ADENOVIRUS ENCODING IL-15

Abstract

A group B adenovirus comprising a sequence of formula 5ITRB.sub.1-B.sub.A-B.sub.2-B.sub.X-B.sub.B-B.sub.Y-B.sub.3-3ITR, wherein: BY comprises a sequence -G1-G2.sub.n-G3.sub.m-G4.sub.p-G5.sub.q. G1 is a first transgene. G2 is a second transgene. G3 is a third transgene. G4 is a fourth transgene. G5 is a fifth transgene and IL-15 is encoded as a transgene in at least one of said locations, and characterised in that BY also encodes a polypeptide comprising the sushi domain of IL-15R alpha.

Claims

1-29. (canceled)

30. A group B adenovirus comprising a sequence of formula (I):
5ITR-B.sub.1-B.sub.A-B.sub.2-B.sub.X-B.sub.B-B.sub.Y-B.sub.3-3ITR(I) wherein: B.sub.1 is a bond or comprises: E1A, E1 B or E1A-E1 B; B.sub.A comprises-E2B-L1-L2-L3-E2A-L4; B.sub.2 is a bond or comprises: E3; B.sub.X is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both; B.sub.B comprises L5; B.sub.Y comprises a sequence -G1-G2.sub.n-G3.sub.m-G4.sub.p-G5.sub.q, wherein: G1 is a first transgene; G2 is a second transgene; G3 is a third transgene; G4 is a fourth transgene; G5 is a fifth transgene, B.sub.3 is a bond or comprises: E4; n is 0 or 1; m is 0 or 1; p is 0 or 1; q is 1; B.sub.3 is a bond or comprises: E4; wherein: IL-15 is encoded as a transgene in position G5, characterised in that B.sub.Y also encodes a polypeptide comprising the sushi domain of IL-15R alpha, and does not comprise a transmembrane or GPI anchor such that it is not membrane anchored when expressed.

31. A group B adenovirus according to claim 30, wherein the polypeptide encoding the sushi domain does not comprise a transmembrane domain or GPI anchor.

32. A group B adenovirus according to claim 30, wherein p is 1 and the polypeptide comprising the IL-15R alpha sushi domain is encoded in G4.

33. A group B adenovirus according to claim 30, wherein the leader sequence as employed for IL-15 is the non-native sequence selected independently from Ig, CD33 or IL-2 leader sequence.

34. A group B adenovirus according to claim 30, wherein the virus also encodes IL-12.

35. A group B adenovirus according to claim 34, wherein IL-12 has a sequence shown in SEQ ID NO: 115.

36. A group B adenovirus according to claim 34, wherein IL-12 is encoded in G1 or G2.

37. A group B adenovirus according to claim 30, where at least one further cytokine is encoded in B.sub.Y.

38. A group B adenovirus according to claim 30, wherein B.sub.Y encodes at least one chemokine.

39. A group B adenovirus according to claim 38, wherein the chemokine is selected from CXCL9, CCL19 and CCL21.

40. A group B adenovirus according to claim 38, wherein the chemokine is encoded in G1, G2 and/or G3.

41. A group B adenovirus according to claim 30, wherein m is 0.

42. A group B adenovirus according to claim 30, wherein the one or more transgenes (such as all transgenes) in B.sub.Y are under the control of the major later promoter.

43. A group B adenovirus according to claim 30 wherein the virus comprises a DNA sequence encoding a protein sequence selected from SEQ ID NO: 175 to 189.

44. A composition comprising a group B adenovirus according to claim 30 and a pharmaceutically acceptable excipient, diluent or carrier.

45. A method of treating cancer, comprising administering a therapeutically effective amount of a group B adenovirus according to claim 30.

46. A combination therapy comprising a virus according to claim 30 and a cellular therapy.

Description

DESCRIPTION OF THE FIGURES

[0282] FIG. 1A shows effect of different combinations of recombinant IL-12, IL15 and IL-18 proteins on IFNg production by cultures of primary breast (T63), colorectal (T64) and kidney (T65) tumour cell preparations.

[0283] FIG. 1B shows expression of the CD25 activation marker on CD4 and CD8 T-cells and NK cells from cultures of primary breast tumour (60) cell preparations treated with different combinations of IL-12, IL15 and IL-18.

[0284] FIG. 1C shows expression of the CD107a marker of activated degranulation on CD4 and CD8 T-cells and NK cells from cultures of primary breast tumour (60) cell preparations treated with different combinations of IL-12, IL15 and IL-18.

[0285] FIG. 1D shows intracellular IFNg expression in CD4 and CD8 T-cells and NK cells from PBMCs treated with different combinations of IL-12, IL15 and IL-18.

[0286] FIG. 2A shows kinetic analysis of PBMC-derived T-cell mediated killing (apoptosis induction) of target cell fibroblasts by a FAP-specific T-cell activator (FAP-TAc) in the presence or absence of combinations of IL-12, IL15 and IL-18.

[0287] FIG. 2B shows enhancement by IFN? of the of PBMC-derived T-cell mediated killing (apoptosis induction) of target cell fibroblasts by a FAP-specific T-cell activator (FAP-TAc) in the presence or absence of combinations of IL-12, IL15 and IL-18.

[0288] FIG. 2C shows kinetic analysis of T-cell mediated killing (apoptosis induction) of target cell fibroblasts by primary tumour-derived lymphocytes (kidney tumour 70) stimulated with a FAP-specific T-cell activator (FAP-TAc) in the presence or absence of combinations of IL-12, IL15 and IL-18.

[0289] FIG. 2D shows the enhancement by IFN? of the T-cell mediated killing (apoptosis induction) of target cell fibroblasts by primary tumour-derived lymphocytes (kidney tumour 70) stimulated with a FAP-specific T-cell activator (FAP-TAc) in the presence or absence of combinations of IL-12, IL15 and IL-18.

[0290] FIG. 2E shows the effect of IL-12 and IL-15 on PBMC-derived NK cell-mediated killing of K562 target tumour cells.

[0291] FIG. 2F shows the effect of IL-12, IL-15 and IFN? on primary tumour-derived NK cell-mediated killing of K562 target tumour cells.

[0292] FIG. 3A shows chemokine stimulated migration of na?ve T-cells prepared from PBMCs in culture using recombinant chemokines.

[0293] FIG. 3B shows chemokine stimulated migration of effector T-cells prepared from PBMCs in culture using recombinant chemokines.

[0294] FIG. 3C shows chemokine stimulated migration of CD45+TILS, prepared from a primary breast tumour sample (53), in culture using recombinant chemokines.

[0295] FIG. 3D shows chemokine stimulated migration of CD3+ T-cells and non-T-cells (CD3-) from primary lymph nodes from breast cancer surgery.

[0296] FIG. 3E shows chemokine stimulated migration of monocyte-derived dendritic cells across a Matrigel coated transwell.

[0297] FIG. 3F shows real-time imaging analysis (Incucyte) of CCL19 and CCL21 chemokine stimulated migration of dendritic cells across a Matrigel coated transwell.

[0298] FIG. 4A shows effect of IL-15 with or without IL-12 on primary lymph node T-cell responses to Muc-1 tumour antigen or CEFT peptides measure by IFNg ELISPOT assay.

[0299] FIG. 4B shows the effect of IL-15 with or without IL-12 on primary lymph node T-cell responses to Her2 tumour antigens (HER2-ECD and HER2-ICD) or CEFT peptides measure by IFNg ELISPOT assay.

[0300] FIG. 5A shows a schematic representation of transgene cassettes encoding human IL-12 as separate p35 and p40 proteins or as single chain IL-12 molecules which use a linker to covalently join the p35 and p40 chains.

[0301] FIG. 5B shows the genome replication of NG-701, NG-702 and NG-703 in A549 cells.

[0302] FIG. 5C shows IL-12 p70 protein production by A549 cells infected with NG-701, NG-702 and NG-703

[0303] FIG. 5D shows RT-qPCR analysis of transgene mRNA expression by NG-701, NG-702 and NG-703 inoculated A549 cells.

[0304] FIG. 5E shows IL-12p40 and IL-12p70 production measured by ELISA assay of supernatants from A549 cells inoculated with NG-701 or NG-702.

[0305] FIG. 5F shows functional activity of IL-12 produced by NG-702 or NG-703 inoculated A549 cells assessed with a HEK-Blue cell IL-12 signaling reporter assay.

[0306] FIG. 5G shows the effect of recombinant human IL-12 or supernatants from NG-702 infected A549 cells on CD107a expression by CD4+ T-cells stimulated with anti-CD3 and/or anti-CD28 antibodies.

[0307] FIG. 5H shows the effect of recombinant human IL-12 or supernatants from NG-702 infected A549 cells on CD107a expression by CD8+ T-cells stimulated with anti-CD3 and/or anti-CD28 antibodies.

[0308] FIG. 6 shows a schematic representation of transgene cassettes encoding the IL12p40-Linker-IL12p35 single chain human IL-12 plus one or more other transgenes. In one embodiment these constructs are inserted in position B.sub.Y.

[0309] FIG. 7A shows genome replication of EnAd, NG-702, NG-704 and NG-706 in A549 cells.

[0310] FIG. 7B shows IL-12p70 protein levels produced by A549 cells inoculated with NG-707, NG-704 or NG-706.

[0311] FIG. 7C shows IFNa protein levels produced by the same A549 cells treated with NG-704 or NG-706.

[0312] FIG. 7D shows the expression of mRNA for Flt3L, MIP1a, IFN?, CXCL9 and IL-12 transgenes in A549 cells inoculated with NG-707.

[0313] FIG. 7E shows the expression of Flt3L, MIP1a, IFN?, CXCL9 and IL-12 transgene proteins by A549 cells inoculated with NG-707.

[0314] FIG. 7F shows the expression of IFN?, CCL19, IL-18 and IL-12 transgene proteins by A549 cells inoculated with NG-709

[0315] FIG. 7G shows functional IL-12 activity in supernatants of A549 cells inoculated with NG-704, NG-706, NG-707 or NG-709.

[0316] FIG. 7H shows production of IL-12, Flt3L and CCL21 transgene proteins by A549 cells inoculated with NG-708.

[0317] FIG. 7I shows encoded transgene protein production by NG-708 and NG-709 inoculated A549 cells measured by specific ELISAs.

[0318] FIG. 7J shows encoded transgene protein production by A549 cells inoculated with different viruses depicted in FIG. 7A above (1?10.sup.6 cells).

[0319] FIG. 8A shows production of IL-12 p70 protein by tumour cell preparations from a primary colorectal (68) and a kidney (70) tumour inoculated with NG-702 or NG-704.

[0320] FIG. 8B shows production of IFNg by a primary kidney tumour cell preparation cultured with different combinations of IL-12, IL-15 and IL-18, with or without inoculation with EnAd, NG-702 or NG-704 viruses.

[0321] FIG. 8C shows time course of IL-12 p70 protein production by tumour cell preparations from a kidney (70) and a colorectal (71) primary tumour cell preparations inoculated with NG-707.

[0322] FIG. 8D shows production of IL-12 p70 protein by primary breast tumour cell preparations (66 and 67) inoculated with NG-702, NG-704, NG-707 or NG-709.

[0323] FIG. 8E shows time course of IL-12 p70 protein production by a primary colorectal tumour (75) cell preparation inoculated with NG-707 at two different dose levels.

[0324] FIG. 8F shows time course of IL-12 p70 protein production by a primary colorectal tumour (76) cell preparation inoculated with NG-707 at two different dose levels.

[0325] FIG. 8G shows time course of IL-12 p70 protein production by a primary colorectal tumour (79) cell preparation inoculated with NG-707 at two different dose levels.

[0326] FIG. 8H shows time course of IL-12 p70 protein production by a primary kidney tumour (tumour 70) cell preparation inoculated with NG-707 at two different dose levels.

[0327] FIG. 9A shows schematic representation of transgene cassettes encoding transmembrane or soluble secreted forms of IL-15 receptor alpha sushi domain with or without IL-15.

[0328] FIG. 9B shows production of IL-15 by NG-740 compared to three viruses not expressing an IL-15 receptor alpha form.

[0329] FIG. 9C shows IL-15 production following co transfection of an IL-15 plasmid with either transmembrane form of IL-15 receptor alpha sushi domain (sushi-TM) or a soluble secreted version (sushi).

[0330] FIG. 9D shows functional activity of IL-15 in samples from FIG. 9C.

[0331] FIG. 9E shows IL-15 production by A549 cells inoculated with NG-740 and NG-748 expressing transmembrane or soluble secreted forms of IL-15 receptor alpha sushi domain, respectively

[0332] FIG. 9F shows IL-15 production by primary colorectal tumour cells inoculated with NG-740 and NG-748 expressing transmembrane or soluble secreted forms of IL-15 receptor alpha sushi domain, respectively.

[0333] FIG. 9G shows IFNg production by primary kidney tumour cells treated with NG-748 or NG-702, in presence of different IL-12 or IL-15 respectively.

[0334] FIG. 10A shows schematic representation of transgene cassettes encoding IL-12 and IL-15 together with transmembrane or soluble secreted forms of IL-15 receptor alpha sushi domain.

[0335] FIG. 10B shows IL-12 and IL-15 production by A549 cells treated with the viruses depicted in FIG. 10A, measured by ELISA.

[0336] FIG. 10C shows functional activity of IL-12 and IL-15 from same samples as FIG. 10B.

[0337] FIG. 10D shows IL-12, IL-15 and IFNg production by A549 cells inoculated with different viruses, with PBMCs added to the cultures after 24h.

[0338] FIG. 10E shows IL-12, IL-15 and IFNg production by A549 cells inoculated with different viruses, with purified CD3+T_cells added to the cultures after 24h.

[0339] FIG. 10F shows IFNg production by T-cells cultured in direct contact with, or separated in a transwell format, from A549 cells treated with different viruses.

