Oncolytic adenovirus encoding protein B7

Abstract

The present disclosure provides a replication-competent oncolytic adenovirus with selectivity for cancer cells, wherein the adenovirus comprises a transgene under the control of an endogenous viral promoter, wherein the transgene comprises a DNA sequence encoding a B7 protein or an active fragment thereof, compositions comprising the same, methods of generating the viruses, and use of the virus and compositions in treatment, particularly in cancer treatment.

Description

1 / 119 “ONCOLYTIC ADENOVIRUS ENCODING PROTEIN B7”

[0001] The present disclosure relates to an oncolytic adenovirus comprising a transgene encoding at least one B7 protein, such as CD80, or an active fragment thereof, compositions comprising the same, and use of the virus and compositions in treatment, particularly in the treatment of cancer. BACKGROUND

[0002] Cancer is still a major social burden in terms of the hardship and suffering of patients and their families, and also in terms of the high financial cost of treatment, care and support for patients. It is currently believed that the immune system of healthy individuals routinely clears cancerous cells. However, in these cancer patients, one or more of the defense mechanisms involved in this clearance is / are reduced or completely switched off.

[0003] It is now known that tumors change their microenvironment to make it more permissive to their growth. This occurs through the release, from the tumor, of extracellular signals that, for example, promote tumor angiogenesis and / or induce local immune suppression or immune tolerance.

[0004] It is clear from many different preclinical and clinical studies that the microenvironment within tumors can suppress the development and activity of antitumor immune responses, with a wide variety of mechanisms potentially demonstrating a role. Particular immunosuppressive mechanisms basically prevent T cell responses from mediating the extermination of tumor cells. Suppressive mechanisms may include preventing T cells from entering tumor tissues, inhibiting the activation of T cells that enter the tumor, and modulating tumor cell proteins, which reduces the ability of T cells to recognize or respond to them. The importance of such pathways Petition 870170082359, dated 10 / 26 / 2017, pp. 495 / 684 2 / 119 immunosuppressive agents in supporting tumor progression have been particularly highlighted by the clinical efficacy shown by antibodies to receptors in two suppressive pathways, CTLA4 and PD-1 / PDL1, which has led to market approval for the treatment of melanoma and other cancers.

[0005] B7 is a type of peripheral membrane protein found on activated antigen-presenting cells (APCs) that, when bound to a CD28 or CD152 surface protein (CTLA-4) on a T cell, can produce a co-stimulatory signal or a coinhibitory signal to enhance or diminish the activity of an MHC-TCR signal between the antigen-presenting cell (APC) and the T cell, respectively. In addition to being present on activated APCs, B7 can also be found on the T cells themselves.

[0006] There are several steps to the activation of the immune system against an antigen. The T cell receptor first interacts with a complex of its specific peptide antigen (Ag) bound to a major histocompatibility complex (MHC) surface protein. CD4 or CD8 proteins on the T cell surface interact with the MHC to help stabilize the MHC / Ag interaction with the T cell receptor complex, which comprises both antigen-binding chain dimers (alpha / beta or gamma / delta) and the CD3 signaling complex (comprising the gamma, delta, epsilon, and zeta chains). This is also called Signal 1, and its main purpose is to provide the initial signaling and ensure the antigen specificity of T cell activation.

[0007] However, MHC binding is insufficient on its own to stimulate differentiation and activation of a full effector T cell. In fact, the absence of additional stimulatory signals can leave the T cell anergic. The co-stimulatory signals needed to continue the immune response can come from B7-CD28 and CD40-CD40L interactions. There are other activation signals that play a role in immune responses. For example, in the TNF family of molecules, the 4-1BB (CD137) protein on the T cell Petition 870170082359, dated 10 / 26 / 2017, pp. 496 / 684 3 / 119 can be connected to 4-1 BBL on the APC.

[0008] The B7 (CD80 / B7-1 and / or CD86 / B7-2) protein is present on the surface of APCs, and it interacts with the CD28 receptor on the surface of the T cell. This is a source of Signal 2 (cytokines can also contribute to T cell activation, which can be termed Signal 3). This interaction produces a series of downstream signals that promote the survival of target T cells, activation, and differentiation into an effector cell that can mediate aspects of the immune response, such as the elimination of virus-infected cells or tumor cells, and the recruitment of inflammatory cells.

[0009] Generally, to initiate a T cell response, the stimulating signal and the co-stimulatory signal are provided by an antigen-presenting cell in order to induce both CD4 and CD8 T cell responses. But effector CD8 T cells recognize their antigen-associated MHC class I molecules that are present on most nucleated cells, including tumor cells. However, the present inventors have reason to believe that the signals to activate T cells do not need to come from the same cell or cell type. Thus, it would be useful to provide one or more of these signals (i.e., the stimulating signal and / or the co-stimulatory signal) to the immune system, for example, on the surface of a cancerous cell.

[0010] Currently, there is much interest in inhibiting PD-1 (programmed cell death protein)

[0011] 1) and / or its PDL1 ligand activity (also known as B7-H1) due to the fact that the pathway is considered to have an important role in the downregulation of immune responses, for example, in cancers.

[0012] However, some studies suggest that CD80 (B7-1) not only acts as a T cell co-stimulator by binding to CD28 on the T cell, but that it may also bind to PDL1, for example, when Petition 870170082359, dated 10 / 26 / 2017, pp. 497 / 684 4 / 119 expressed on the same cell membrane, and block inhibitory signaling interactions of PDL1-PD1. Thus, acting on two different pathways, CD80 may be a viable and potentially more useful molecule for restoring or enhancing human T cell activation. Soluble forms of CD80 also appear to have the capacity to counteract PDL1-PD1 mediated T cell inhibition, see, for example, Haile et al. Soluble CD80 Restores T Cell Activation and Overcomes Tumor Cell Programmed Death Ligand 1-Mediated Immune Suppression J Immunol 2013; 191:2829-2836. A CD80-Fc fusion protein has been generated and is being tested for safety and efficacy, see Journal of Immunology, 2014, 193:3835-3841.

