised using the readings for wells containing FaDu cells or FaDu.sup.CD44−/−+ATCs without addition of cell culture media containing BiTE (=100% cell viability), and wells lacking cells (=0% cell viability).

[0477] The results are shown in FIG. 3. Culture in the presence of CD44v6-specific BiTE was found to increase the cell killing of FaDu cells substantially more than FaDu.sup.CD44−/− cells.

[0478] To determine whether circulating CD44v6 BiTE induces “on target, off tumor” toxicity to immune cells in blood, cell culture media containing BiTE was added to PBMCs from healthy donors in culture in vitro. After 72 hours in culture, PBMCs were analysed by flow cytometry. Briefly, cells were stained with antibodies specific for CD3, CD56, CD33, CD14, CD19 in order to permit the delineation of different immune cell subsets within the PBMC population.

[0479] The results are shown in FIGS. 4A to 4C. Culture in the presence of CD44v6-specific BiTE was found not to influence affect the numbers of T cells, NK cells, monocytes of B cells. Culture in the presence of CD19-specific BiTE was found not to influence affect the numbers of T cells, NK cells or monocytes, but significantly reduced the number of B cells.

Example 5: Characterisation of HDAds In Vitro

5.1 Analysis of HDAd Transgene Expression

[0480] Firefly luciferase-labelled FaDu and FaDu.sup.HER2−/− cells were infected with 200 viral particles/cell with HDAdCD44v6BiTE, HDAdIL-12_PD-L1 or HDAd Trio (see Example 1.2) by addition of viral particles to cell culture medium of the cells in culture. At 24 hours post-infection, HER2-specific CAR-T cells (see Example 1.1) were added at an effector:target cell ratio of 1:10. Cell culture supernatant was collected at 48 hours post-infection.

[0481] 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).

[0482] At 72 hours after initiation of the co-culture, the residual FaDu cells were detected by analysis of firefly luciferase activity.

[0483] The results are shown in FIGS. 5A to 5F.

[0484] IL-12 was detected in the cell culture supernatant of cells infected with HDAdIL-12_PD-L1 or HDAd Trio. Anti-PD-L1 minibody was also detected in the cell culture supernatant of cells infected with HDAdIL-12 PD-L1 or HDAd Trio.

[0485] The HER2-specific CAR-T cells killed significantly more FaDu cells than FaDu.sup.HER2−/− cells. Cell killing of FaDu cells and FaDu.sup.HER2−/− cells was greater in the presence of CD44v6BiTE. Cell killing of FaDu cells and FaDu.sup.HER2−/− cells was greater in the presence of IL-12 and anti-PD-L1 minibody. Cell killing of FaDu cells and FaDu.sup.HER2−/− cells was greatest in the presence of CD44v6BiTE, IL-12 and anti-PD-L1 minibody.

[0486] The ability of Onc5/3Ad2E1Δ24 (Onc.Ad), the combination of Onc5/3Ad2E1Δ24+HDAd Trio (referred to in the Figures as “CAd Trio”) and HDAd Trio to cause cell killing of FaDu cells and FaDu.sup.HER2−/− cells was analysed.

[0487] Briefly, FaDu cells or FaDu.sup.HER2−/− cells were seeded in wells of 96-well plates and infected with Onc.Ad (alone), Onc5/3Ad2E1Δ24+HDAd Trio (at a ratio of Onc5/3Ad2E1Δ24 to HDAd Trio of 1:20) or HDAd Trio (alone) at various different viral particle/cell concentrations. Cells were cultured for 4 days, and then MTS reagents (Promega) were added to each well, with cells being incubated at 37° C. for 2 hours. Live cells were then detected by measuring the absorbance at 490 nm with a plate reader. Readings were normalised using the readings for untreated cells (=100% cell viability), and wells lacking cells (=0% cell viability).

[0488] Cell culture supernatants from 100 viral particles/cell conditions were also collected and secretion of IL-12 into the cell culture supernatant was analysed by ELISA.

[0489] The results are shown in FIGS. 6A to 6D.

Example 6: Analysis of the Anticancer Effects of OncAd and HDAds In Vivo

6.1 Ectopic FaDu Cell-Derived Model of Squamous Cell Head and Neck Carcinoma

[0490] FaDu cells were transplanted subcutaneously into the right flank of NSG mice, and mice were untreated (control), or administered with: [0491] (i) 1×10.sup.8 viral particles of Onc5/3Ad2E1Δ24 and HDAdCD44v6BiTE, at a ratio of Onc5/3Ad2E1Δ24 to HDAdCD44v6BiTE of 1:20 (referred to as “BiTE+CART” in the Figures); [0492] (ii) 1×10.sup.8 viral particles of Onc5/3Ad2E1Δ24 and HDAdIL-12_PD-L1, at a ratio of Onc5/3Ad2E1Δ24 to HDAdIL-12_PD-L1 of 1:20 (referred to as “12_PD+CART” in the Figures); or [0493] (iii) 1×10.sup.8 viral particles of Onc5/3Ad2E1Δ24 and HDAd Trio, at a ratio of Onc5/3Ad2E1Δ24 to HDAd Trio of 1:20 (referred to as “Trio+CART” in the Figures).