[0340] FIG. 10G shows IL-15 and IFNg production by A549 cells inoculated with different viruses, with PBMCs or purified T-cells added to the cultures after 24h.

[0341] FIG. 11A shows production of IFNg, IL-12 and IL-15 by primary colorectal tumour cells (tumour 98) 72h following treatment with different viruses.

[0342] FIG. 11B shows production of IFNg by primary colorectal tumour cells (tumour 99) 96h following treatment with different viruses.

[0343] FIG. 12A shows a schematic representation of transgene cassettes encoding IL-12 and IL-15 together with transmembrane or soluble secreted forms of IL-15 receptor alpha sushi domain and a further chemokine transgene (CXCL9 or CCL21).

[0344] FIG. 12B shows the production of IL-12p70, IL-15, CXCL9 and CCL21 by A549 cells treated with different viruses.

[0345] FIG. 12C shows the production of IL-15 by A549 cells treated with different viruses.

[0346] FIG. 12D shows the production of IL-12p70, IL-15 and CXCL9 transgene proteins and IFNg cytokine secretion by primary tumour cells (tumour 105) treated with different viruses.

[0347] FIG. 12E shows the production of IL-12p70, IL-15, CXCL9 and IFNg by primary colorectal tumour cells (tumour 105) treated with different viruses.

[0348] FIG. 12F shows the production of IL-12p70, IL-15, CXCL9 CCL21 and IFNg by primary colorectal tumour cells (tumour 107) treated with different viruses.

[0349] FIG. 12G shows the migration of monocyte-derived dendritic cells stimulated by CCL21 transgene protein in supernatants from A549 cells treated with NG-795A and migration inhibition in the presence of added anti-CCL21 antibody.

[0350] FIG. 12H shows the migration of monocyte-derived dendritic cells stimulated by CCL21 transgene protein in supernatants from A549 cells treated with NG-795A and selective migration inhibition in the presence of added anti-CCL21 antibody.

[0351] FIG. 13 shows the levels of IL-12 p70 in the plasma of human tumour xenograft-bearing mice injected with NG-786A, NG-791A or NG-796A compared with those dosed with EnAd or untreated mice.

[0352] FIG. 14 shows the generic structure of chimeric antigen receptors.

[0353] FIG. 15 shows a graph of the migration of monocyte-derived dendritic cells stimulated by CCL21 transgene protein in supernatants from A549 cells treated with NG-796A and migration inhibition in the presence of added anti-CCL21 antibody.

[0354] FIG. 16A shows the control of A549 tumour xenograft growth by IV dosed NG-704 prior to transfer of Her2-specific CAR-T cells compared to CAR-T alone of CAR-T plus EnAd pre-dosing.

[0355] FIG. 16B shows the control of A549 tumour xenograft growth by IV dosed NG-796A prior to transfer of HER2-specific CAR-T cells compared to CAR-T alone of CAR-T plus EnAd pre-dosing.

[0356] FIG. 16C shows the levels of CCL21 in A549 xenograft tumours following IV dosing with NG-796A or EnAd.

[0357] FIG. 17 shows the levels of IL-12, CCL21, IL-15 and IL-15Ra sushi domain protein in supernatants of A549 cells infected with NG-796A.

[0358] FIG. 18A shows the levels of IL-15 detected in supernatants of A549 cells transfected with pUC-796A or pRES-128 with or without different concentrations of recombinant IL-15Ra sushi domain protein added to the cultures.

[0359] FIG. 18B shows a schematic representation of transgene cassettes in CMV pUC vectors encoding single chain IL-12 (scIL12), CCL21 and IL-15 with (pUC-796A) or without (pRES-128) a sequence encoding secreted IL-15Ra sushi domain