[0013] The present inventors believe that B7 proteins or an active fragment thereof delivered and expressed by an oncolytic virus, for example, on the surface of a cancerous cell, would be useful in activating the patient's own system to fight cancer. Furthermore, B7 proteins, such as CD80, if simply administered systemically, have the potential to stimulate immune responses systemically in an undesirable way. The present inventors believe that a more sophisticated delivery of these proteins is necessary to create an appropriate therapeutic range, in which beneficial therapeutic effects are achieved and undesirable effects are minimized. SUMMARY OF REVELATION

[0014] Thus, an oncolytic adenovirus with selectivity for cancer cells is provided, wherein the adenovirus comprises a transgene under the control of an endogenous viral promoter, wherein the transgene comprises a DNA sequence encoding a B7 protein or an active fragment thereof. This is beneficial because the oncolytic virus according to the present disclosure preferentially infects cancer cells and thus penetrates the microenvironment created by cancer. Once in cancer cells, the virus-encoded B7 proteins can be expressed, for example, on the cell surface (i.e., Petition 870170082359, dated 10 / 26 / 2017, pp. 498 / 684 5 / 119 cancerous cell surface). This is advantageous because the B7 protein is then in the desired location where it can be biologically active.

[0015] In one embodiment, the encoded B7 protein comprises a sequence capable of anchoring the protein to the surface of a cell, for example, a transmembrane domain sequence, GPI anchor, or similar.

[0016] Thus, in one embodiment, the cancer cell is infected with a virus of the present disclosure that expresses a particular B7 protein or molecule on the surface of the cancer cell, wherein the B7 protein is suitable to provide at least the co-stimulatory signal, i.e., signal 2 to activate a T cell, and / or can bind to and inhibit the activity of PD-L1 expressed on the surface of the cancer cell or other cells in the local microenvironment.

[0017] In one embodiment, the B7 sequence comprises a transmembrane element of a

[0018] B7 protein, for example, a transmembrane element native to the particular B7 protein or a transmembrane domain of a “different” B7 protein from the one that is particularly expressed.

[0019] B7 proteins are surface-expressed proteins, and can also be used to carry additional proteins to the surface of cancer cells, for example, where at least the transmembrane domain of a B7 protein is attached to an additional protein.

[0020] Thus, in one aspect, a replication-competent oncolytic adenovirus is provided with selectivity for cancer cells, wherein the adenovirus comprises a transgene under the control of an endogenous viral promoter, wherein the transgene comprises a DNA sequence encoding a B7 protein or an active fragment thereof.

[0021] An oncolytic virus is also provided. Petition 870170082359, dated 10 / 26 / 2017, pp. 499 / 684 6 / 119 competent for replication according to claim 1, wherein the B7 protein or an active fragment thereof is independently selected from the group comprising B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7H5 and B7-H6, in particular, wherein the B7 protein is B7-1 (CD80) or an active fragment thereof.

[0022] In one embodiment, the oncolytic virus competent for replication is a group B adenovirus.

[0023] In one embodiment, the replication-competent oncolytic virus is a chimeric virus. In one embodiment, the replication-competent oncolytic virus has a main structure that is enadenotucirev (also called EnAd).

[0024] In one embodiment, the oncolytic virus competent for replication has a formula (I): 5'ITR-B1-BA-B2-BX-BB-BY-B3-3'ITR (I)

[0025] Bi includes: E1A, E1B or E1A-E1B;

[0026] Ba is E2B-L1-L2-L3-E2A-L4;

[0027] B2 is a linkage or comprises E3 or a transgene, for example, under an exogenous or endogenous promoter;

[0028] Bx is a linkage or a DNA sequence comprising: a restriction site, one or more transgenes, or both;

[0029] Bb includes L5;

[0030] By comprises a transgene that encodes a B7 protein or an active fragment thereof; and

[0031] B3 is a connection or comprises E4.

[0032] In one embodiment, the oncolytic virus competent for replication according to any one of claims 1 to 7, wherein the B7 protein or active fragment thereof comprises a transmembrane sequence, for example, a transmembrane domain of a PDGF receptor, or GPI anchor suitable for anchoring the protein or fragment to a cell membrane. Petition 870170082359, dated 10 / 26 / 2017, pp. 500 / 684 7 / 119

[0033] In one embodiment, the replication-competent oncolytic virus additionally comprises a second transgene, for example, encoding a polypeptide selected from the group comprising a cytokine, a chemokine, an antagonistic antibody molecule or fragment thereof, and an agonistic antibody molecule or fragment thereof.

[0034] In one embodiment, the second and third transgenes, for example, encoding two different polypeptides selected from the group comprising a cytokine, a chemokine, an antibody, such as an antagonistic antibody molecule or fragment thereof, or an agonistic antibody molecule or fragment thereof.

[0035] In one embodiment, the second or third transgene encodes a cytokine selected from the group comprising IL-2, IFN-alpha, IFN-beta, IFN-gamma, Flt3 ligand, GM-CSF, IL-15, and IL-12.

[0036] In one embodiment, the second or third transgene encodes a chemokine selected from the group comprising MIPI1α, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, and CCL21.

[0037] In one embodiment, a combination of a cytokine and a chemokine is encoded by the virus selected from the group comprising Mipla and Flt3 ligand, and Mipla and IFNa.

[0038] In one embodiment, the virus encodes an antibody molecule or fragment thereof, for example, comprising a transmembrane sequence or GPI anchor such that it is a cell membrane-anchored form or a transmembrane domain, for example, of a PDGF receptor. In another embodiment, the antibody molecule or binding fragment thereof comprises an anti-human CD3 antigen-binding domain.

[0039] In one embodiment, the antibody molecule Petition 870170082359, dated 10 / 26 / 2017, pp. 501 / 684 8 / 119 is an inhibitor, for example, selected from the group comprising an inhibitor of an angiogenesis factor, such as an anti-VEGF antibody molecule, and an inhibitor of T cell deactivating factors, such as an anti-CTLA-4 antibody molecule.

[0040] In one embodiment, the antibody molecule is an agonist, for example, from one or more selected from the group comprising CD40, GITR, 0X40, CD27 and 4-1 BB.

[0041] In one embodiment, an exogenous protein or virus-encoded proteins is / are a suitable form for expression on a cancerous cell surface. SUMMARY OF FIGURES

[0042] Figure 1 shows some of the main molecules involved in T cell recognition of antigen-presenting cells or tumor cells, and some of the signaling events induced in the responding T cell. The structure of PD1 and its interaction with the IgV domain of PD1 is also illustrated.