[0494] Three days later, the mice were administered with 1×10.sup.6 firefly luciferase-labelled HER2-specific CAR-T cells.

[0495] Tumor volumes were measured on days 3, 7, 11, 14 and 21 after viral particle administration. The end point was established at tumor volume of >1,500 mm.sup.3. The expansion and localisation of the HER-2 specific CAR-T cells was monitored by analysis of luciferase activity by intraperitoneal injection of D-Luciferin (1.5 mg per mouse), and imaging of the mice 10 min later using an IVIS imager (Xenogen).

[0496] The results are shown in FIGS. 7A to 7C.

[0497] The HER2-specific CAR-T cells were shown to localise to the FaDu tumors.

6.2 Orthotopic FaDu Cell-Derived Model of Squamous Cell Head and Neck Carcinoma

[0498] In a first experiment, 0.5×10.sup.6 firefly luciferase-labelled FaDu cells were injected orthotopically into NSG male mice, and six days later mice were untreated, or administered with: [0499] (i) 1×10.sup.8 viral particles of Onc5/3Ad2E1Δ24 and HDAdIL-12_PD-L1, at a ratio of Onc5/3Ad2E1Δ24 to HDAdIL-12_PD-L1 of 1:20 (referred to as “12_PDL1+CART” in the Figures); or [0500] (ii) 1×10.sup.8 viral particles of Onc5/3Ad2E1Δ24 and HDAd Trio, at a ratio of Onc5/3Ad2E1Δ24 to HDAd Trio of 1:20 (referred to as “Trio+CART” in the Figures).

[0501] Three days later, the mice were administered with 0.2×10.sup.6 HER2-specific CAR-T cells.

[0502] Tumors were monitored by analysis of luciferase activity by intraperitoneal injection of D-Luciferin (1.5 mg per mouse), and imaging of the mice 10 min later using an IVIS imager (Xenogen).

[0503] The results are shown in FIGS. 8A and 8B. Treatment with the combination of Onc5/3Ad2E1Δ24+HDAd Trio plus HER2-specific CAR-T cells was found to lead to earlier tumor control as compared to treatment with HER2-specific CAR-T cells only, and also as compared to treatment with the combination of Onc5/3Ad2E1Δ24+HDAdIL-12_PD-L1 plus HER2-specific CAR-T cells.

[0504] In a separate experiment, 0.5×10.sup.6 FaDu cells were injected orthotopically into NSG male mice, and six days later mice were untreated (control), or administered with: [0505] (i) 1×10.sup.8 viral particles of Onc5/3Ad2E1Δ24 and HDAdIL-12_PD-L1, at a ratio of Onc5/3Ad2E1Δ24 to HDAdIL-12_PD-L1 of 1:20 (referred to as “12_PDL1+CART” in the Figures); or [0506] (ii) 1×10.sup.8 viral particles of Onc5/3Ad2E1Δ24 and HDAd Trio, at a ratio of Onc5/3Ad2E1Δ24 to HDAd Trio of 1:20 (referred to as “Trio+CART” in the Figures).

[0507] Three days later, the mice were administered with 0.2×10.sup.6 or 1×10.sup.6 firefly luciferase-labelled HER2-specific CAR-T cells.

[0508] Tumor volumes and survival were monitored over time. The end point was established at tumor volume of >1,500 mm.sup.3. The expansion and localisation of the HER-2 specific CAR-T cells was monitored by analysis of luciferase activity by intraperitoneal injection of D-Luciferin (1.5 mg per mouse), and imaging of the mice 10 min later using an IVIS imager (Xenogen).

[0509] The results are shown in FIGS. 9A to 9C. The combination of Onc5/3Ad2E1Δ24+HDAd Trio was found to restrict CAR-T cell expansion, and attenuate early mouse death.

[0510] The phenotype of the HER2-specific CAR-T cells was analysed by flow cytometry prior to infusion into mice, and at day 120 after being harvested from the tongue and lymph nodes of mice that had been treated according to (i) or (ii) above.

[0511] The results are shown in FIG. 10. CD4+ CAR-T cells were found to persist more than CD8+ CAR-T cells.