SEQUENCES

[0360] SEQ ID NO: 1 Short splice acceptor (SSA) DNA sequence -CAGG [0361] SEQ ID NO: 2 Splice acceptor (SA) DNA sequence [0362] SEQ ID NO: 3 Splice acceptor DNA sequence [0363] SEQ ID NO: 4 High efficiency self-cleavable P2A peptide sequence [0364] SEQ ID NO: 5 High efficiency self-cleavable T2A peptide sequence [0365] SEQ ID NO: 6 High efficiency self-cleavable E2A peptide sequence [0366] SEQ ID NO: 7 High efficiency self-cleavable F2A peptide sequence [0367] SEQ ID NO: 8 Poly adenylation (PA) sequence (SV40 late polyA sequence) [0368] SEQ ID NO: 9 Human IL-12 p35 protein with signal sequence (IL12p35) [0369] SEQ ID NO: 10 Human IL-12 p40 protein with signal sequence (IL12p40) [0370] SEQ ID NO: 11 Human IL-12 p70 single chain protein with a (Gly.sub.4Ser).sub.3 linker joining the IL12p40 protein with its signal sequence N-terminal to the IL12p35 protein without its signal sequence (IL12p40LinkerIL12p35) [0371] SEQ ID NO: 12 Human IL-12 p70 single chain protein with a (Gly.sub.4Ser).sub.3 linker joining the IL12p35 protein with its signal sequence N-terminal to the IL12p40 protein without its signal sequence (IL12p35LinkerIL12p40) [0372] SEQ ID NO: 13 Human Interferon alpha 2 protein sequence [0373] SEQ ID NO: 14 Human Fms-related tyrosine kinase 3 ligand (Flt3L) soluble extracellular domain protein sequence [0374] SEQ ID NO: 15 Human MIP1alpha LD78beta (MIP1a) protein sequence [0375] SEQ ID NO: 16 Human CXCL9 protein sequence [0376] SEQ ID NO: 17 Human CCL21 protein sequence [0377] SEQ ID NO: 18 Human CCL5 protein sequence [0378] SEQ ID NO: 19 Human CCL19 protein sequence [0379] SEQ ID NO: 20 Human IL-18 long isoform [0380] SEQ ID NO: 21 Human IL-15 protein with N-terminal human Ig leader sequence [0381] SEQ ID NO: 22 Human Ig leader peptide sequence [0382] SEQ ID NO: 23 Human IL-15 protein sequence without leader sequence [0383] SEQ ID NO: 24 Human CCL21 C-terminally truncated protein sequence (CCL21t) [0384] SEQ ID NO: 25 Human IL-15 receptor alpha Sushi domain protein sequence, with IL-15 receptor alpha leader sequence at N-terminus, with a C-terminal myc peptide linking it to a PDGF receptor transmembrane domain (IL15RsushimycTM) [0385] SEQ ID NO: 26 Human IL-15 receptor alpha Sushi domain protein sequence with IL-15 receptor alpha leader sequence at N-terminus [0386] SEQ ID NO: 27 Myc peptide sequence [0387] SEQ ID NO: 28 PDGF receptor transmembrane domain protein sequence [0388] SEQ ID NO: 29 Human IL-15 receptor alpha Sushi domain protein sequence, with IL-15 receptor alpha leader sequence at N-terminus, with a Gly.sub.4Ser C-terminal peptide sequence linking it to a PDGF receptor transmembrane domain (IL15RsushiG4STM) [0389] SEQ ID NO: 30 Gly.sub.4Ser peptide linker sequence [0390] SEQ ID NO: 31 (Gly.sub.4Ser).sub.3 peptide linker sequence [0391] SEQ ID NO: 32 Human CCL21 coding DNA sequence encoding SEQ ID NO: 17 [0392] SEQ ID NO: 33 Human CCL21 modified codon sequence (CCL21mod) encoding same protein sequence as encoded by SEQ ID NO: 32 [0393] SEQ ID NO: 34 Human CCL21 coding sequence truncated at the 3 end to produce a C-terminally truncated protein sequence (SEQ ID NO: 24) [0394] SEQ ID NO: 35 Human CCL21 modified coding sequence truncated at the 3 end (CCL21tmod) to produce a C-terminally truncated protein sequence (SEQ ID NO: 24), the same protein sequence as encoded by SEQ ID NO: 34 [0395] SEQ ID NO: 36 Protein sequence encoded by NG-704 transgene cassette [0396] SEQ ID NO: 37 Protein sequence encoded by NG-706 transgene cassette [0397] SEQ ID NO: 38 Protein sequence encoded by NG-707 transgene cassette [0398] SEQ ID NO: 39 Protein sequence encoded by NG-708 transgene cassette [0399] SEQ ID NO: 40 Protein sequence encoded by NG-709 transgene cassette [0400] SEQ ID NO: 41 Protein sequence encoded by NG-720 transgene cassette [0401] SEQ ID NO: 42 Protein sequence encoded by NG-721 transgene cassette [0402] SEQ ID NO: 43 Protein sequence encoded by NG-722 transgene cassette [0403] SEQ ID NO: 44 Protein sequence encoded by NG-723 transgene cassette [0404] SEQ ID NO: 45 Protein sequence encoded by NG-724 transgene cassette [0405] SEQ ID NO: 46 Protein sequence encoded by NG-725 transgene cassette [0406] SEQ ID NO: 47 Protein sequence encoded by NG-726 transgene cassette [0407] SEQ ID NO: 48 Protein sequence encoded by NG-740 transgene cassette [0408] SEQ ID NO: 49 Protein sequence encoded by NG-742 transgene cassette [0409] SEQ ID NO: 50 Protein sequence encoded by NG-744 transgene cassette [0410] SEQ ID NO: 51 Protein sequence encoded by NG-746 transgene cassette [0411] SEQ ID NO: 52 Protein sequence encoded by NG-750 transgene cassette [0412] SEQ ID NO: 53 Protein sequence encoded by NG-751 transgene cassette [0413] SEQ ID NO: 54 Protein sequence encoded by NG-752 transgene cassette [0414] SEQ ID NO: 55 Protein sequence encoded by NG-753 transgene cassette [0415] SEQ ID NO: 56 Protein sequence encoded by NG-754 transgene cassette [0416] SEQ ID NO: 57 Protein sequence encoded by NG-755 transgene cassette [0417] SEQ ID NO: 58 Protein sequence encoded by NG-756 transgene cassette [0418] SEQ ID NO: 59 Protein sequence encoded by NG-757 transgene cassette [0419] SEQ ID NO: 60 Protein sequence encoded by NG-758 transgene cassette [0420] SEQ ID NO: 61 Protein sequence encoded by NG-759 transgene cassette [0421] SEQ ID NO: 62 Protein sequence encoded by NG-760 transgene cassette [0422] SEQ ID NO: 63 Protein sequence encoded by NG-761 transgene cassette [0423] SEQ ID NO: 64 Protein sequence encoded by NG-762 transgene cassette [0424] SEQ ID NO: 65 Protein sequence encoded by NG-763 transgene cassette [0425] SEQ ID NO: 66 Protein sequence encoded by NG-764 transgene cassette [0426] SEQ ID NO: 67 Protein sequence encoded by NG-765 transgene cassette [0427] SEQ ID NO: 68 Protein sequence encoded by NG-768 transgene cassette [0428] SEQ ID NO: 69 Protein sequence encoded by NG-769 transgene cassette [0429] SEQ ID NO: 70 Protein sequence encoded by NG-770 transgene cassette [0430] SEQ ID NO: 71 Protein sequence encoded by NG-771 transgene cassette [0431] SEQ ID NO: 72 Protein sequence encoded by NG-772 transgene cassette [0432] SEQ ID NO: 73 Protein sequence encoded by NG-773 transgene cassette [0433] SEQ ID NO: 74 Genome sequence of NG-701 virus [0434] SEQ ID NO: 75 Genome sequence of NG-702 virus [0435] SEQ ID NO: 76 Genome sequence of NG-703 virus [0436] SEQ ID NO: 77 Genome sequence of NG-704 virus [0437] SEQ ID NO: 78 Genome sequence of NG-706 virus [0438] SEQ ID NO: 79 Genome sequence of NG-707 virus [0439] SEQ ID NO: 80 Genome sequence of NG-708 virus [0440] SEQ ID NO: 81 Genome sequence of NG-709 virus [0441] SEQ ID NO: 82 Genome sequence of NG-720 virus [0442] SEQ ID NO: 83 Genome sequence of NG-721 virus [0443] SEQ ID NO: 84 Genome sequence of NG-722 virus [0444] SEQ ID NO: 85 Genome sequence of NG-723 virus [0445] SEQ ID NO: 86 Genome sequence of NG-724 virus [0446] SEQ ID NO: 87 Genome sequence of NG-725 virus [0447] SEQ ID NO: 88 Genome sequence of NG-726 virus [0448] SEQ ID NO: 89 Genome sequence of NG-740 virus [0449] SEQ ID NO: 90 Genome sequence of NG-742 virus [0450] SEQ ID NO: 91 Genome sequence of NG-744 virus [0451] SEQ ID NO: 92 Genome sequence of NG-746 virus [0452] SEQ ID NO: 93 Genome sequence of NG-750 virus [0453] SEQ ID NO: 94 Genome sequence of NG-751 virus [0454] SEQ ID NO: 95 Genome sequence of NG-752 virus [0455] SEQ ID NO: 96 Genome sequence of NG-753 virus [0456] SEQ ID NO: 97 Genome sequence of NG-754 virus [0457] SEQ ID NO: 98 Genome sequence of NG-755 virus [0458] SEQ ID NO: 99 Genome sequence of NG-756 virus [0459] SEQ ID NO: 100 Genome sequence of NG-757 virus [0460] SEQ ID NO: 101 Genome sequence of NG-758 virus [0461] SEQ ID NO: 102 Genome sequence of NG-759 virus [0462] SEQ ID NO: 103 Genome sequence of NG-760 virus [0463] SEQ ID NO: 104 Genome sequence of NG-761 virus [0464] SEQ ID NO: 105 Genome sequence of NG-762 virus [0465] SEQ ID NO: 106 Genome sequence of NG-763 virus [0466] SEQ ID NO: 107 Genome sequence of NG-764 virus [0467] SEQ ID NO: 108 Genome sequence of NG-765 virus [0468] SEQ ID NO: 109 Genome sequence of NG-768 virus [0469] SEQ ID NO: 110 Genome sequence of NG-769 virus [0470] SEQ ID NO: 111 Genome sequence of NG-770 virus [0471] SEQ ID NO: 112 Genome sequence of NG-771 virus [0472] SEQ ID NO: 113 Genome sequence of NG-772 virus [0473] SEQ ID NO: 114 Genome sequence of NG-773 virus [0474] SEQ ID NO: 115 Mature human IL-12 p70 single chain protein with a (Gly.sub.4Ser).sub.3 linker joining the IL12p40 protein without its signal sequence N-terminal to the IL12p35 protein without its signal sequence (mature IL12p40LinkerIL12p35) [0475] SEQ ID NO: 116 DNA sequence for the protein coding sequence of the NG-701 transgene cassette, including the stop codon [0476] SEQ ID NO: 117 DNA sequence for the protein coding sequence of the NG-702 transgene cassette, including the stop codon [0477] SEQ ID NO: 118 DNA sequence for the protein coding sequence of the NG-703 transgene cassette, including the stop codon [0478] SEQ ID NO: 119 DNA sequence for the protein coding sequence of the NG-704 transgene cassette, including the stop codon [0479] SEQ ID NO: 120 DNA sequence for the protein coding sequence of the NG-706 transgene cassette, including the stop codon [0480] SEQ ID NO: 121 DNA sequence for the protein coding sequence of the NG-707 transgene cassette, including the stop codon [0481] SEQ ID NO: 122 DNA sequence for the protein coding sequence of the NG-708 transgene cassette, including the stop codon [0482] SEQ ID NO: 123 DNA sequence for the protein coding sequence of the NG-709 transgene cassette, including the stop codon [0483] SEQ ID NO: 124 DNA sequence for the protein coding sequence of the NG-720 transgene cassette, including the stop codon [0484] SEQ ID NO: 125 DNA sequence for the protein coding sequence of the NG-721 transgene cassette, including the stop codon [0485] SEQ ID NO: 126 DNA sequence for the protein coding sequence of the NG-722 transgene cassette, including the stop codon [0486] SEQ ID NO: 127 DNA sequence for the protein coding sequence of the NG-723 transgene cassette, including the stop codon [0487] SEQ ID NO: 128 DNA sequence for the protein coding sequence of the NG-724 transgene cassette, including the stop codon [0488] SEQ ID NO: 129 DNA sequence for the protein coding sequence of the NG-725 transgene cassette, including the stop codon [0489] SEQ ID NO: 130 DNA sequence for the protein coding sequence of the NG-726 transgene cassette, including the stop codon [0490] SEQ ID NO: 131 DNA sequence for the protein coding sequence of the NG-740 transgene cassette, including the stop codon [0491] SEQ ID NO: 132 DNA sequence for the protein coding sequence of the NG-742 transgene cassette, including the stop codon [0492] SEQ ID NO: 133 DNA sequence for the protein coding sequence of the NG-744 transgene cassette, including the stop codon [0493] SEQ ID NO: 134 DNA sequence for the protein coding sequence of the NG-746 transgene cassette, including the stop codon [0494] SEQ ID NO: 135 DNA sequence for the protein coding sequence of the NG-750 transgene cassette, including the stop codon [0495] SEQ ID NO: 136 DNA sequence for the protein coding sequence of the NG-751 transgene cassette, including the stop codon [0496] SEQ ID NO: 137 DNA sequence for the protein coding sequence of the NG-752 transgene cassette, including the stop codon [0497] SEQ ID NO: 138 DNA sequence for the protein coding sequence of the NG-753 transgene cassette, including the stop codon [0498] SEQ ID NO: 139 DNA sequence for the protein coding sequence of the NG-754 transgene cassette, including the stop codon [0499] SEQ ID NO: 140 DNA sequence for the protein coding sequence of the NG-755 transgene cassette, including the stop codon [0500] SEQ ID NO: 141 DNA sequence for the protein coding sequence of the NG-756 transgene cassette, including the stop codon [0501] SEQ ID NO: 142 DNA sequence for the protein coding sequence of the NG-757 transgene cassette, including the stop codon [0502] SEQ ID NO: 143 DNA sequence for the protein coding sequence of the NG-758 transgene cassette, including the stop codon [0503] SEQ ID NO: 144 DNA sequence for the protein coding sequence of the NG-759 transgene cassette, including the stop codon [0504] SEQ ID NO: 145 DNA sequence for the protein coding sequence of the NG-760 transgene cassette, including the stop codon [0505] SEQ ID NO: 146 DNA sequence for the protein coding sequence of the NG-761 transgene cassette, including the stop codon [0506] SEQ ID NO: 147 DNA sequence for the protein coding sequence of the NG-762 transgene cassette, including the stop codon [0507] SEQ ID NO: 148 DNA sequence for the protein coding sequence of the NG-763 transgene cassette, including the stop codon [0508] SEQ ID NO: 149 DNA sequence for the protein coding sequence of the NG-764 transgene cassette, including the stop codon [0509] SEQ ID NO: 150 DNA sequence for the protein coding sequence of the NG-765 transgene cassette, including the stop codon [0510] SEQ ID NO: 151 DNA sequence for the protein coding sequence of the NG-768 transgene cassette, including the stop codon [0511] SEQ ID NO: 152 DNA sequence for the protein coding sequence of the NG-769 transgene cassette, including the stop codon [0512] SEQ ID NO: 153 DNA sequence for the protein coding sequence of the NG-770 transgene cassette, including the stop codon [0513] SEQ ID NO: 154 DNA sequence for the protein coding sequence of the NG-771 transgene cassette, including the stop codon [0514] SEQ ID NO: 155 DNA sequence for the protein coding sequence of the NG-772 transgene cassette, including the stop codon [0515] SEQ ID NO: 156 DNA sequence for the protein coding sequence of the NG-773 transgene cassette, including the stop codon [0516] SEQ ID NO: 157 PDGF receptor A transmembrane domain amino acid sequence [0517] SEQ ID NO: 158 PDGF receptor B transmembrane domain amino acid sequence [0518] SEQ ID NO: 159 Insulin-like growth factor 1 receptor transmembrane domain amino acid sequence [0519] SEQ ID NO: 160 IL-6 receptor transmembrane domain amino acid sequence [0520] SEQ ID NO: 161 CD28 transmembrane domain amino acid sequence [0521] SEQ ID NO: 162 Sequence comprising a start codon(gcc)gccRccAUGg [0522] SEQ ID NO: 163 Human IL-15 protein with N-terminal human CD33 leader sequence [0523] SEQ ID NO: 164 Human CD33 leader peptide sequence [0524] SEQ ID NO: 165 Human IL-15 protein with N-terminal human IL-2 leader sequence [0525] SEQ ID NO: 166 Human IL-2 leader peptide sequence [0526] SEQ ID NO: 167 Protein sequence encoded by NG-748 transgene cassette [0527] SEQ ID NO: 168 Protein sequence encoded by NG-774 transgene cassette [0528] SEQ ID NO: 169 Protein sequence encoded by NG-775 transgene cassette [0529] SEQ ID NO: 170 Protein sequence encoded by NG-776 transgene cassette [0530] SEQ ID NO: 171 Protein sequence encoded by NG-777 transgene cassette [0531] SEQ ID NO: 172 Protein sequence encoded by NG-781 transgene cassette [0532] SEQ ID NO: 173 Protein sequence encoded by NG-782 transgene cassette [0533] SEQ ID NO: 174 Protein sequence encoded by NG-784 transgene cassette [0534] SEQ ID NO: 175 Protein sequence encoded by NG-785 transgene cassette [0535] SEQ ID NO: 176 Protein sequence encoded by NG-785A transgene cassette [0536] SEQ ID NO: 177 Protein sequence encoded by NG-786A transgene cassette [0537] SEQ ID NO: 178 Protein sequence encoded by NG-787 transgene cassette [0538] SEQ ID NO: 179 Protein sequence encoded by NG-787A transgene cassette [0539] SEQ ID NO: 180 Protein sequence encoded by NG-788P transgene cassette [0540] SEQ ID NO: 181 Protein sequence encoded by NG-789P transgene cassette [0541] SEQ ID NO: 182 Protein sequence encoded by NG-790P transgene cassette [0542] SEQ ID NO: 183 Protein sequence encoded by NG-791A transgene cassette [0543] SEQ ID NO: 184 Protein sequence encoded by NG-792A transgene cassette [0544] SEQ ID NO: 185 Protein sequence encoded by NG-794A transgene cassette [0545] SEQ ID NO: 186 Protein sequence encoded by NG-795A transgene cassette [0546] SEQ ID NO: 187 Protein sequence encoded by NG-796A transgene cassette [0547] SEQ ID NO: 188 Protein sequence encoded by NG-799A transgene cassette [0548] SEQ ID NO: 189 DNA sequence for the protein coding sequence of the NG-748 transgene cassette, including the stop codon [0549] SEQ ID NO: 190 DNA sequence for the protein coding sequence of the NG-774 transgene cassette, including the stop codon [0550] SEQ ID NO: 191 DNA sequence for the protein coding sequence of the NG-775 transgene cassette, including the stop codon [0551] SEQ ID NO: 192 DNA sequence for the protein coding sequence of the NG-776 transgene cassette, including the stop codon [0552] SEQ ID NO: 193 DNA sequence for the protein coding sequence of the NG-777 transgene cassette, including the stop codon [0553] SEQ ID NO: 194 DNA sequence for the protein coding sequence of the NG-781 transgene cassette, including the stop codon [0554] SEQ ID NO: 195 DNA sequence for the protein coding sequence of the NG-782 transgene cassette, including the stop codon [0555] SEQ ID NO: 196 DNA sequence for the protein coding sequence of the NG-784 transgene cassette, including the stop codon [0556] SEQ ID NO: 197 DNA sequence for the protein coding sequence of the NG-785 transgene cassette, including the stop codon [0557] SEQ ID NO: 198 DNA sequence for the protein coding sequence of the NG-785A transgene cassette, including the stop codon [0558] SEQ ID NO: 199 DNA sequence for the protein coding sequence of the NG-786A transgene cassette, including the stop codon [0559] SEQ ID NO: 200 DNA sequence for the protein coding sequence of the NG-787 transgene cassette, including the stop codon [0560] SEQ ID NO: 201 DNA sequence for the protein coding sequence of the NG-787A transgene cassette, including the stop codon [0561] SEQ ID NO: 202 DNA sequence for the protein coding sequence of the NG-788P transgene cassette, including the stop codon [0562] SEQ ID NO: 203 DNA sequence for the protein coding sequence of the NG-789P transgene cassette, including the stop codon [0563] SEQ ID NO: 204 DNA sequence for the protein coding sequence of the NG-790P transgene cassette, including the stop codon [0564] SEQ ID NO: 205 DNA sequence for the protein coding sequence of the NG-791A transgene cassette, including the stop codon [0565] SEQ ID NO: 206 DNA sequence for the protein coding sequence of the NG-792A transgene cassette, including the stop codon [0566] SEQ ID NO: 207 DNA sequence for the protein coding sequence of the NG-794A transgene cassette, including the stop codon [0567] SEQ ID NO: 208 DNA sequence for the protein coding sequence of the NG-795A transgene cassette, including the stop codon [0568] SEQ ID NO: 209 DNA sequence for the protein coding sequence of the NG-796A transgene cassette, including the stop codon [0569] SEQ ID NO: 210 DNA sequence for the protein coding sequence of the NG-799A transgene cassette, including the stop codon [0570] SEQ ID NO: 211 Genome sequence of NG-748 virus [0571] SEQ ID NO: 212 Genome sequence of NG-774 virus [0572] SEQ ID NO: 213 Genome sequence of NG-775 virus [0573] SEQ ID NO: 214 Genome sequence of NG-776 virus [0574] SEQ ID NO: 215 Genome sequence of NG-777 virus [0575] SEQ ID NO: 216 Genome sequence of NG-781 virus [0576] SEQ ID NO: 217 Genome sequence of NG-782 virus [0577] SEQ ID NO: 218 Genome sequence of NG-784 virus [0578] SEQ ID NO: 219 Genome sequence of NG-785 virus [0579] SEQ ID NO: 220 Genome sequence of NG-785A virus [0580] SEQ ID NO: 221 Genome sequence of NG-786A virus [0581] SEQ ID NO: 222 Genome sequence of NG-787 virus [0582] SEQ ID NO: 223 Genome sequence of NG-787A virus [0583] SEQ ID NO: 224 Genome sequence of NG-788P virus [0584] SEQ ID NO: 225 Genome sequence of NG-789P virus [0585] SEQ ID NO: 226 Genome sequence of NG-790P virus [0586] SEQ ID NO: 227 Genome sequence of NG-791A virus [0587] SEQ ID NO: 228 Genome sequence of NG-792A virus [0588] SEQ ID NO: 229 Genome sequence of NG-794A virus [0589] SEQ ID NO: 230 Genome sequence of NG-795A virus [0590] SEQ ID NO: 231 Genome sequence of NG-796A virus [0591] SEQ ID NO: 232 Genome sequence of NG-799A virus [0592] SEQ ID NO: 233 Protein sequence encoded by NG-701 transgene cassette [0593] SEQ ID NO: 234 Protein sequence encoded by NG-702 transgene cassette [0594] SEQ ID NO: 235 Protein sequence encoded by NG-703 transgene cassette [0595] SEQ ID NO: 236 Human IL-15 protein with N-terminal human Ig leader sequence with a peptide linker joining it to the Human IL-15 receptor alpha Sushi domain without its' signal sequence [0596] SEQ ID NO: 237 Linker peptide joining IL-15 to IL-15 receptor alpha sushi domain in NG-787 and NG-787A [0597] SEQ ID NO: 238 Human PDGFR Receptor A transmembrane region peptide [0598] SEQ ID NO: 239 Human PDGFR Receptor B transmembrane region peptide [0599] SEQ ID NO: 240 Human Insulin-Like Growth Factor 1 transmembrane region peptide [0600] SEQ ID NO: 241 Human IL6-R transmembrane region peptide [0601] SEQ ID NO: 242 Human CD28 transmembrane region peptide [0602] SEQ ID NO: 243 Human IL-15 receptor alpha Sushi domain protein sequence [0603] SEQ ID NO: 244 Protein sequence of recombinant N-terminally His-tagged IL-15Ra sushi domain protein with C-terminal P2A peptide sequence [0604] SEQ ID NO: 245 DNA sequence of CMV pUC vector pUC-796A [0605] SEQ ID NO: 246 DNA sequence of CMV pUC vector pRES-128