[0043] Figure 2 shows some of the B7 family ligands and CD28 family binding partners of receptors;

[0044] Figure 3 shows a schematic of transgene cassettes for viruses that express human CD80 (Figure 3A), that co-express human IFNα and human CD80 (Figure 3B), that co-express human OKT3 scFv and human CD80 (Figure 3C), that co-express human Flt3L, human MI1α and human IFNα (Figure 3D), that co-express human Flt3L, human MI1α and human CD80 (Figure 3E), that co-express human IFNα, human MI1α and human CD80 (Figure 3F) and a schematic of the open reading frame (ORF) or OKT3 scFv (Figure 3G);

[0045] Figure 4 shows replication of EnAd (ColoAdl) and human CD80-encoding virus NG-330 in HT-29 (Figure 4A) and A549 (Figure 4B) tumor cells; Petition 870170082359, dated 10 / 26 / 2017, pp. 502 / 684 9 / 119

[0046] Figure 5 shows CD80 expression on the membrane of A549 (Figure 5A) or HT-29 (Figure 5B) tumor cells by fluorescent immunostaining at different times after infection with NG-330; No CD80 expression on the cell membrane was observed with EnAd or uninfected tumor cells (UIC);

[0047] Figure 6 shows comparable oncolytic potencies of EnAd and NG-330 in an HT-29 cytotoxicity assay; thus, NG-330 retains its oncolytic properties while also carrying a transgene.

[0048] Figure 7 shows the comparable oncolytic potency of EnAd and CD80+ IFN α-expressing NG-343 virus (Figure 7 A) and IFNα secretion by NG-343-infected HT-29 and A549 tumor cells over a period of up to 72 hours;

[0049] Figure 8 shows CD80 expression and tumor cell kill at 48 or 72 hours post-infection by FACS analysis using anti-CD8 immunostaining along with a cell viability stain. CD80 could be detected on the cell surface of both live and dead NG-343 treated cells, but not EnAd or uninfected control A549 tumor cells (Figure 8A-D). Similar CD80 expression was seen with both HT-29 and A549 tumor cells (Figure 8E).

[0050] Figure 9 shows comparable virus replication with both EnAd and NG-343 in tumor cells (HT-29) and non-tumor cells (MRC5, WI38 and bronchial epithelial cells), with the latter showing much lower levels of replication (Figure 9A), IFN α secretion (Figure 9B) and CD80 expression (Figure 9C) following infection with NG343 detected only in HT-29 tumor cells;

[0051] Figure 10 shows that A549 tumor cells infected with NG-343 can induce increased surface levels of both Petition 870170082359, dated 10 / 26 / 2017, pp. 503 / 684 10 / 119 CD80 levels of PD-L1 on the surface of DCs in PBMC co-cultures, when compared to tumor cell culture infected with EnAd or not infected;

[0052] Figure 11 shows the expression of IFNα and CD80 by HT-29 Figure 11 A and C) and A549 tumor cells (Figure 11 B and C) infected with NG-347 virus;

[0053] Figure 12 shows the expression of MIPI1 α (Figure 12A), IFNa (Figure 12B) and Flt3L (Figure 12C) by A549 tumor cells infected with NG-345 virus;

[0054] Figure 13 shows comparable oncolytic potency (Figures 13 A and B) and infectivity (Figure 13 C) of EnAd, NG347 and NG-348 viruses in an HT-29 cytotoxicity assay;

[0055] Figure 14 shows high CD80 expression for 48 hours on the cell surface of A549 tumor cells infected with NG-347 or NG-348 virus, but little or no CD80 expression after infection with EnAd;

[0056] Figure 15 shows high CD80 expression for 48 hours on the cell surface of DLD-1 tumor cells infected with NG-347 or NG-348 virus, but little or no CD80 expression after infection with EnAd;

[0057] Figure 16 shows CD80 expression in EpCam+A549 cells infected with NG-348 and co-cultured with human CD3+ T cells, but not when the infection was with EnAd;

[0058] Figure 17 shows that CD25 is upregulated in human CD3+ T cells following co-culture with A549 cells infected with NG-348, but not when the infection was with EnAd (Figure 17A), with both the percentage of CD25+ cells (Figure 17B) and the level of CD25 expression per cell (Figure 17C) being increased;

[0059] Figure 18 shows CD25 is regulated in such a way Petition 870170082359, dated 10 / 26 / 2017, pp. 504 / 684 11 / 119 increasing in both the CD4+ and CD4- (mainly CD8) and CD3+ human T cell subset following co-culture with A549 cells infected with NG-348, but not when the infection was with EnAd;

[0060] Figure 19 shows low levels of HLA-DR expression in human CD3+T cells following co-culture with A549 cells infected with NG-348 or EnAd;

[0061] Figure 20 shows the induction of expression of CD107a on the surface of live CD3+T cells following co-culture with A549 cells infected with NG-348, but not when the infection was with EnAd;

[0062] Figure 21 shows expression induction of CD107a was found on the surface of both CD4+ and CD4- T cell subsets, as well as CD3+ T cells, following co-culture with A549 cells infected with NG-348, but not when the infection was with EnAd;

[0063] Figure 22 shows induction of IL2 (Figure 22A) and IFNγ (Figure 22B) production by CD3+ T cells following co-culture with A549 cells infected with NG-348, but not with IL-2, and only low levels of IFNγ when the infection was with EnAd;

[0064] Figure 23 shows production induction of IFNγ was produced by both CD4+ and CD8+ T cells (Figure 22B) and CD3+ cells following co-culture with A549 cells infected with NG-348, but there was no production (CD4+ cells) or the production was low (CD8+ cells) of IFNγ when the infection was with EnAd;

[0065] Figure 24 shows CD69 is upregulated in more human CD3+ T cells following co-culture with NG-347-infected A549 cells than when infection was with EnAd;

[0066] Figure 25 shows production induction of IFNγ by human CD3+ T cells following co-culture with A549 cells infected with NG-347, but not when the infection was with EnAd; Petition 870170082359, dated 10 / 26 / 2017, pp. 505 / 684 12 / 119

[0067] Figure 26 shows a schematic of the cassettes NG-348A, NG-420 and NG-420A transgene;