Example 7: Analysis of the OncAd and HDAd Trio In Vivo in Prostate and Pancreatic Cancers

7.1 Ectopic PC-3 Cell-Derived Model of Prostate Adenocarcinoma

[0512] 4×10.sup.6 PC-3 cells were injected subcutaneously into the right flank of NSG mice, and six days later mice were untreated (control), or administered with: [0513] (i) 1×10.sup.8 viral particles of Onc5/3Ad2E1Δ24 and HDAd Trio, at a ratio of Onc5/3Ad2E1Δ24 to HDAd Trio of 1:20 (referred to as “CAdVEC” in the Figures).

[0514] Three days later, the mice were administered with 1×10.sup.6 firefly luciferase-labelled HER2-specific CAR-T cells, or were not administered with luciferase-labelled HER2-specific CAR-T cells.

[0515] Tumor volumes and survival were monitored over time. The end point was established at tumor volume of >1,500 mm.sup.3. The expansion and localisation of the HER-2 specific CAR-T cells was monitored by analysis of luciferase activity by intraperitoneal injection of D-Luciferin (1.5 mg per mouse), and imaging of the mice 10 min later using an IVIS imager (Xenogen).

[0516] The results are shown in FIGS. 11A to 11C. Treatment with Onc5/3Ad2E1Δ24+HDAd Trio+HER-2 specific CAR-T cells (referred to in the Figures as “CAd+CART”) was found to reduced tumor volumes and increase survival to a greater extent than treatment with Onc5/3Ad2E1Δ24+HDAd Trio (referred to in the Figures as “CAdVEC”), or treatment with HER2-specific CAR-T cells only (referred to in the Figures as “CART”).

7.2 Ectopic CAPAN-1 Cell-Derived Model of Pancreatic Adenocarcinoma

[0517] In a separate experiment, 5×10.sup.6 CAPAN-1 cells were injected subcutaneously into the right flank of NSG mice, and six days later mice were untreated (control), or administered with: [0518] (i) 1×10.sup.8 viral particles of Onc5/3Ad2E1Δ24 and HDAd Trio, at a ratio of Onc5/3Ad2E1Δ24 to HDAd Trio of 1:20 (referred to as “CAdVEC” in the Figures).

[0519] Three days later, the mice were administered with 1×10.sup.6 firefly luciferase-labelled PSCA-specific CAR-T cells (see Example 1.1), or were not administered with luciferase-labelled PSCA-specific CAR-T cells.

[0520] Tumor volumes and survival were monitored over time. The end point was established at tumor volume of >1,500 mm.sup.3. The expansion and localisation of the PSCA specific CAR-T cells was monitored by analysis of luciferase activity by intraperitoneal injection of D-Luciferin (1.5 mg per mouse), and imaging of the mice 10 min later using an IVIS imager (Xenogen).

[0521] The results are shown in FIGS. 12A to 12C. Treatment with Onc5/3Ad2E1Δ24+HDAd Trio+PSCA specific CAR-T cells (referred to in the Figures as “CAd+CART”) was found to reduced tumor volumes and increase survival to a greater extent than treatment with Onc5/3Ad2E1Δ24+HDAd Trio (referred to in the Figures as “CAdVEC”), or treatment with PSCA-specific CAR-T cells only (referred to in the Figures as “CART”).

Example 8: Analysis of the Ability of HDAd-Encoded BiTEs to Induce Cell Killing of Cancer Cells by Adenovirus Specific T Cells In Vitro

[0522] Firefly luciferase-labelled FaDu cells FaDu.sup.CD44−/− cells or FaDu.sup.HER2−/− cells (see Example 1.5) were infected with 100 viral particles/cell of HDAdCD19BiTE, HDAdHER2BiTE, HDAdCD44v6BiTE or HD2xBiTEs (see Example 1.2) by addition of viral particles to cell culture medium of the cells in culture.

[0523] At 24 hours post-infection, AdVSTs (see Example 1.7) were added at an effector:target cell ratio of 1:10.

[0524] After 72 hours, cell killing was analysed by Luciferase assay. Briefly, cells were washed with PBS, and lysis buffer was added. Cell lysates were collected, and the residual cancer cells were determined by measuring ffLuc activity using plate reader. Readings were normalised using the readings for wells containing FaDu cells, FaDu.sup.CD44−/− or FaDu.sup.HER2−/−+AdVSTs without infection by HDAds (=100% cell viability), and wells lacking cells (=0% cell viability).

[0525] The results are shown in FIG. 13. HER2-specific and CD44v6-specific BiTEs were found to be very effective at inducing cell killing of FaDu cells by AdVSTs, irrespective of adenovirus infection.