EXAMPLES

EXAMPLE 1: Activity of Recombinant IL-15, IL-18 and IFN? Proteins in Primary Human Tumour Samples

[0606] To evaluate potential additive or synergistic activities of different combinations of cytokines and/or chemokines on primary tumour cell culture responses, recombinant proteins were used to model molecules encoded as transgenes in viruses. For effects on primary human tumour cells, surgically excised tumour samples were placed in Aqix organ transportation medium (supplemented with amphotericin B, penicillin, streptomycin, gentamycin and metronidazole) and shipped from the clinical site at 4? C. and obtained for processing in the laboratory within 24h. Samples were cut into small pieces using scalpels and then enzymatically dissociated using a tumour dissociation mix (Miltenyi Biotech) on a Gentle MACS tissue disruptor (Miltenyi Biotech). Single cell suspensions were obtained by filtering and plated into 96 well or 24 well plates, depending on the cell yield in either RPMI (Gibco) supplemented with foetal bovine serum, L-glutamine, sodium pyruvate and non-essential amino acids, or Cancer Cell Line Medium XF (PromoCell). Both media formulations were additionally supplemented with amphotericin B, penicillin and streptomycin. Cell types contained in these suspensions were routinely characterized by flow cytometry and shown to include tumour cells and different immune cell subsets, including T cells, B cells and NK cells.

[0607] Three primary tumour samples, tumour 63 (breast), tumour 64 (CRC) and tumour 65 (kidney) were received and processed as described above. Recombinant IL-12 p70 (15 ng/mL, R&D Systems), IL-18 (50 ng/mL, RnD Systems) and IL-15 (50 ng/mL, InvivoGen) were added either alone or in combination to the dissociated tumour cell cultures. Supernatant samples were harvested 72 hours after stimulation and were clarified as described previously. IFN? protein was quantified by ELISA. Both IL-12 and IL-15 individually stimulated IFN? production from tumour 64 and 65 cultures (but not 63), but the combination of IL-12+IL-15, or IL-12+IL-15+IL-18 stimulated the production of higher IFN? levels than these stimuli alone, including from tumour 63 where no other combination produced detectable levels (FIG. 1A). In these tumour cell cultures, recombinant IL-18 did not stimulate IFN? production and had little or no effect on the production of IFN? triggered by IL-15 or the combination of IL-12 and IL-15.

[0608] In a second experiment, a breast tumour sample (tumour 60) was dissociated and cultured as described above with recombinant proteins. Live cells were harvested 72h post infection by scraping the monolayer and pipetting gently. Cells were pelleted by centrifugation at 300?g before adding 200 ?L PBS containing 1 ?L of Live-Dead Fixable Aqua (Life Technologies) and incubating on ice in the dark for 10 minutes. Samples were then pelleted by centrifugation before adding a cocktail of antibodies targeting several cell surface proteins (CD45, CD3, CD4, CD8, CD56, CD107a and CD25) in 50 ?L cold PBS containing 2% FBS (flow buffer). Samples were incubated on ice in the dark for 20-30 minutes before being pelleted by centrifugation, washed twice in flow buffer and resuspended in flow buffer. Samples were then analysed by flow cytometry using an Attune NxT flow cytometer. Data showed that IL-15 alone, or in combination with IL-12 and IL-18, increased expression of the CD25 activation marker on CD4 and CD8 T cells, and NK cells (FIG. 1B). CD107a expression on NK cells was increased by the presence of IL-12, IL-15, IL-18 or combinations thereof. CD107a expression on CD4 and CD8 T cells was increased by the presence of IL-15, which was further enhanced by IL-12 or IL-12 plus IL-18. baseline (FIG. 1C).

[0609] In a third experiment, PBMCs from a healthy donor were cultured in RPMI (Gibco) supplemented with foetal bovine serum, L-glutamine, Na-Pyruvate and non-essential amino acids with the indicated recombinant proteins. Live cells were harvested 48 hours post stimulation and analysed by flow cytometry. However, in this case, 12 hours before harvesting, cells were treated with brefeldin A and an additional step of fixation/permeabilization was performed after the described above extracellular staining (using BD Cytofix/Cytoperm? kit), after which cells were incubated with anti-IFN? antibodies on ice in the dark for 30 minutes. Then cells were pelleted by centrifugation, washed twice in flow buffer and resuspended in flow buffer. Samples were analysed by flow cytometry using an Attune NxT flow cytometer. Data showed that IL-12 in combination with IL-15 increased the production of the IFN? by CD4 and CD8 T cells and by NK cells, and this was further enhanced by the presence of IL-18 (FIG. 1D).

EXAMPLE 2: Enhancement of T-Cell Mediated Killing of Target Cells by IL-12 with and without Other Recombinant Cytokines

[0610] To evaluate the potential for IL-12, IL-15, IL-18 and IFN? (alone or in combination) to enhance the T-cell mediated killing of target cell, we used a virus encoding a bispecific T cell activator (TAc), targeting both fibroblast activation protein (FAP) present on the surface of tumour-associated fibroblasts, and CD3 on T-cells (FAP-TAc) to drive T-cell mediated killing of FAP-expressing cells (Freedman et al, 2018 An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells. Cancer Res Nov 18:1-14; WO2018/041838 and WO2018/041827).

[0611] A549 cells were infected with 1ppc NG-617, which expresses the FAP-TAc (also described in the literature as a bispecific T-cell engager, BiTE). Supernatants were harvested 11 days post infection and clarified by centrifugation at 300?g for 5 minutes and then aliquoted and frozen at ?80? C. The FAP expressing lung fibroblast cell line MRC-5 was seeded into 96 well plates at a density of 1?10.sup.4 cells per well which were incubated at 37? C., 5% CO.sub.2 for 24 hours before staining with 5?M Caspase Green reagent (IncuCyte; Essen Bioscience). Media was removed and replaced with freshly thawed NG-617 treated cell supernatant containing FAP-TAc protein, along with T cells isolated from human PBMC donors at 6?10.sup.5 cells per well, and recombinant IL-12 p70 (15 ng/mL, R&D Systems) was added, either alone or in combination with IL-18 (50 ng/mL, R&D Systems), IL-15 (50 ng/mL, InvivoGen) and/or IFN?(1,500U/mL, InvivoGen). Plates were then incubated at 37? C., 5% CO.sub.2 for 24 hours in an IncuCyte live cell imager taking 4 images per well every 30 minutes. Data showed that IL-12 alone did not enhance FAP-TAc mediated T cell killing of MRC5 cells above that stimulated by FAP-TAc alone (observed as an increase in caspase positive MRC5 cells), while IL-12 and IL-15 together with or without IL-18 providing enhancement of killing (FIG. 2A). In the same experiment, IFN? further enhanced T-cell mediated killing with or without added cytokines (FIG. 2B).

[0612] In a second experiment, CD45+tumour infiltrating leukocytes (TILs) were isolated from tumour 70 using a TIL isolation kit (Miltenyi) on an AutoMACS cell isolator (Miltenyi). A T-cell mediated killing assay was then carried out as described above, using TILs in place of PBMC-derived T cells. Data showed that IL-12 alone did not enhance T cell killing of MRC5 cells above the FAP-TAc alone while IL-12+IL-15 and IL-12+IL-15+IL-18 did enhance killing, with IL-12+IL-15 providing the greatest enhancement (FIG. 2C). In the same experiment, IFN? enhanced killing when added to any of the combinations except IL-12 alone (FIG. 2D).

[0613] In another experiment, NK cells were isolated from PBMCs and pre-stimulated with IL-12 and/or IL-15 for 24 hours and then rested for 12 hours before incubating them with the low MHC class I expressing K562 cell line. K562 cells (target cells) were pre-labelled with Violet Cell tracker (ThermoFisher) according to the manufacturers protocol and then incubated at 37? C., 5% CO.sub.2 with NK cells at a 1:1 ratio (5?10.sup.4 cells per well in a 96-well plate) in the presence of 5?M Caspase Green reagent for 3 hours. Cells were then spun at 300?g for 5 minutes and washed twice with PBS and resuspended in flow buffer. Samples were analysed by flow cytometry using an Attune NxT flow cytometer and percentage of caspase-positive K562 cells were plotted (FIG. 2E). Data showed that both IL-12 and IL_15 can individually enhance NK-mediated killing, which is further increased by combining these two cytokines.

[0614] In a similar experiment, K562 cells were incubated as described above with TILs derived from a primary kidney tumour. Dissociated tumour cells containing ?16% NK as a proportion of total live cells were seeded in a collagen-coated 24-well plate and after 4 hours non-adherent cells (TIL-enriched cells) was transferred to another collagen-coated plate and stimulated or not with the indicated cytokines for 18 hours. Cells were then counted and incubated with labelled K562 target cells for 4 hours (ratio effectors:target cells 2:1 based on NK numbers at day 0). Primary tumour-infiltrating NK cells stimulated with IL-12 or IL-15 alone showed enhanced killing of K562 target cells, with a combination of the two cytokines giving a greater enhancement of killing (FIG. 2F).

EXAMPLE 3: Migration of Immune Cells in Response to Recombinant Chemokines

[0615] Several experiments were carried out to evaluate chemokine effects on the migration of the immune cell subsets in PBMC preparations as well as TILs isolated from primary tumour samples. Migration assays were carried out using the Transwell assay system (Corning), whereby chemokines were added to media in a 96 or 24 well plate, before adding a plate insert on top with a permeable membrane containing holes of 3 ?m in diameter, into which the cells were added. T cells and their subsets were magnetically isolated from PBMCs using MACS beads (Miltenyi), and TILs were isolated from tumour samples dissociated as described in Example 1. Media either alone, or containing CXCL9, CXCL10, CCL19 or CCL21 at 50 nM, was added into the bottom of Transwell plates, before adding na?ve T cells (100,000 cells per well) or effector CD8+ T cells (80,000 cells per well) into the upper compartment Plates were incubated at 37? C., 5% CO2 for 3 hours and 30 minutes before counting the cells in the lower compartment by flow cytometry using an Attune NxT (Thermofisher). All chemokines stimulated migration of both na?ve and effector T-cells above the medium only control, but more na?ve T cells were stimulated to migrate by CCL19 and CCL21 than by CXCL9 or CXCL10 (FIG. 3A), and the inverse was true for effector T cells, with CXCL9 and CXCL10 stimulated cells migrating in larger numbers (FIG. 3B).

[0616] In a second experiment, TILs isolated from a breast tumour (tumour 53) were rested for approximately 24h and then added to Transwell plates as described previously. All four chemokines stimulated migration above background (media only control), with CCL19 and CCL21 stimulating the migration of the largest number of TILs (FIG. 3C).

[0617] In a third experiment, leukocytes from dissociated primary lymph nodes removed as part of breast cancer surgery (same procedure used for primary tumours and described above in Example 1) were left to adhere on plastic in a flask (at 37? C., 5% C02) for 3 hours before removing non-adherent cells for the migration assay. A transwell migration assay was performed as described above in a 24-well plate, where 1.2?10.sup.5 cells were added to the upper compartment. Each condition was run in duplicate and cells in the bottom compartment after migration were counted and stained/analysed by flow cytometry using an Attune NxT (Thermofisher)(FIG. 3D). Data showed that both CXCL9 and CCL21 induced migration of lymph node-derived T cells (CD3+) and non-T cells (CD3-lymphocytes).

[0618] In another experiment, monocyte-derived dendritic cells from a healthy donor were prepared by culturing them with 50 ng/mL of recombinant GM-CSF plus IL-4 for 7 days and then matured with LPS for 24h before testing them in migration assays. Transwell assay was performed using 24-well plates, with inserts whose permeable membrane contains holes of 8?m in diameter, which were previously coated with 100 ?g/mL of Matrigel. Migration assay were then run for 6 hours at 37? C., 5% CO2 and analysed as described above. As shown in FIG. 3E, monocyte-derived dendritic cells strongly respond to both CCL19 and CCL21. An Incucyte chemotaxis assay was also performed following the manufacture's protocol using 96-well plates (Essen Bioscience) with 8?m-pore membrane coated with Matrigel (FIG. 3F). Cells on top of the membrane were scanned and quantified over time using the Chemotaxis analysis software. As shown in FIG. 3F, dendritic cell numbers in the top compartment decreased over time in response to recombinant CCL19 or CCL21 compared to media control, demonstrating their chemotactic response to these chemokines.