[0068] Figure 27 shows genome replication and exon gene expression (mRNA levels) for EnAd, NG-347 and NG-348 in MRC-5 fibroblast cells compared with A549 tumor cells;

[0069] Figure 28 shows CD80 transgene mRNA and anti-CD3 scFv and CD80 transgene protein (flow cytometry) for NG-348 virus in MRC-5 fibroblast cells compared with A549 tumor cells;

[0070] Figure 29 shows transgene mRNA from CD80 and CD80 transgene protein for NG-347 virus in MRC-5 fibroblast cells compared with A549 tumor cells;

[0071] Figure 30 shows mRNA and secreted protein levels of MlP1 α and IFNa generated by NG-347 virus in MRC-5 fibroblast cells compared with A549 tumor cells;

[0072] Figure 31 shows genome replication and exon gene expression (mRNA levels) for EnAd, NG-347 and NG-348 in purified human T cell cultures;

[0073] Figure 32 shows transgene mRNA from CD80 and scFv anti-CD3 and protein expression (flow cytometry) for NG-348 virus in human T cells compared with A549 tumor cells;

[0074] Figure 33 shows transgene mRNA from CD80 and CD80 transgene protein for NG-347 virus in purified human T cells compared with A549 tumor cells;

[0075] Figure 34 shows transgene mRNA from IFNa and MlP1a generated by NG-347 virus in T cells compared to A549 tumor cells;

[0076] Figure 35 shows genome replication of NG-347 and NG-348 and exon gene expression by human PBMCs in Petition 870170082359, dated 10 / 26 / 2017, pp. 506 / 684 13 / 119 comparison with A549 tumor cells;

[0077] Figure 36 shows CD80 and scFv anti-CD3 mRNA generated by NG-348 virus by PBMCs compared to A549 tumor cells;

[0078] Figure 37 shows CD80, IFNa and MlP1a mRNA generated by NG-347 virus by PBMCs compared to A549 tumor cells;

[0079] Figure 38 shows the similar activation of human dendritic cells by EnAd, NG-347 and NG-348 virus particles, as measured by downregulation of CD14 expression and upregulation of CD80 on the cell surface;

[0080] Figure 39 shows protein secretion MI1α and particle-mediated PBMCs grown with NG-348 (A) or NG-347 (B) compared with EnAd;

[0081] Figure 40 shows genome replication of NG-347 or NG-348 in cocultures of T cells or PBMCs with MRC-5 fibroblast cells compared with cocultures with A549 tumor cells;

[0082] Figure 41 shows INFy secreted by PBMCs or T cells co-cultured with MRC-5 fibroblast cells compared with A549 tumor cells, and treated with EnAd or NG-348 virus;

[0083] Figure 42 shows MIPI1α and IFNa secreted by human dendritic cells treated with EnAd, NG-347 or NG-348 virus particles;

[0084] Figure 43 shows the activation of NFKB and IFN reporter genes in JurkatDual reporter T cells co-cultured with A549 tumor cells infected with EnAd, NG-347 or NG-348;

[0085] Figure 44 shows the reporter activity of NF-κB luciferase generated by co-cultured JurkatDual reporter T cells Petition 870170082359, dated 10 / 26 / 2017, pp. 507 / 684 14 / 119 with A549 HCT-116, DLD and HT29 tumor cells treated with EnAd, NG-347, NG-348 or NG-420;

[0086] Figure 45 shows the reporter activity of NF-κB-luciferase generated by JurkatDual cells co-cultured with A549 or HT29 tumor cells infected with NG-348 and NG-420 viruses as a function of added virus particles;

[0087] Figure 46 shows the pharmacokinetics of EnAd and NG-348 virus in the blood; cytokine levels in the blood after exposure to EnAd or NG-348 virus; tissue biodistribution of EnAd or NG-348 virus 6 or 24 hours after IV administration in CD1 mice;

[0088] Figure 47 shows the pharmacokinetics in blood of EnAd, NG-347 and NG-348 viruses following IV administration to CB17-SCID mice bearing a subcutaneous HCT-116 tumor xenograft;

[0089] Figure 48 shows the tissue distribution of EnAd, NG-347, and NG-348 viruses 6 hours after intravenous dosing in CB17-SCID tumor-bearing mice and virus genomes in HCT-116 tumor xenografts on day 7 and days 14 to 21 following intravenous or intratumoral dosing of EnAd, NG-347, and NG-348;

[0090] Figure 49 shows viral exon mRNA generated in HCT-116 tumor xenografts by EnAd, NG-347, or NG-348 viruses on day 7 or 14 to 21 following intravenous or intratumoral dosing;

[0091] Figure 50 shows mRNA levels for CD80 exon and transgene in HCT-116 tumor xenografts 7 or 21 days after intravenous dosing with NG-348 virus;

[0092] Figure 51 shows mRNA levels for a transgene encoding ScFv anti-CD3 and CD80 in HCT116 tumor xenografts 7 or 14 to 21 days after IV dosing with NG-348 virus;

[0093] Figure 52 shows mRNA levels of MIPI1α IFNa transgenes in HCT-116 tumor xenografts 7 or 14 to 21 Petition 870170082359, dated 10 / 26 / 2017, pp. 508 / 684 15 / 119 days following intravenous dosing with NG-347 virus;

[0094] Figure 53 shows protein expression of CD80 in HCT-116 tumor xenografts 7 and 21 days after an intravenous dose of NG-348 virus; and shows expression of MIPI1α protein and CD80 in HCT-116 tumors after an intravenous dose of NG-347 virus; SUMMARY OF SEQUENCE LISTING

[0095] SEQ ID NO: 1 shows DNA sequence By corresponding to ae which includes bp 29,345 to 29,379 of the EnAd genome.