EXAMPLE 4: Cytokine-Mediated Enhancement of Tumour Ag-Specific Responses by Lymph Node Derived T-Cells from Breast Cancer Surgery

[0619] To assess if IL-12 and IL-15 cytokines can enhance an antigen-specific anti-tumour T cell response, an IFN? ELISpot assay with primary lymph nodes derived cells from breast cancer surgery was performed. After dissociation (as described above in Example 3), lymph node cells were seeded in a U-bottom 96-well plate and treated with either breast cancer-associated peptide pools, or CEFT (Clostridium tetani, Epstein-Barr virus (HHV-4), Human cytomegalovirus (HHV-5), Influenza A) peptide pool or just DMSO-containing media as controls. The following breast cancer-associated peptide pools (purchased from JPT) were used in this assay: MUC-1 (mucin-1), HER2-ECD (receptor tyrosine-protein kinase erbB-2-extracellular domain) and HER2-ICD (receptor tyrosine-protein kinase erbB-2-intracellular domain). After 1 hour, recombinant IL-12 or IL-15, or combination of those cytokines, was added to the cultures at the indicated concentrations and cells were left for 6 days at 37? C., 5% CO2. The day before the IFN? ELISpot assay, cells were rested (by removing cell culture media and replacing it just with complete media). On the day of ELISpot, cells were counted and plated (2.5?10.sup.4 per well) and boosted with the same peptides they were stimulated with. Also, at the same time, previously frozen cells (on the day of lymph node dissociation) from the same sample were thawed and added (1.85?10.sup.5 per well) as antigen presenting cells. IFN? ELISpot plates were developed according to the manufacture's protocol (Mabtech) and then spot numbers read using an ELISpot plate reader (CTL Europe GmbH). As shown in FIG. 4, IL-15 significantly increased T-cell responses to MUC-1 (FIG. 4A) and HER2 (FIG. 4B).

EXAMPLE 5: Production of Viruses Encoding Human IL-12 p35 and IL-12 p40 Either as Separate Proteins or Joined by a Flexible Linker

[0620] Three viruses (NG-701, NG-702, NG-703) were generated that differently encode human IL-12 transgenes (Table 1, FIG. 5A).

TABLE-US-00002 Table1 VirusID TransgeneCassette NG-701 SSA.sup.1-IL12p35.sup.2-P2A.sup.3-IL12p40.sup.4-PA.sup.5 (SEQIDNO:74) (SEQIDNO:233) NG-702 SSA.sup.1-IL12p40LinkerIL12p35.sup.6-PA.sup.5 (SEQIDNO:75) (SEQIDNO:234) NG-703 SSA.sup.1-IL12p35LinkerIL12p40.sup.7-PA.sup.5 (SEQIDNO:76) (SEQIDNO:235) .sup.1SEQ ID NO. 1; .sup.2SEQ ID NO. 9; .sup.3SEQ ID NO. 4; .sup.4SEQ ID NO. 10; .sup.5SEQ ID NO. 8; .sup.6SEQ ID NO. 11; .sup.7SEQ ID NO. 12;

[0621] In each transgene cassette, the cDNA encoding the IL-12 sequences was flanked at the 5 end with a short splice acceptor sequence (SSA, SEQ ID NO: 1-CAGG). At the 3 end of the IL-12 sequences, a SV40 late poly(A) sequence (PA, SEQ ID NO: 8) was encoded. In virus NG-701 the individual IL-12 p35 and IL-12 p40 sequences were linked with a P2A ribosome skipping sequence (SEQ ID NO: 4) to enable both IL-12 chains to be translated and produced as separate chains. In viruses NG-702 and NG-703 the IL-12 transgene encoded a single chain variant created by linking the sequences for the two individual p35 and p40 IL-12 chains with a sequence encoding a flexible linker (Gly.sub.4Ser).

Virus Production

[0622] The plasmid pColoAd2.4 (WO2015/097220) was used to generate the plasmids pNG-701, pNG-702 and pNG-703 by direct insertion of synthesised transgene cassettes encoding the transgene proteins. The pColoAd2.4 plasmid and transgene cassette were digested using AsiSI and SbfI restriction enzymes. Each digested transgene cassette was directly ligated into the digested pColoAd2.4 plasmid. The pNG-701 transgene cassette encodes for IL-12 p35 and IL-12 p40 as two separate transgene proteins (SEQ IDs NOs: 9 and 10), the pNG-702 transgene cassette encodes a single chain IL-12 molecule (SEQ ID NO. 11) comprising the IL-12 p40 protein covalently linked to the N-terminus of the IL-12 p35 protein with a (Gly.sub.4Ser).sub.3 linker (SEQ ID NO: 31) and the pNG-703 transgene cassette encodes a single chain IL-12 molecule (SEQ ID NO: 12) comprising the IL-12 p35 protein covalently linked to the N-terminus of the IL-12 p40 protein with a (Gly.sub.4Ser).sub.3 linker. Schematics of the transgene cassettes are shown in FIG. 5A. Construction of plasmid DNA was confirmed by restriction analysis and Sanger sequencing.

[0623] The plasmids, pNG-701, pNG-702 and pNG-703 were linearised by restriction digest with the enzyme AscI to produce the virus genomes. The viruses were amplified and purified according to methods given below.

[0624] Digested DNA was purified by phenol/chloroform extraction and precipitated for 16?2 hrs, ?20? C. in 600 ?l >95% molecular biology grade ethanol and 15 ?l 3M Sodium Acetate. The precipitated DNA was pelleted by centrifuging at 13000 rpm, 5 mins and was washed twice in 500 ?l 70% ethanol. The clean DNA pellet was air dried, resuspended in 500 ?l OptiMEM containing 15 ?l lipofectamine transfection reagent and incubated for 30 mins, RT. The transfection mixture was then added drop wise to a T-25 flask containing 293 cells grown to 70% confluency. After incubation of the cells with the transfection mix for approximately 2 hrs at 37? C., 5% CO.sub.2 4mls of cell media (DMEM high glucose with glutamine supplemented with 2% FBS) was added to the cells and the flasks was incubated 37? C., 5% CO.sub.2.

[0625] The transfected 293 cells were monitored every 24 hrs and were supplemented with additional media as required. The production of virus was monitored by observation of a significant cytopathic effect (CPE) in the cell monolayer. Once extensive CPE was observed the virus was harvested from 293 cells by three freeze-thaw cycles. The harvested viruses were used to re-infect 293 cells in order to amplify the virus stocks. Viable virus production during amplification was confirmed by observation of significant CPE in the cell monolayer. Once CPE was observed the virus was harvested from 293 cells by three freeze-thaw cycles. The amplified stocks of viruses were used for further amplification before the viruses were purified by double caesium chloride banding to produce purified virus stocks.

[0626] Viral Particle Infectivity & IL-12 p70 Quantification

[0627] A549 human lung adenocarcinoma cells were seeded in 12 well plates at a cell density of 7.5?10.sup.5 cells/well and infected with either EnAd, NG-701, NG-702 or NG-703 at 0.01 particles per cell (ppc). Plates were incubated at 37? C., 5% CO.sub.2 before harvesting cells and supernatants 3,4 or 7 days later. Supernatant samples were clarified by centrifugation at 300?g for 5 minutes. The cell fraction was obtained by adding RLT lysis buffer (Qiagen)+beta-mercaptoethanol (Sigma) to each well and pipetting to ensure maximal recovery. The cell fraction was then pooled with the pellet obtained during supernatant clarification and viral genomes were quantified for each time point by qPCR. Data demonstrated that each of the tested viruses produced similar quantities of viral genomes with similar kinetics (FIG. 5B). IL-12 p70 protein was quantified by ELISA from supernatant samples from the same experiment. ELISA data showed that all three viruses encoding IL-12 produced IL-12 p70 protein, with NG-701 and NG-702 producing more than NG-703 (FIG. 5C). A similar second study, using different ppc levels, RT-qPCR analysis demonstrated similar transgene mRNA expression by NG-701, NG-702 and NG-703 (FIG. 5D).

[0628] In a third experiment, A549 human lung adenocarcinoma cells were seeded in T175 flasks and infected with 10ppc of NG-701 or NG-702 at a cell density of 1.45?10.sup.7 cells/flask. Flasks were incubated at 37? C., 5% CO.sub.2 before harvesting cells and supernatant 72 hours later. Cells and supernatant were separated by centrifugation at 300?g, as described above. IL-12 p40 and IL-12 p70 proteins were quantified in supernatant samples by ELISA. Data showed that NG-701, encoding IL-12 p40 and IL-12 p35 as separate proteins, predominantly produced the IL-12 p40 protein with little IL-12 p70, while NG-702, encoding the two IL-12 subunits joined by a flexible linker, produced primarily the IL-12 p70 protein (FIG. 5E).

[0629] In another experiment, A549 human lung adenocarcinoma cells were seeded in T175 flasks and infected with 10ppc of NG-702 or NG-703 at a cell density of 2.6?10.sup.7 cells/flask. Flasks were incubated at 37? C., 5% CO.sub.2 before harvesting supernatant 72 hours later. Cells and supernatant were separated by centrifugation at 300?g, as described above. The functional activity of the produced IL-12 p70 protein was assessed using a HEK-Blue reporter cell line. HEK-Blue? IL-12 (InvivoGen) cells stably express the human IL-12 receptor and genes of the IL-12 signalling pathway along with a STAT4-inducible secreted alkaline phosphatase (SEAP) reporter gene. HEK-Blue IL-12 cells were seeded at 5?10.sup.4 cells per well in 96 well plates before being stimulated with supernatants or recombinant IL-12 (InvivoGen) at 100 ng/mL, followed by incubation at 37? C., 5% CO2 for 18-24 hours. Assay plates were centrifuged at 300?g for 5 minutes before removing clarified supernatant and transferring 20 ?L into a separate 96 well plate along with 180 ?L of Quanti-Blue reagent. The plate was incubated for 1 hour at 37? C. before analysing the plate on SpectraMax i3x plate reader with absorbance set to 620 nm. Data showed that supernatant from NG-702 infected cells led to the secretion of more SEAP from the reporter cells than supernatants from NG-703 infected cells (FIG. 5F).

[0630] In a further study, purified human CD4+ or CD8+ T-cells were activated with anti-CD3, anti-CD28 or both anti-CD3 and anti-CD28, or left unactivated, and cultured in the presence of recombinant human IL-12 (rhIL-12) or supernatants (SN) from NG-702 infected A549 cells for 6 days. Cells were then harvested and analysed by flow cytometry for the expression of NG-107a as a measure activation of cytotoxic T-cell effector function. NG-702 supernatants enhanced the expression of CD107a to a similar degree to that seen with rhIL-12 (FIGS. 5G&H).

EXAMPLE 6: Production of Viruses Encoding a Single Chain IL-12 Together with Other Transgenes

[0631] A set of viruses with transgene cassettes comprising a single chain IL-12 transgene as well as one or more additional transgenes were designed, produced and purified. Viruses NG-704, NG-706, NG-707, NG-708 and NG-709 were generated according to the methods of Example 5. For the remaining viruses (NG-720 and higher numbers), the pColoAd2.4 plasmid was digested using AsiSI and SbfI restriction enzymes and each synthesised transgene cassette was amplified by PCR using primers to add a 20 bp sequence to the 5 and the 3 ends of the amplified PCR product. The added sequences were complementary to sequences flanking the transgene cassette insertion site of the pColoAd2.4 plasmid and enabled direct assembly of the PCR amplified transgene cassette into the digested pColoAd2.4 plasmid. Subsequent steps were the same as described in Example 5. The viruses encoding a single chain IL-12 plus at least one other transgene are listed in Table 2 and illustrated in FIG. 6. For some virus preparations, smaller scale purifications were run using Optiprep (Iodixanol) density gradients, centrifuging at 155,000 g for 1 hour at 10? C., instead of using caesium chloride.

TABLE-US-00003 TABLE2 ViruseswithtransgenecassetteshavingasinglechainIL-12 (IL12p40LinkerIL12p35)plusoneormoreothertransgenes VirusID TransgeneCassette NG-704(SEQIDNO.77) SSA.sup.1-IL12p40LinkerIL12p35.sup.6-P2A.sup.3-IFNa.sup.8-PA.sup.5(SEQIDNO.36) NG-706(SEQIDNO.78) SSA.sup.1-IFNa.sup.8-P2A.sup.3-IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.37) NG-707(SEQIDNO.79) SSA.sup.1-Flt3L.sup.9-P2A.sup.3-MIP1a.sup.10-T2A.sup.11-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13-F2A.sup.14- IL12p40Linkerp35.sup.6-PA.sup.5(SEQIDNO.38) NG-708(SEQIDNO.80) SSA.sup.1-IL12p40Linkerp35.sup.6-P2A.sup.3-CCL21.sup.15-T2A.sup.11-CCL5.sup.16-E2A.sup.12- Flt3L.sup.9-PA.sup.5(SEQIDNO.39) NG-709(SEQIDNO.81) SSA.sup.1-IFNa.sup.8-P2A.sup.3-CCL19.sup.17-T2A.sup.11-IL18.sup.18-E2A.sup.12- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.40) NG-720(SEQIDNO.82) SSA.sup.1-IL12p40LinkerIL12p35.sup.6-P2A.sup.3-IL15.sup.19-T2A.sup.11-CXCL9.sup.13-E2A.sup.12- CCL21.sup.15-F2A.sup.14-IFNa.sup.8-PA.sup.5(SEQIDNO.41) NG-721(SEQIDNO.83) SSA.sup.1-IL12p40LinkerIL12p35.sup.6-P2A.sup.3-IL15.sup.19-T2A.sup.11-CXCL9.sup.13-E2A.sup.12- CCL21trunc.sup.20-F2A.sup.14-IFNa.sup.8-PA.sup.5(SEQIDNO.42) NG-722(SEQIDNO.84) SSA.sup.1-IL12p40LinkerIL12p35.sup.6-P2A.sup.3-CXCL9.sup.13-E2A.sup.12-IL15.sup.19-T2A.sup.11- CCL21.sup.15-F2A.sup.14-IFNa.sup.8-PA.sup.5(SEQIDNO.43) NG-723(SEQIDNO.85) SSA.sup.1-IL12p40LinkerIL12p35.sup.6-P2A.sup.3-CXCL9.sup.13-E2A.sup.12-IL15.sup.19-T2A.sup.11- IFNa.sup.8-F2A.sup.14-CCL21.sup.15-PA.sup.5(SEQIDNO.44) NG-724(SEQIDNO.86) SSA.sup.1-IL12p40LinkerIL12p35.sup.6-P2A.sup.3-CXCL9.sup.13-E2A.sup.12-IL15.sup.19-T2A.sup.11- IFNa.sup.8-F2A.sup.14-CCL21trunc.sup.20-PA.sup.5(SEQIDNO.45) NG-725(SEQIDNO.87) SSA.sup.1-IL12p40LinkerIL12p35.sup.6-P2A.sup.3-IL15.sup.19-E2A.sup.12-CCL21.sup.15-F2A.sup.14- CXCL9.sup.13-T2A.sup.11-IFNa.sup.8-PA.sup.5(SEQIDNO.46) NG-726(SEQIDNO.88) SSA.sup.1-CCL21.sup.15-E2A.sup.12-IL15.sup.19-F2A.sup.14-IL12p40LinkerIL12p35.sup.6-T2A.sup.11- CXCL9.sup.13-P2A.sup.3-IFNa.sup.8-PA.sup.5(SEQIDNO.47) NG-750(SEQIDNO.93) SSA.sup.1-CCL21.sup.15-P2A.sup.3-IL15.sup.19-T2A.sup.11-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13-F2A.sup.14- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.52) NG-751(SEQIDNO.94) SSA.sup.1-IL15.sup.19-P2A.sup.3-CCL21.sup.15-T2A.sup.11-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13-F2A.sup.14- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.53) NG-752(SEQIDNO.95) SSA.sup.1-CCL21.sup.15-T2A.sup.11-IL15.sup.19-P2A.sup.3-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13-F2A.sup.14- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.54) NG-753(SEQIDNO.96) SSA.sup.1-IL15.sup.19-T2A.sup.11-CCL21.sup.15-P2A.sup.3-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13-F2A.sup.14- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.55) NG-754(SEQIDNO.97) SSA.sup.1-CXCL9.sup.13-T2A.sup.11-CCL21.sup.15-P2A.sup.3-IFNa.sup.8-E2A.sup.12-IL15.sup.19-F2A.sup.14- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.56) NG-755(SEQIDNO.98) SSA.sup.1-IL15.sup.19-P2A.sup.3-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13-F2A.sup.14- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.57) NG-756(SEQIDNO.99) SSA.sup.1-IL15.sup.19-T2A.sup.11-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13-F2A.sup.14- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.58) NG-757(SEQIDNO.100) SSA.sup.1-CXCL9.sup.13-F2A.sup.14-IFNa.sup.8-E2A.sup.12-IL15.sup.19-T2A.sup.11- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.59) NG-758(SEQIDNO.101) SSA.sup.1-CXCL9.sup.13-F2A.sup.14-IFNa.sup.8-E2A.sup.12-IL15.sup.19-P2A.sup.3- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.60) NG-759(SEQIDNO.102) SSA.sup.1-IL12p40LinkerIL12p35.sup.6-T2A.sup.11-CXCL9.sup.13-F2A.sup.14-IFNa.sup.8-E2A.sup.12- IL15.sup.19-PA.sup.5(SEQIDNO.61) NG-760(SEQIDNO.103) SSA.sup.1-IFNa.sup.8-E2A.sup.12-CCL21.sup.15-T2A.sup.11-IL15.sup.19-P2A.sup.3-CXCL9.sup.13-F2A.sup.14- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.62) NG-761(SEQIDNO.104) SSA.sup.1-IL15.sup.19-E2A.sup.12-CCL21.sup.15-T2A.sup.11-IFNa.sup.8-P2A.sup.3-CXCL9.sup.13-F2A.sup.14- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.63) NG-762(SEQIDNO.105) SSA.sup.1-IL15.sup.19-P2A.sup.3-IFNa.sup.8-E2A.sup.12-IL12p40LinkerIL12p35.sup.6-PA.sup.5 (SEQIDNO.64) NG-763(SEQIDNO.106) SSA.sup.1-IL15.sup.19-T2A.sup.11-IFNa.sup.8-E2A.sup.12-IL12p40LinkerIL12p35.sup.6-PA.sup.5 (SEQIDNO.65) NG-764(SEQIDNO.107) SSA.sup.1-IL15.sup.19-F2A.sup.14-IFNa.sup.8-E2A.sup.12-IL12p40LinkerIL12p35.sup.6-PA.sup.5 (SEQIDNO.66) NG-765(SEQIDNO.108) SSA.sup.1-IL15.sup.19-P2A.sup.3-CXCL9.sup.13-F2A.sup.14-IL12p40LinkerIL12p35.sup.6-PA.sup.5 (SEQIDNO.67) NG-768(SEQIDNO.109) SSA.sup.1-CXCL9.sup.13-T2A.sup.11-CCL21mod.sup.23-P2A.sup.3-IFNa.sup.8-E2A.sup.12-IL15.sup.19-F2A.sup.14- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.68) NG-769(SEQIDNO.110) SSA.sup.1-CCL21tmod.sup.24-P2A.sup.3-IL15.sup.19-T2A.sup.11-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13- F2A.sup.14-IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.69) NG-770(SEQIDNO.111) SSA.sup.1-IL15.sup.19-P2A.sup.3-CCL21tmod.sup.24-T2A.sup.11-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13- F2A.sup.14-IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.70) NG-771(SEQIDNO.112) SSA.sup.1-CCL21tmod.sup.24-T2A.sup.11-IL15.sup.19-P2A.sup.3-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13- F2A.sup.14-IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.71) NG-772(SEQIDNO.113) SSA.sup.1-IL15.sup.19-T2A.sup.11-CCL21tmod.sup.24-P2A.sup.3-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13-F2A.sup.9- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.72) NG-773(SEQIDNO.114) SSA.sup.1-CXCL9.sup.13-T2A.sup.11-CCL21tmod.sup.24-P2A.sup.3-IFNa.sup.8-E2A.sup.12-IL15.sup.19- F2A.sup.14-IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.73) NG-774(SEQIDNo.212) SSA.sup.1-IL15.sup.25-T2A.sup.11-IFNa.sup.8-E2A.sup.12-IL12p40LinkerIL12p35.sup.6-PA.sup.5 (SEQIDNO.168) NG-775(SEQIDNO.213) SSA.sup.1-IL15.sup.25-P2A.sup.3-IFNa.sup.8-E2A.sup.12-IL12p40LinkerIL12p35.sup.6-PA.sup.5 (SEQIDNO.169) NG-776(SEQIDNO.214) SSA.sup.1-IL15.sup.26-T2A.sup.11-IFNa.sup.8-E2A.sup.12-IL12p40LinkerIL12p35.sup.6-PA.sup.5 (SEQIDNO.170) NG-777(SEQIDNO.215) SSA.sup.1-IL15.sup.26-P2A.sup.3-IFNa.sup.8-E2A.sup.12-IL12p40LinkerIL12p35.sup.6-PA.sup.5 (SEQIDNO.171) NG-781(SEQIDNO.216) SSA.sup.1-IL15.sup.25-T2A.sup.11-IFNa.sup.8-E2A.sup.12-CCL21.sup.28-F2A.sup.9- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.172) NG-782(SEQIDNO.217) SSA.sup.1-IL15.sup.25-T2A.sup.11-IFNa.sup.8-E2A.sup.12-CXCL9.sup.13-F2A.sup.9- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.173) NG-784(SEQIDNO.218) SSA.sup.1-CXCL9.sup.13-F2A.sup.9-IFNa.sup.8-E2A.sup.12-IL15.sup.25-T2A.sup.11- IL12p40LinkerIL12p35.sup.6-PA.sup.5(SEQIDNO.174) .sup.1SEQ ID NO. 1; .sup.3SEQ ID NO. 4; .sup.5SEQ ID NO. 8; .sup.6SEQ ID NO. 11; .sup.8SEQ ID NO. 13; .sup.9SEQ ID NO. 14; .sup.10SEQ ID NO. 15; .sup.11SEQ ID NO. 5; .sup.12SEQ ID NO. 6; .sup.13SEQ ID NO. 16; .sup.14SEQ ID NO. 7; .sup.15SEQ ID NO. 17; .sup.16SEQ ID NO. 18; .sup.17SEQ ID NO. 19; .sup.18SEQ ID NO. 20; .sup.19SEQ ID NO. 21; .sup.20SEQ ID NO. 24; .sup.23SEQ ID NO. 33; .sup.24SEQ ID NO. 35; .sup.25SEQ ID NO. 163; .sup.26SEQ ID NO. 165;

EXAMPLE 7: Production and Activity of Viruses Encoding Human IL-12 and Other Transgenes

Viral Particle Infectivity & Quantification of Transgene Protein Production

[0632] A549 cells were infected as described in Example 5, with either EnAd, NG-702, NG-704 or NG-706 at 100ppc. Supernatants and cells were harvested and clarified 24 or 48 hours later. Viral genomes were quantified for each time point by qPCR. Data demonstrated that each of the tested viruses produced similar quantities of viral genomes with similar kinetics (FIG. 7A). IL-12 p70 protein was quantified by ELISA from supernatant samples from the same experiment. ELISA data showed that all three viruses encoding single chain IL-12 produced IL-12 p70 protein, with NG-706 producing the most, and NG-704 the least (FIG. 7B). Quantification of IFN?? levels in the same supernatants by ELISA showed that NG-706 infected tumour cells produced less IFN? transgene protein than NG-704 infected cells (FIG. 7C).

[0633] In a second experiment, A549 human lung adenocarcinoma cells were seeded in 96 well plates at a cell density of 5?10.sup.4 cells/well and infected with either EnAd or NG-707 at 1ppc. Plates were incubated at 37? C., 5% CO2 before harvesting cells and supernatants 4 days later. Cells and supernatant samples were clarified and lysed as described previously. The cell fraction was analysed by RT-qPCR using primers targeting each of the encoded transgenes and data demonstrated that NG-707, but not EnAd, produced similar quantities of mRNA encoding each of the 5 encoded genes (FIG. 7D). IL-12 p70, Flt3 ligand (Flt3L), MIP1a, IFN? and CXCL9 transgene protein production was quantified by ELISA using supernatant samples from the same experiment ELISA data showed that NG-707 produced each of the 5 encoded proteins (FIG. 7E).

[0634] In a third experiment, A549 lung carcinoma cells were seeded and infected with NG-709, and supernatants harvested as described above. IL-12 p70, IFN?, MIP1a, CCL19 and IL-18 transgene protein production was quantified by ELISA, which showed that NG-709 produced each of the 4 encoded proteins (FIG. 7F).

[0635] In a fourth experiment, A549 lung carcinoma cells were seeded and infected with EnAd, NG-704, NG-706, NG-707 and NG-709 at 1ppc before harvesting and clarifying supernatants 72 hours later as described above. HEK-Blue IL-12 cells were seeded at 5?10.sup.4 cells per well in 96 well plates before being stimulated with supernatants (each pre-diluted 10-fold), or recombinant IL-12 (InvivoGen) at concentrations ranging from 100 to 1.6 ng/mL. Assay plates were incubated at 37? C., 5% CO2 for 20-24 hours before being centrifuged at 300?g for 5 minutes and removing clarified supernatant of which 20 ?L was then transferred into a separate 96 well plate along with 180 ?L of Quanti-Blue reagent Assay plates were incubated for 1 hour at 37? C. before analysing plates on a SpectraMax i3x plate reader with absorbance set to 620 nm. Data showed that NG-704 produced less functional IL-12 than the other IL-12 encoding viruses (FIG. 7G).

[0636] In a fifth experiment, A549 lung carcinoma cells were seeded and infected with NG-708 at 1ppc, and supernatants were harvested and clarified 96h later as described above. IL-12 p70, CCL21 and Flt3L transgene protein production was quantified by ELISA, which showed that NG-708 produced each of these 3 encoded proteins (FIG. 7H).

[0637] In a sixth experiment, A549 cells were seeded and infected with NG-708 or NG-709 at 10ppc, and supernatants were harvested and clarified after 72 hours as described above. IL-12, Flt3L, CCL19, CCL21, CCL5, IFN? and IL-18 transgene protein production was quantified by ELISA, which showed that NG-709 produced each of the 4 encoded proteins and NG-708 produced IL-12, CCL21 and Flt3L but CCL5 was not detected (ND) in this experiment (FIG. 7I).

[0638] In further experiments, other viruses depicted in FIG. 6 were used to infect A549 cells at 1ppc, and supernatants were harvested and clarified 96h later as described above and transgene protein production was quantified by ELISA. Levels of the different transgene proteins varied between different viruses, but IL-15 levels were consistently low or undetectable (FIG. 7J). For some of the viruses, some of the transgene protein levels were not determined (ND), or were tested but found to be below the lower limit of quantitation (LLOQ) of the assay.