[0096] SEQ ID NO: 2 PDGF TM domain

[0097] SEQ ID NO: 3 JUNCTION ACCEPTOR SEQUENCE

[0098] SEQ ID NO: 4 JUNCTION ACCEPTOR SEQUENCE

[0099] SEQ ID NO: (posterior polyA sequence SV40) 5 Polyadenylation sequence

[00100] Inner Ribosome (IRES) SEQ ID NO 1: 6 High-efficiency autocleavable

[00101] Entry Sequence SEQ ID NO: 7 High-efficiency autocleavable

[00102] Peptide Sequence SEQ ID NO: 8 High-efficiency autocleavable

[00103] Peptide Sequence SEQ ID NO: 9 High-efficiency autocleavable

[00104] Peptide Sequence SEQ ID NO: 10 Peptide sequence T2A

[00105] SEQ ID NO: 11 Amino acid sequence CD80 humans

[00106] SEQ ID NO: 12 Human interferon amino acid sequence Petition 870170082359, dated 10 / 26 / 2017, pp. 509 / 684 16 / 119

[00107] SEQ ID NO: 13 Amino acid sequence of human soluble Flt3 ligand

[00108] SEQ ID NO: 14 Amino acid sequence of human macrophage inflammatory protein 1a (LD78b isoform)

[00109] SEQ ID NO: 15 Anti-human CD3 single-chain Fv membrane-anchored form

[00110] SEQ ID NO: 16 Genome sequence of NG-330 virus comprising the EnAd genome with a transgene cassette encoding the T-lymphocyte activating antigen, CD80, inserted in the By region. The transgene cassette contains a 5' SSA human CD80 cDNA sequence and a 3' poly(A)

[00111] SEQ ID NO: 17 Genome sequence of NG-343 virus comprising the EnAd genome with a transgene cassette encoding IFNα and CD80 inserted in the By region. The transgene cassette contains a 5' SSA IFNα cDNA sequence, P2A peptide, CD80 cDNA sequence, and a 3' poly(A)

[00112] SEQ ID NO: 18 Genome sequence of NG-345 virus comprising the EnAd genome with a transgene cassette encoding Flt3 ligand, MlP1a and IFNa, inserted in the By region. The transgene cassette contains a 5' SSA Flt3 ligand cDNA, P2A peptide sequence, MlP1a cDNA sequence

[00113] SEQ ID NO: 19 Genome sequence of NG-346 virus comprising the EnAd genome with a transgene cassette encoding Flt3 ligand, MlP1a and CD80, inserted in the By region. The transgene cassette contains a 5' SSA Flt3 ligand cDNA sequence, P2A peptide sequence, MlP1a cDNA

[00114] SEQ ID NO: 20 Genome sequence of NG-347 virus comprising the EnAd genome with a transgene cassette encoding IFNa, MlP1a and CD80, inserted in the By region. The transgene cassette Petition 870170082359, dated 10 / 26 / 2017, pp. 510 / 684 17 / 119 contains a 5' SSA IFNα cDNA sequence, a P2A peptide sequence, an MlP1α cDNA sequence, and T2A.

[00115] SEQ ID NO: 21 EnAd Genome

[00116] SEQ ID NO: 22 Genome region E2B of EnAd (BP 10355-5068)

[00117] SEQ ID NO: 23 REGION E3 OF EnAd

[00118] S ID NO: 24 A non-encoding sequence for inclusion in Bx

[00119] SEQ ID NO: 25 A non-encoding sequence for inclusion in By

[00120] SEQ ID NO: 26 to 34 Plug connector sequences

[00121] SEQ ID NO: 35 to 74 Flexible connector sequence

[00122] SEQ ID NO: 75 and 76 Rigid connector sequence

[00123] SEQ ID NO: 77 to 90 Connector sequence

[00124] SEQ ID NO: 91 PDGFR A Receptor

[00125] SEQ ID NO: 92 PDGFR B Receptor

[00126] SEQ ID NO: 93 Insulin-like growth factor 1

[00127] SEQ ID NO: 94 IL6-R

[00128] SEQ ID NO: 95 CD28

[00129] SEQ ID NO: 96 Virus genome sequence NG-348 comprising the EnAd genome with a transgene cassette encoding a membrane-anchored chimeric form of the single-chain anti-human Fv CD3e and the T-lymphocyte activating antigen, CD80, inserted into the By region.

[00130] SEQ ID NO: 97 Nucleic acid that encodes Petition 870170082359, dated 10 / 26 / 2017, pp. 511 / 684 18 / 119 scFv-OKT3 bound to the membrane

[00131] SEQ ID NO: 98 Transgene cassette sequence for NG-348

[00132] SEQ ID NO: 99 Anti-human CD3 scFv membrane-anchored form with C-terminal V5 tag

[00133] SEQ ID NO: 100 V5 Tag (9 amino acid variants)

[00134] SEQ ID NO: 101 Genome sequence of NG-348A virus comprising the EnAd genome with a transgene cassette encoding a membrane-anchored chimeric form of single-stranded anti-human Fv CD3e with C-terminal V5 tag and T-lymphocyte activating antigen, CD80 inserted in the region

[00135] SEQ ID NO: 102 Genome sequence of NG-420 virus comprising the EnAd genome with a transgene cassette encoding a membrane-anchored chimeric form of the single-stranded anti-human Fv CD3e inserted into the By region. The transgene cassette contains a 5' SSA

[00136] SEQ ID NO: 103 Genome sequence of NG-420A virus comprising the EnAd genome with a transgene cassette encoding a membrane-anchored chimeric form of the single-stranded anti-human Fv CD3e and a C-terminal V5 tag inserted in the BY region. The transgene cassette contains a

[00137] SEQ ID NO: 104 Linker

[00138] SEQ ID NO: 105 Sequence comprising a start codon

[00139] SEQ ID NO: 106 c-myc tag

[00140] SEQ ID NO: 107 c-myc label with amino acid spacer at the N and C terminus

[00141] SEQ ID NO: 108 PDGF domain of spacer TM - tag c-myc-spacer Petition 870170082359, dated 10 / 26 / 2017, pp. 512 / 684 19 / 119

[00142] SEQ ID NO: 109 Fully synthetic EnAd genome with embedded cloning site for transgene cassette insertion as in pEnAd2.4 plasmid DETAILED DESCRIPTION OF THE REVELATION

[00143] B7 is a family of proteins.

[00144] A B7 protein encoded in an oncolytic virus of the present discovery may be useful due to the fact that the extracellular domain of the protein family member generally modulates a biological function, for example, the extracellular domain of B7-1 may be employed to trigger or stimulate T cells. The actual biological function is specific to the extracellular domain of each given B7 protein (i.e., generally different protein members of the B7 family have different functions). Other functions of B7 proteins, such as B7-1 and / or B7-2, may include the ability to bind to CD28 and / or CTLA-4 and, in particular, to signal or activate the relevant signaling cascade or cascades.