EXAMPLE 8: Production of IL-12 p70 by Viruses in Primary Human Tumour Samples

[0639] To evaluate virus and transgene protein activities using primary human tumour cells, surgically excised tumour samples were placed in Aqix organ transportation medium (supplemented with amphotericin B, penicillin, streptomycin, gentamycin and metronidazole) and shipped from the clinical site at 4? C. and obtained for processing in the laboratory within 24h. In two separate experiments, a colorectal tumour (CRC, tumour 68) and a kidney tumour (tumour 70) sample were cut into small pieces using scalpels and then enzymatically dissociated using a tumour dissociation mix (Miltenyi Biotech) on a Gentle MACS tissue disruptor (Miltenyi Biotech). Single cell suspensions were obtained by filtering and plated into 96 well or 24 well plates, depending on the cell yield in either RPMI (Gibco) supplemented with foetal bovine serum and insulin-transferrin or Cancer Cell Line Medium XF (PromoCell). Both media formulations were additionally supplemented with amphotericin B, penicillin and streptomycin. Cell types contained in these suspensions were routinely characterized by flow cytometry and shown to include tumour cells and different immune cell subsets, including T cells, B cells and NK cells. Single cell suspensions obtained from each tumour sample were either left uninfected, or infected with EnAd, NG-702 or NG-704 at 1000ppc. Recombinant proteins (IL-12 p70 at 15 ng/mL, IL-15 at 50 ng/mL, IL-18 at 50 ng/mL) were also added either alone, in combination or excluded to cover different permutations. Wells containing viruses expressing IL-12 were not supplemented with recombinant IL-12 p70. Supernatant samples were harvested and clarified as described in Example 7 at 48h (tumour 68) or 96h (tumour 70) post infection. IL-12 p70 protein was quantified by ELISA from supernatant samples from both experiments. ELISA data showed that both viruses encoding IL-12 produced IL-12 p70 protein from tumour 70, with NG-702 producing the most, while only NG-702 produced quantifiable levels from tumour 68 (FIG. 8A). Supernatant samples from tumour 70 cultures were also analysed by ELISA for the presence of IFN? protein. The combination of IL-12+IL-15 stimulated the production of IFN? both with recombinant IL-12, and where IL-12 was produced by cultures infected with NG-702 or NG-704. IL-12 alone, recombinant or encoded by NG-702 or NG-704, did not stimulate the production of IFN?. In some cases, the addition of recombinant IL-18 to these cultures boosted IFN? production further (FIG. 8B). No IL-15 alone control was included in this experiment, however the study summarised in FIG. 1A demonstrated that IL-15 alone induces similar levels of IFN? to IL-12 alone, which in this study induced low or unquantifiable levels. The lower production of IFN? from NG-704 infected wells correlate with the lower level of IL-12 production as compared to NG-702 in this experiment (FIG. 8A).

[0640] In a second experiment, tumour 70 cells and those from an additional dissociated CRC tumour (tumour 71) were both cultured and infected with NG-707 at 1000ppc. Supernatant and cell samples were harvested 3, 4, 5, 7 and 10 days post infection. Cells and supernatant samples were clarified and lysed as described previously. IL-12 p70 protein was quantified by ELISA from supernatant samples. ELISA data showed that NG-707 produced IL-12 p70 protein from both tumour samples at all measured time points (FIG. 8C).

[0641] In a third experiment, two breast tumour samples (tumour 66 and 67) were dissociated as described previously, cultured and either left uninfected (UIC), or infected with EnAd, NG-702, NG-704, NG-707 or NG-709 at 1000ppc. Supernatant and cell samples were harvested 4 days post infection. Supernatant samples were clarified as described previously. IL-12 p70 protein was quantified by ELISA from supernatant samples. ELISA data showed that NG-707 and NG-709 produced detectable IL-12 p70 protein from both tumour samples, while NG-702 only produced detectable protein from tumour 67, and NG-704 failed to produce detectable protein from either tumour sample (FIG. 8D).

[0642] In a fourth experiment, a colorectal tumour sample (tumour 75) was dissociated and cultured as described earlier and either left uninfected (UIC) or infected with NG-707 at 1ppc or 100ppc. Supernatant samples were harvested 1, 4, 6, 8 and 11 days post inoculation, clarified as described previously and IL-12 p70 protein quantified by ELISA. ELISA data showed that NG-707 produced detectable IL-12 p70 protein from day 4 at 100ppc, and from day 8 with 1ppc (FIG. 8E). Note: IL-12 protein detected at day 4 in the uninfected control is likely due to low level production by immune cells present in the heterogeneous mix of cells present in the tumour samples.

[0643] In a fifth experiment, a colorectal tumour sample (tumour 76) was dissociated and cultured as described earlier and either left uninfected (UIC) or infected with NG-707 at 1ppc or 1000ppc. Supernatant samples were harvested 1, 4, 6, 8 and 11 days post inoculation, clarified as described previously and IL-12 p70 protein quantified by ELISA. ELISA data showed that NG-707 produced detectable IL-12 p70 protein from day 1 at 1000ppc, and from day 4 with 1ppc (FIG. 8F).

[0644] In a sixth experiment, a colorectal tumour sample (tumour 79) was dissociated and cultured as described earlier and either left uninfected (UIC) or infected with NG-707 at 1ppc or 1000ppc. Supernatant samples were harvested 1, 4, 6, 8- and 11-days post inoculation, clarified as described previously and IL-12 p70 protein quantified by ELISA. ELISA data showed that NG-707 produced detectable IL-12 p70 protein from day 4 at 1000ppc, with 1ppc not leading to the production of detectable IL-12 at any time point (FIG. 8G).

[0645] In a seventh experiment, a primary renal cell carcinoma sample (tumour 70) was dissociated as described earlier and either left uninfected (UIC) or infected with NG-707 at 100ppc or 1000ppc. Supernatant samples were harvested 3, 4, 5, 7- and 10-days post inoculation, clarified as described previously and IL-12 p70 protein quantified by ELISA. Data showed that NG-707 produced detectable IL-12 p70 protein at higher levels with 1000ppc than with 100ppc (FIG. 8H).

EXAMPLE 9: Activity of Viruses Encoding Human IL-15 Together with the IL-15 Binding Region of the Human IL-15 Receptor Alpha (IL-15Ra) in the Transgene Cassette

[0646] Functional signalling by IL-15 involves it binding first to IL-15Ra molecules which then together bind to cells bearing receptors comprising the common gamma chain (gc) and IL-2 receptor beta (IL-2Rb). To investigate the effect of encoding an IL-15 binding domain on the functional activity of an IL-15 transgene, a membrane-anchored form of the main IL-15 binding region of the IL-15Ra (the Sushi domain) was created by linking the sequence to the transmembrane region of the PDGF receptor via either a cMyc peptide or a Gly4Ser linker. These transgene sequences were used to create viruses NG-744 and NG-746, as well as NG-740 and NG-742 which also encode IL-15 (Table 3, FIG. 9A).

TABLE-US-00004 TABLE3 NG-740 SSA.sup.1-IL15RsushimycTM.sup.27- (SEQIDNO.89) P2A.sup.3-IL15.sup.19-PA.sup.5 (SEQIDNO.48) NG-742 SSA.sup.1-IL15RsushiG4STM.sup.28- (SEQIDNO.90) P2A.sup.3-IL15.sup.19-PA.sup.5 (SEQIDNO.49) NG-744 SSA.sup.1-IL15RsushiG4STM.sup.28- (SEQIDNO.91) PA.sup.5(SEQIDNO.50) NG-746 SSA.sup.1-IL15RsushimycTM.sup.27- (SEQIDNO.92) PA.sup.5(SEQIDNO.51) NG-748 SSA.sup.1-IL15Rsushi.sup.29-P2A.sup.3- (SEQIDNO.211) IL15.sup.19-PA.sup.5 (SEQIDNO.167) .sup.1SEQ ID NO. 1; .sup.3SEQ ID NO. 4; .sup.5SEQ ID NO. 8; .sup.19SEQ ID NO. 21; .sup.27SEQ ID NO. 25; .sup.28SEQ ID NO. 29; .sup.29SEQ ID NO. 26

[0647] Viruses were produced and purified using the protocol described in Example 6.

[0648] In a first experiment, NG-740 inoculation of A549 cells led to increased IL-15 production by ELISA compared to other viruses (NG-757, NG-758, NG-759) encoding an IL-15 transgene but no IL-15Ra (FIG. 9B).

[0649] To evaluate the effect on IL_15 production of expressing either transmembrane-anchored or soluble secreted forms of IL-15Ra sushi domain, a transfection approach was first undertaken using pUC vectors expressing gene products under control of a CMV promoter. Transfection of HEK-293 cells with an IL-15 pUC vector alone or together with a separate vector expressing either the soluble secreted or transmembrane anchored forms of IL-15Ra sushi domain showed that either form of IL-15Ra increased production of IL-15 as measured by ELISA (FIG. 9C) or using a functional reporter assay (FIG. 9D). This reporter assay used HEK-BlueIL-2 cells (InVivogen) which, because HEK293 cells constitutively express native IL-15Ra on their cell surface, respond to IL-15 as well as IL-2 to produce the secreted alkaline phosphatase reporter protein that is measured using a colorimetric enzyme assay.

[0650] A further virus, NG-748, was then constructed and prepared (as in Example 6) that produced a soluble (secreted) version of the IL-15Ra sushi domain together with IL-15 (Table 3, FIG. 9A). Inoculation of A549 cells (FIG. 9E) or a primary colorectal tumour cell sample (FIG. 9F) with either NG-740 or NG-748 showed that both led to production of IL-15, as measured by ELISA, with higher levels produced with NG-748 which encodes the soluble secreted IL-15Ra sushi domain.

[0651] In another study, primary kidney tumour cells (tumour 101) were treated with NG-748 or NG-702, in the presence of different levels of added recombinant IL-12 or IL-15, respectively. Production of IFNg by the primary TILs in the cultures was measured by ELISA of culture supernatants. The data (FIG. 9G) show a strong, dose-dependent synergy of IFNg induction by the IL-15 producing NG-748 virus with added IL-12, and a similar dose-dependent synergy of the IL-12 producing virus NG-702 with added IL-15.

EXAMPLE 10: Viruses Encoding IL-15Rsushi Designed to Secrete Both IL-12 and IL-15

[0652] Further viruses were designed to incorporated different forms of the IL-15Ra sushi domain together with both IL-12 and IL-15 transgenes. Viruses NG-785, NG-785A, NG-786A, NG-787 and NG-787A (Table 4; FIG. 10A) were produced and purified as described in Example 6.

TABLE-US-00005 TABLE4 NG-785 SSA.sup.1-IL12p40LinkerIL12p35.sup.6- (SEQIDNO.219) T2A.sup.11-IL15RsushimycTM.sup.27- P2A.sup.3-IL15.sup.19-PA.sup.5(SEQIDNO.175) NG-785A SSA.sup.1-IL12p40LinkerIL12p35.sup.6- (SEQIDNO.20) T2A.sup.11-IL15RsushimycTM.sup.27- P2A.sup.3-IL15.sup.19-PA.sup.5(SEQIDNO.176) NG-786A SSA.sup.1-IL-12p40LinkerIL12p35.sup.6- (SEQIDNO.221) T2A.sup.11-IL15Rsushi.sup.29-P2A.sup.3- IL15.sup.19-PA.sup.5(SEQIDNO.177) NG-787 SSA.sup.1-IL12p40LinkerIL12p35.sup.6- (SEQIDNO.222) T2A.sup.11-IL15Rsushi-Linker- IL15.sup.30-PA.sup.5(SEQIDNO.178) NG-787A SSA.sup.1-IL12p40LinkerIL12p35.sup.6- (SEQIDNO223) T2A.sup.11-IL15Rsushi-Linker- IL15.sup.30-PA.sup.5(SEQIDNO.179) .sup.1SEQ ID NO. 1; .sup.3SEQ ID NO. 4; .sup.5SEQ ID NO. 8; .sup.6SEQ ID NO. 11; .sup.11SEQ ID NO. 5; .sup.19SEQ ID NO. 21; .sup.27SEQ ID NO. 25; .sup.29SEQ ID NO. 26; .sup.30SEQ ID NO. 236

[0653] To assess transgene protein production by these viruses, A549 cells were infected with 1ppc of viruses and supernatants collected after 96h for testing in ELISA and functional reporter assays for IL-12 and IL-15. ELISA data showed good expression levels of IL-12 and IL-15 from all five viruses, with the exception of a very low level of IL-12 being produced by NG-785 (FIG. 10B). Testing different dilutions of these same supernatants in IL-12 and IL-15 functional reporter assays (as described in Example 5 and Example 9) generated data that aligned with the ELISA results, demonstrating functionality of the transgene proteins made (FIG. 10C).

[0654] In a second study, A549 cells left untreated or inoculated with 1ppc of EnAd, NG-740, NG-748 or the viruses listed in Table 4 and PBMCs added after 24h to assess the ability of cytokines produced by the viruses to stimulate immune cell responses. Supernatants were then collected after 72h and assessed by ELISA for levels of IL-12 and IL-15 transgene protein as well as IFNg produced by the added immune cells. The data showed that the five viruses expressing both IL-12 and IL-15 with IL-15Rsushi variants induced much higher IFNg production than NG-740 and NG-748 which only express IL-15 with IL-15Rsushi variants not IL-12 (FIG. 10D).

[0655] A further experiment, A549 cells were inoculated with 1ppc of NG-785A, NG-786A or NG-787A or controls and after 24 hours culture, human CD3+ T-cells (purified from PBMCs) were added and at 72 hours culture supernatants were assessed for IFNg levels by ELISA as a functional assessment of the transgene protein production by the viruses. All three transgene bearing viruses produced their encoded IL-12 and IL-15 proteins leading to the production of high levels of IFNg, indicating activation of the added T-cells, whereas the control virus EnAd did not (FIG. 10E).

[0656] A further experiment with these same three viruses compared culture conditions where the virus-inoculated A549 tumour cells were in contact with the added responder T-cells with culturing in a transwell plate where the T-cells are separated from the virus inoculated tumour cells to remove direct contact between them. This enabled a comparison of the T-cell activation achieved when the IL-15Rsushi transgene protein can move across the transwell filter along with the IL-12 and IL-15 transgene proteins (NG-786A) with the IL-15Rsushimyc? which cannot (NG-785A) as it stays in the membrane of the incoculated tumour cells. EnAd or EnAd with recombinant IL-12 and IL-15 added were used as comparator controls. Viruses were used at 1ppc and T-cells added to the two culture types 24h later. IFNg in the T-cell culture supernatants measured by ELISA shows that when the T-cells are separated from the transgene protein producing tumour cells, using a secreted version of the IL-15Ra sushi domain (NG-786A) led to higher levels of activation compared to the transmembrane IL-15Ra sushi domain form (NG-785A) and also higher than achieved without an IL-15Ra transgene as shown using EnAd inoculation with recombinant IL-12 and IL-15 (FIG. 10F).