[00145] Alternatively, the transmembrane domain of B7 proteins can be used to target proteins encoded by a virus of the present disclosure to the surface of a cancer cell, for example, by fusing the transmembrane domain to the C-terminus of the relevant protein.

[00146] The B7 protein, as used herein, unless the context indicates otherwise, refers to the full-length sequence of a protein from the B7 family or a sequence that is at least 95% similar or identical to it (such as 96%, 97%, 98%, 99%, or 100% similar or identical to it throughout the entire relevant sequence). The B7 family includes B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7H3, B7-H4, B7-H5, B7-H6, B7-H7. When the full-length protein is used, then at least one normal biological function of the protein will generally be present.

[00147] Full-length protein, Petition 870170082359, dated 10 / 26 / 2017, pp. 513 / 684 20 / 119 as employed in relation to the B7 family, refers to at least the extracellular domain, which includes chimeric B7 proteins in which the chimeric sequence has the structure and function of a B7 protein and in which the sequences constituting the chimera are selected from proteins in the B7 family. The elements in a full-length B7 protein fragment or fragment may be from the same or different B7 proteins. Thus, in one embodiment, the B7 protein or fragment is chimeric.

[00148] Chimeric B7 proteins, as used herein, refer to those where substantially all sequences constituting the chimera are from a single B7 protein, for example, at least 98% of the chimeric sequence are fragments of B7 proteins fused together. Thus, a chimeric fragment, as used herein, refers to a fragment comprising a sequence from two or more different B7 proteins.

[00149] In one embodiment, the full-length B7 protein comprises the extracellular domain, for example, of a single B7 protein, such as B7-1 and / or B7-2.

[00150] In one embodiment, the full-length B7 protein comprises the extracellular domain and the transmembrane domain, for example, of the same B7 protein or, alternatively, the extracellular domain of a B7 protein (such as B7-1 and / or B7-2) and a transmembrane domain or equivalent, such as a lipid membrane anchor, of a completely different protein.

[00151] In one embodiment, a full-length chimeric B7 protein may comprise an extracellular domain of a B7 protein (such as B7-1 and / or B7-2) and the transmembrane domain of a different B7 protein.

[00152] In one embodiment, the full-length B7 protein comprises the extracellular domain, the transmembrane domain, and the intracellular domain, for example, all of the same protein. Petition 870170082359, dated 10 / 26 / 2017, pp. 514 / 684 21 / 119 B7 or two or more different B7 proteins.

[00153] The active fragment of a B7 protein, as used herein, refers to a fragment that has at least one function of a B7 protein, for example, to facilitate expression on the surface of a cancerous cell or other biological function of a B7 protein.

[00154] In one embodiment, the fragment has at least 50% of the activity of the full-length protein, such as 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the activity of the full-length protein.

[00155] In one embodiment, the active fragment comprises or consists of an extracellular B7 domain or a sequence at least 95% similar or identical thereto, such as 96, 97, 98, 99 or 100% similar or identical.

[00156] In one embodiment, the B7 fragment comprises or consists of a transmembrane domain of a particular B7 protein, such as B7-1, described herein. Employing the latter is considered to contribute to expression on the cell surface.

[00157] In one embodiment, the active B7 fragment may be part of an extracellular domain. An active fragment, for example, a transmembrane fragment or a larger fragment comprising more B7 domains, may be employed in a fusion protein with an additional protein, for example, to facilitate the expression of the additional protein on the surface of a cancerous cell.

[00158] Larger fragment, as used in this document, does not refer to size or weight per se, but to a larger repertoire of sequence information (i.e., the fragment comprises sequences from at least two B7 domains) which, in turn, can provide more functionality.

[00159] In one modality, the larger fragment Petition 870170082359, dated 10 / 26 / 2017, pp. 515 / 684 22 / 119 comprises some relevant B7 protein activity. In one embodiment, an active B7 fragment is a fragment that retains the essential biological activity of the full-length protein, for example, the ability to trigger or activate T cells.

[00160] The activity of a given protein fragment can be analyzed in a relevant in vitro assay, for example, using a full-length protein as a comparator, for example, employing an assay described in the Examples in this document. When the active fragment is a transmembrane domain, the activity can be evaluated by analyzing the surface expression in cells of the relevant protein to which the transmembrane domain is attached, for example, using an assay described in the Examples in this document.

[00161] When the full-length B7 protein is part of a fusion protein, then the B7 portion may be linked to the additional protein by an amide bond between the end of one sequence and the beginning of the next protein sequence or connected by a linker. Examples of linkers are given below.

[00162] A full-length B7 protein comprising a transmembrane domain can be employed to present the extracellular domain of the B7 protein and the protein or fragment fused or bound to it onto the surface of the infected cancer cell. In general, in this embodiment, the B7 protein will be fixed to the surface of the cancer cell, and the other protein will be at the N-terminus and on the extracellular side of the cancer cell surface.

[00163] That said, proteins can be arranged as desired, for example, with the extracellular B7 domain at the N-terminus, fused or linked at its C-terminus to the next protein or fragment which, in turn, is fused or linked at the C-terminus to the transmembrane domain, for example, a transmembrane domain of a B7 protein.

[00164] Generally, when a protein of Petition 870170082359, dated 10 / 26 / 2017, pp. 516 / 684 23 / 119 full-length B7 is employed in a fusion protein, so both the B7 protein and the additional protein will have a biological function.

[00165] The fusion protein, as used in this document, refers to at least two proteins or fragments or a combination of at least one protein and at least one fragment that are directly fused or connected to each other, for example, by a linker.

[00166] Melt, as used in this document, generally refers to an amide bond between the end of one polypeptide (or protein / fragment) and the beginning of the next polypeptide (or protein / fragment).

[00167] Linked, unless the context indicates otherwise, refers to two entities, such as two polypeptide sequences that are connected via a linker. A linker is a sequence that is not naturally present in the polypeptide or a sequence that is not present at that particular position relative to both polypeptides. In one embodiment, the fusion protein comprises a B7 protein or an active fragment thereof. Fusion proteins comprising B7 protein or fragments and additional proteins are not referred to as chimeric proteins in this document. In general, the fusion protein, as used in this document, refers to a combination of a B7 protein or fragment thereof and another non-B7 protein / fragment.

[00168] Only proteins containing fragments of different B7 proteins are referred to as chimeric in this document, as described above. In one embodiment, the fusion proteins of the present disclosure do not comprise a B7 protein or an active fragment thereof and are encoded by a virus of the present disclosure in addition to the B7 protein or a fragment thereof.

[00169] Thus, viruses of the present disclosure may encode entities in addition to the B7 protein or an active fragment thereof, such entities include additional proteins. Petition 870170082359, dated 10 / 26 / 2017, pp. 517 / 684 24 / 119 B7 FAMILY

[00170] In one embodiment, B7 is independently selected from B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, active fragments thereof and combinations thereof. In one embodiment, the B7 protein is B7-1 (CD80), B7-2 (CD86) or an active fragment of any of the same and combinations thereof, in particular, B7-1 or an active fragment thereof.

[00171] B7 proteins include B7-1 (also known as CD80 uniprot number P33681), B7-2 (also known as CD86 uniprot number P42081). These proteins bind CD28 and CTLA-4.

[00172] In one mode, CD80 has the following sequence: MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHV TKEVKEVATLSCGHNVSVEELAQTRIY WQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVL KYEKDAFKRE HLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAI NTTVSQDPETE LYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAI TLISVNGIFVIC CLTYCFAPRCRERRNERLRRESVRPV SEQ ID NO: 11

[00173] Other B7 proteins include B7-DC (also known as PDCD1LG2 and PD-L2 uniprot number Q9BQ51), B7-H1 (also known as PD-L1 and CD274: uniprot number Q9NZQ7). Both proteins bind PD-1.

[00174] Programmed death ligand 1 (PD-Ll) is a 40kDa type 1 transmembrane protein that has been speculated to play a key role in suppressing the immune system. Apparently, upregulation of PD-Ll may allow cancers to escape the host immune system. An analysis of 196 tumor specimens of Petition 870170082359, dated 10 / 26 / 2017, pp. 518 / 684 A study of 25 / 119 patients with renal cell carcinoma found that high PD-Ll tumor expression was associated with increased tumor aggressiveness and a 4.5-fold increased risk of death. Ovarian cancer patients with higher PD-Ll expression had a significantly worse prognosis than those with lower expression. PD-Ll expression correlated inversely with intraepithelial CD8+ T lymphocyte count, suggesting that PD-Ll in tumor cells may suppress antitumor CD8+ T cells. The effect is likely tumor type dependent; a study in patients with non-small cell lung cancer showed that higher PD-Ll protein and mRNA expression are associated with increased local lymphocytic infiltrate and longer survival. Several anti-PD-Ll antibodies have shown promise in treating various cancers in clinical trials.

[00175] In one embodiment, the B7-DC and / or B7-H1 protein or a fragment thereof employed in the virus of the present disclosure does not stimulate immunosuppression, for example, it is modified to remove the immunosuppressive function.

[00176] Alternatively, a virus encoding the extracellular domain of B7-H1 in an unmodified form could be employed to treat appropriate cancers where upregulation of PD-L1 is associated with a good / improved prognosis, such as lung cancer. In one embodiment at least the cytoplasmic (intracellular domain) of B7-DC and / or B7-H1 is deleted or non-functional. While not wishing to be theory-bound, there is evidence suggesting that removal of the intracellular domain reduces the resistance of cancer cells to lysis Blood 2008, April 1; 111(7) 3635–3643.

[00177] In one embodiment, only the transmembrane domain fragment of B7-DC and / or B7-H1 is employed. In another embodiment, the following proteins are not provided as full-length B7-DC and B7-H1 proteins with relevant biological activity.

[00178] Other B7 proteins include B7-H2 (also Petition 870170082359, dated 10 / 26 / 2017, pp. 519 / 684 26 / 119 known as ICOSLG, B7RP1, CD275: uniprot number 075144) which binds ICOS, B7-H3 (also known as CD276: uniprot number Q5ZPR3), B7H4 (also known as VTCN1: uniprot number Q727D3), B7-H5 (also known as VISTA, Gi24 platelet receptor, SISP1), B7-H6 (also known as NCR3LG1, NR3L1) which binds NKp30, B7-H7 (also known as HHLA2) which binds CD28H.

[00179] In one embodiment, the fragment only comprises the transmembrane domain of any one of B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7.

[00180] Individual proteins include unique proteins, that is, proteins or active fragments thereof that are not part of a fusion protein (which includes chimeric proteins) and also fusion proteins. In one embodiment, individual proteins are unique proteins (which includes active fragments thereof).

[00181] In one embodiment, the cytoplasmic domain of the B7 protein is present. In another embodiment, the cytoplasmic domain is absent. The absence of the cytoplasmic domain may reduce or eliminate intracellular signaling to the cancer cell, which is relevant to one or more of the embodiments discussed below. TRANSMEMBRANE DOMAINS

[00182] In one embodiment, a transmembrane domain other than that derived from a B7 protein is employed to express a protein (including a fusion protein) encoded by a virus of the present disclosure on the surface of an infected cancer cell, for example, the transmembrane domain may be employed to present an active B7 protein fragment or other protein of interest on the surface of the infected cancer cell. Alternatively, the same may be employed to present a fusion protein, for example, comprising a B7 protein or an active fragment thereof on said surface. In one embodiment, the transmembrane domain of a receptor of Petition 870170082359, dated 10 / 26 / 2017, pp. 520 / 684 27 / 119 PDGF or a fragment thereof is used to express a B7 and / or other protein on the surface of a cancer cell.

[00183] In one embodiment, a transmembrane agglutination or anchor sequence employed in the present disclosure comprises a PDGFR TM domain (e.g., ala513-arg561), such as AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKPR (SEQ ID NO: 2).

[00184] In one embodiment, the agglutination or anchor sequence employed in the present disclosure comprises a label attached, for example, to a PDGF receptor or fragment thereof, such as the PDGFR TM domain, in particular, SEQ ID NO: 2.

[00185] Suitable tags include His tags, flag tags, c-myc tags and the like. More specifically, the aggregation or anchor may comprise a c-myc tag, for example, of SEQ ID NO: 106 EQKLISEEDL followed by a PDGFR TM domain that is employed, (for example, ala513-arg561), as shown in SEQ ID NO: 2 AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR.