[0657] In a similar study to that shown in FIG. 10D, either PBMCs or purified T-cells were added to assess IL-12/IL-15 mediated immune cell activation. The data again showed that all five transgene-bearing viruses tested led to activation of IFNg production (FIG. 10G).

EXAMPLE 11: Evaluation of Activity of Viruses IL-12, IL-15 and IL-5Rsushi in Primary Human Tumour Cell Cultures

[0658] Primary human tumour samples from two colorectal cancer patients were processed and cultured as described in Example 1. Replicate cultures were inoculated with 1000ppc of EnAd, NG-785A, NG-786A or NG-787A, or left untreated. After 72h (Tumour 98, Figure A) or 96h (Tumour 99, FIG. 11B), supernatants were removed and levels IFNg were measured by ELISA as a measure of activation of the endogenous tumour infiltrating lymphocytes in the tumour samples. As shown in FIG. 11, all the transgene-expressing viruses led to production of IL-12 and IL-15 by the primary tumour cells and all led to stimulation of IFNg production.

EXAMPLE 12: Generation and Characterization of IL-5Rsushi, IL-12 and IL-15 Viruses Additionally Expressing a Fourth Transgene

[0659] A further set of viruses having IFN?, CXCL9 or CCL21 as an additional one or two transgenes encoded in the transgene cassette along with IL-5Rsushi, IL-12 and IL-15 were designed produced as described in Example 6 (Table 5; FIG. 12A).

TABLE-US-00006 TABLE5 NG-788P SSA.sup.1-IL12p40LinkerIL12p35.sup.6-T2A.sup.11- (SEQIDNO.224) CCL21.sup.28-E2A.sup.12-IL15Rsushi.sup.29-P2A.sup.3- IL15.sup.25-PA.sup.5(SEQIDNO.180) NG-789P SSA.sup.1-IL12p40LinkerIL12p35.sup.6-T2A.sup.11- (SEQIDNO.225) CXCL9.sup.13-E2A.sup.12-IL15Rsushi.sup.29-P2A.sup.3- IL15.sup.25-PA.sup.5(SEQIDNO.181) NG-790P SSA.sup.1-IL12p40LinkerIL12p35.sup.6-T2A.sup.11 (SEQIDNO.226) -CXCL9.sup.13-F2A.sup.14-CCL21.sup.28-E2A.sup.12- IL15Rsushi.sup.29-P2A.sup.3-IL15.sup.25-PA.sup.5 (SEQIDNO.182) NG-791A SSA.sup.1-IL12p40LinkerIL12p35.sup.6-T2A.sup.11- (SEQIDNO.227) CXCL9.sup.13-E2A.sup.12-IL15Rsushi.sup.29-P2A.sup.3- IL15.sup.19-PA.sup.5(SEQIDNO.183) NG-792A SSA.sup.1-IL12p40LinkerIL12p35.sup.6-T2A.sup.11- (SEQIDNO.228) IFNa.sup.8-E2A.sup.12-IL15Rsushi.sup.29-P2A3_ IL15.sup.19-PA.sup.5(SEQIDNO.184) NG-794A SSA.sup.1-CXCL9.sup.13-E2A.sup.12-IL12p40Linker (SEQIDNO.229) IL12p35.sup.6-T2A.sup.11-IL15Rsushi.sup.29-P2A3_ IL15.sup.19-PA.sup.5(SEQIDNO.185) NG-795A SSA.sup.1-IL12p40LinkerIL12p35.sup.6-T2A.sup.11- (SEQIDNO.230) CCL21.sup.28-E2A.sup.12- IL15RsushimycTM.sup.27-P2A.sup.3-IL15.sup.19-PA.sup.5 (SEQIDNO.186) NG-796A SSA.sup.1-IL12p40LinkerIL12p35.sup.6-T2A.sup.11- (SEQIDNO.231) CCL21.sup.28-E2A.sup.12-IL15Rsushi.sup.29-P2A.sup.3- IL15.sup.19-PA.sup.5(SEQIDNO.187) NG-799A SSA.sup.1-CCL21.sup.28-E2A.sup.12-IL15Rsushi.sup.29- (SEQIDNO232) P2A.sup.3-IL15.sup.19-T2A.sup.11- IL12p40LinkerIL12p35.sup.6-PA.sup.5 (SEQIDNO.188) .sup.1SEQ ID NO. 1; .sup.3SEQ ID NO. 4; .sup.5SEQ ID NO. 8; .sup.6SEQ ID NO. 11; .sup.8SEQ ID NO. 13; .sup.11SEQ ID NO. 5; 12 SEQ ID NO. 6; .sup.13SEQ ID NO. 16; .sup.14SEQ ID NO. 7; .sup.19SEQ ID NO. 21; .sup.25SEQ ID NO. 163; .sup.27SEQ ID NO. 25; .sup.28SEQ ID NO. 17; .sup.29SEQ ID NO. 26

[0660] Viruses NG-788P, NG-794A, NG-795A, NG-796A and NG-799A were characterized for production of their transgene proteins, by specific ELISA assays, by infecting A549 cells, with uninfected (UIC) or EnAd infected A549 cells serving as controls. With the exception of NG-799A, all viruses made detectable levels of all their encoded transgene proteins, with IL-15 levels being notably higher with NG-794A and NG-796A (FIG. 12B). NG-799A did not make detectable levels of CCL21 and the level of IL-15 was also low.

[0661] In a similar second study, the same 5 viruses were tested alongside the viruses listed in Table 4, Example 10 to compare the levels of IL-15 transgene protein produced by viruses co-expressing IL-12 and IL-15 with IL-15Rsushi. The IL-15 ELISA data from the A549 cell culture supernatants (FIG. 12C) shows detectable IL-15 from all the viruses, with the highest levels from using NG-794A and NG-796A.

[0662] NG-794A and NG-796A were then tested for their effects on primary human tumour cell cultures established as described in Example 1 using a colorectal tumour sample (tumour 105) inoculated with viruses at 1000ppc and supernatants collected for cytokine analyses by ELISA after 48h. The data (FIG. 12D) show that both transgene-bearing viruses produced their respective transgenes and led to the activation of IFNg production.

[0663] In a similar study, a different colorectal tumour sample (tumour 107) was treated with NG-786A or NG-796A and compared to EnAd or no treatment. As shown in FIG. 12E, both transgene-bearing viruses produced their transgene proteins and led to activation of IFNg production by these primary tumour cells.

[0664] In a further study, another colorectal tumour sample (tumour 112) was treated with NG-786A, NG-791A, NG-794A or NG-796A and compared to EnAd or no treatment. As shown in FIG. 12F, all transgene-bearing viruses produced their transgene proteins and led to activation of IFNg production by these primary tumour cells.

[0665] The production of IFNg by PBMC-derived T-cells or primary tumour cell cultures served to test the functionality of the IL12 and IL-15/IL-15Rsushi transgene proteins. To demonstrate functionality of the CCL21 transgene product, monocyte-derived dendritic cells prepared from PBMCs (by culturing them with GM-CSF and IL-4 as described in Example 3) were stimulated with LPS for 24 before using in a transwell migration assay. The assay was run using an 8 mm pore-size transwell coated with 500 mg/mL Matrigel, as described in Example 3. Culture supernatants from uninfected A549 cells or A549 cells treated with EnAd or NG-795A were placed in the bottom well, with or without a blocking antibody to CCL21 (pre-incubated for 1 hour at room temperature) and dendritic cells added to the top wells. After 12h of culture, the numbers of dendritic cells that had migrated into the lower chamber were measured by flow cytometry. The data in FIG. 12G show that dendritic cell migration was increased selectively by the CCL21-containing supernatant from NG-795A and this was inhibited by the anti-CCL21 antibody, showing that the CCL21 transgene product is functional as a chemokine for dendritic cells. Similar data were also obtained from a repeat experiment where the dendritic cells were labelled with 1 mM Cell Tracer Violet (Invitrogen) after LPS stimulation and prior to use in the migration assay, and an isotype matched antibody was also included as an irrelevant antibody control to demonstrate the specificity of the migration inhibition by anti-CCL21 (FIG. 12H).

EXAMPLE 13: Production of IL-12 p70 by Viruses in In Vivo Mouse Studies

[0666] To systemically evaluate virus and transgene protein production and activities we set up in vivo study in which SCID mice were subcutaneously implanted in one flank with A549 cells (2 million cells mixed with Matrigel, 50:50 ratio). Once tumours reached a volume of ?200 m m.sup.3, mice were randomised into different groups (7mice per group) and treated intravenously on each of day 0, 2 and 5, with 5?10.sup.9 virus particles of EnAd, NG-786A, NG-791A or NG-796A viruses (or PBS in the no virus control). On day 7, a cocktail of labelled cells (effector memory and na?ve T cells isolated from PBMCs from healthy donorsusing Miltenyi specific kits) responsive to CXCL9 and CCL21 chemokine gradients was injected intravenously. After 48 hours tumour xenografts were analysed for T cell infiltration, transgene RNA expression and chemokine protein expression by ELISA and plasma samples were analysed for IL-12 transgene protein production by ELISA.

[0667] The data in FIG. 13 show that IL-12p70 was found in the blood of all mice treated with IL-12 transgene-encoding viruses, indicating that the viruses had infected tumour cells and expressed the encoded transgenes.

EXAMPLE 14: In Vitro Migration of Dendritic Cells in Response to CCL21

[0668] To demonstrate functionality of the CCL21 transgene produced by NG-796A virus, monocyte-derived dendritic cells prepared from PBMCs (by culturing them with GM-CSF and IL-4 as described in Example 3) were stimulated with LPS for 24 before using in a transwell migration assay as described for NG-795A in Example 12. The data in FIG. 15 show that dendritic cell migration was selectively increased by the CCL21-containing supernatant from NG-796A, compared to supernatants from uninfected (UIC) or EnAd infected cells and this was inhibited by the presence of anti-CCL21 blocking antibody (but not by its isotype control).

EXAMPLE 15: NG-704 and NG-796A Synergy with CAR-T Cells in an In Vivo Mouse Tumour Xenograft Model

[0669] To evaluate virus and transgene activities in a human tumor xenograft system, we set up an in vivo study in which NSG mice were subcutaneously implanted in one flank with 5?10.sup.6 A549 cells (HER-2 positive). Once tumours reached a volume of ?200 mm.sup.3, mice were randomised into different groups (5 mice per group) and treated intravenously on each of day 0, 3 with 5?10.sup.9 virus particles of EnAd, NG-704 or NG-796A viruses (or PBS in the CAR-T only control). On day 6, 1?10.sup.7 HER-2 specific CAR-T cells (ProMab) were injected intravenously. Tumours were measured twice per week and tumour growth was followed until the end of the study (?90 days). The data in FIGS. 16A&B show that CAR-T cells synergised with both NG-704 and NG-796A, respectively, leading to long-term clearance of the tumours. EnAd-treatment only delayed tumour growth compared to CAR-T cells only, indicating that the transgenes encoded by NG-704 and NG-796A are important for anti-tumour activity in the presence of tumour antigen (HER-2)-specific CAR-T cells

EXAMPLE 16: NG-796A Mediated Production of CCL21 in Human Tumour Xenografts

[0670] SCID mice were subcutaneously implanted in one flank with A549 cells (2 million cells mixed with Matrigel, 50:50 ratio). Once tumours reached a volume of ?200 mm.sup.3, mice were randomised into two different groups (8 mice per group) and treated intravenously on each of day 0, 1 and 3, with 5?10.sup.9 virus particles of EnAd or NG-796A viruses. On day 15, plasma and tumours were collected, tumours lysed and samples analysed for CCL21. by ELISA. The data in FIG. 16C show that CCL21 was found in all of the tumours from mice treated with NG-796A, indicating that the viruses had infected tumour cells and expressed the encoded transgenes. No CCL21 was detected in any plasma samples, indicating that a CCL21 chemokine gradient had been established in the tumours of NG-796A treated mice).

EXAMPLE 17: Production of IL15-Ra Sushi Domain in Tumour Cell Cultures Infected with NG-796A Virus

[0671] A recombinant IL-15Ra sushi domain protein, including the C-terminal P2A peptide and an added N-terminal His tag (SEQ ID NO: 244), was produced and purified by standard E. coli protein expression and purification techniques (Native Antigen Company, Oxford, UK). This recombinant protein was used as a standard in ELISAs for detecting IL-15Ra sushi domain production in transfection and virus infection experiments.

[0672] Secretion of IL-15Ra sushi domain (alongside the other encoded transgenes) in supernatants of A549 cells infected with NG-796A virus (or EnAd as a control) was characterised by ELISA (as described in Example 12) using a standard curve of recombinant IL-15Ra sushi domain protein. Results shown in FIG. 17 show that the IL-15Ra sushi domain protein is produced and secreted at similar levels to those of IL-15 cytokine.

EXAMPLE 18: IL15-Ra Sushi Domain Enhances IL-15 Secretion

[0673] To assess the role of IL-15Ra sushi domain and support its role in the NG-796A virus sequence, we performed transfection experiments using pUC vectors bearing transgene cassettes under control of a CMV promoter, following a similar approach to that described in Example 9. A549 cells were transfected with either pUC-796A (having the transgene cassette sequence of NG-796A; SEQ ID NO: 245) or pRES-128 (a version which lacks the IL-15Ra sushi domain sequence; SEQ ID NO: 246) (see FIG. 18B). Secreted IL-15 cytokine in supernatants was quantified by ELISA. Results shown in FIG. 18A show that encoding the IL-15Ra sushi domain for production by the virus infected cells markedly enhances IL-15 secretion. Addition of recombinant IL-15Ra sushi domain to the cultures, enabling it to interact with any IL-15 released by the cells, led to a small increase in detectable IL-15 indicating that in addition to promoting IL-15 secretion from the cells it may also increase IL-15 stability.