[00186] In one embodiment, the c-myc tag comprises a spacer or spacer amino acids at the 3' and / or 5' end, for example, gsEQKLISEEDLn (SEQ ID NO: 107 wherein the lowercase letters represent the amino acids that are added to the tag as spacers).

[00187] In one embodiment, the agglutination or anchor sequence employed is gsEQKLISEEDLnAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ KKPR (SEQ ID NO: 108) where lowercase letters represent amino acid spacers).

[00188] In general, the protein / polypeptide to which the agglutination or anchor is attached does not contain a stop codon.

[00189] An exogenous protein or proteins Petition 870170082359, dated 10 / 26 / 2017, pp. 521 / 684 28 / 119 encoded by the virus according to the present disclosure will generally comprise a leader sequence (also called a signal peptide). A leader sequence is, for example, a sequence of about 5 to 30 amino acids in length located at the N-terminus of the protein or polypeptide.

[00190] In one embodiment, the leader sequence for the protein to be expressed on the surface of a cancerous cell is human, for example, HuVHSS.

[00191] In one embodiment, the structure of the ORF cassette is as follows: LS-POLY-TAG-TM_D

[00192] where

[00193] LS is a leader sequence, for example, a human leader sequence;

[00194] POLY is a polynucleotide that codes for polypeptide or proteins of interest, in particular, one disclosed in this document;

[00195] TAG is a label, for example, one disclosed in this document, such as c-myc, in particular, SEQ ID NO: 100 or 106;

[00196] TM_D is a TM domain, for example, a PDGFR TM domain, for example, SEQ ID NO: 2.

[00197] When the polypeptide is a scFv, then the ORF can be as follows: LS-VARi-LINK-VAR2-TAG-TM_D

[00198] where

[00199] LS is a leader sequence, for example, a human leader sequence;

[00200] VAR1 is a polynucleotide that codes for a variable region such as the VH region; Petition 870170082359, dated 10 / 26 / 2017, pp. 522 / 684 29 / 119

[00201] LINK is a linker, for example, as disclosed in this document, such as a linker based on G4S units, in particular, SEQ ID NO: 104 GGGGSGGGGSGGGGS;

[00202] VAR2 is a polynucleotide that codes for a variable region, such as a VL region;

[00203] TAG is a label, for example, one disclosed in this document, such as c-myc, in particular, SEQ ID NO: 100 or 106;

[00204] TM_D is a TM domain, for example, a PDGFR TM domain, for example, SEQ ID NO: 2.

[00205] The disclosure also extends to embodiments, in particular those specifically described herein, which comprise a tag on the N or C termini of the polypeptide chains, so that they remain inside or outside the membrane. Thus, a C-terminal tag located inside the membrane is advantageous because it is unlikely to interfere with the binding or function of the polypeptide.

[00206] That said, expressing the tag on the N-terminus of a surface-expressed protein can be useful in some situations because it can facilitate the isolation, identification, and purification of cells expressing the protein.

[00207] In one embodiment, a combination of a transmembrane domain and a secretion signal sequence is employed to express a virus-encoded protein (for example, as described in the present document) on the surface of an infected cancer cell. The present inventors have demonstrated that the encoded proteins are expressed only on cells that are permissive to infection by oncolytic viruses, i.e., cancer cells.

[00208] In one embodiment, the fragment used to express the protein on the surface of the infected cancer cell (such Petition 870170082359, dated 10 / 26 / 2017, pp. 523 / 684 30 / 119 (as the transmembrane fragment) is selected from approximately 20 to 25 hydrophobic amino acids that form a transmembrane alpha helix, for example, from proteins, which includes PDGF receptor, insulin-like growth factor receptor, IL-6 receptor, CD28, glycophorin, LDL receptor, influenza HA protein, insulin receptor, asialoglycoprotein receptor, transferrin receptor.

[00209] In one embodiment, the fragment used to express the protein on the surface of the infected cancer cell (such as the transmembrane fragment) is selected from the group comprising TM domain sequences (minimal portions) determined in SEQ ID NO: 91, 92, 93, 94 or 95: SEQ ID NO: Name SEQUENCE 91 PDGFR Receptor A AVLVLLVIVJJSLJVLVVJW 92 PDGFR Receptor B VV1SAILALVVLTIISLJ1LI 93 INSULIN-LIKE GROWTH FACTOR 1 IIIGPPLJFVFLFSVVIGSJYLFL 94 IL6-R SSSVP LPTFLVAGGS LAFGTI LG IAJVL 95 CD28 F WVLVWGG VLACYSI .LVTVAFIIFWV

[00210] In one embodiment, the transmembrane domain employed is derived from a G protein-coupled receptor or hepatitis B S antigen.

[00211] In one embodiment, the fusion protein comprising a full-length extracellular domain of a B7 protein or fragment and also a transmembrane domain derived from a protein other than B7 is arranged so that the B7 protein is located at the terminal end of the fusion protein distal to the cancer cell surface, which is on the outside of the cancer cell facing Petition 870170082359, dated 10 / 26 / 2017, pp. 524 / 684 31 / 119 to the extracellular space. VIRUS

[00212] Having the DNA sequence encoding a B7 protein or an active fragment under the control of an endogenous promoter is also advantageous because the protein is expressed according to the virus's life cycle as opposed to being expressed constitutively. In the present situation, continuous expression under an exogenous promoter, for example, a strong promoter like the CMV promoter, can produce more B7 protein than is needed for a therapeutic effect and may result in undesirable effects.

[00213] Alternatives to transmembrane domains for expressing proteins on the surface of infected cancer cells include approaches employing glycophospholipid anchors (also termed GPI anchors) attached to the C-terminal amino acid of the extracellular protein or fragment (Low et al 1986, Cross 1987, Low and Saltiel 1988, Ferguson and William 1988). Suitable glycophospholipid anchors for use in the present discovery include those of Thy-1, N-CAM, and DAF.

[00214] In one embodiment, the oncolytic virus according to the present disclosure is an adenovirus, for example, a group B adenovirus. In one embodiment, the virus according to the present disclosure is a chimeric virus, for example, EnAd. In one embodiment, the adenovirus is competent for replication.

[00215] In one embodiment, the virus is replication-deficient and supplied as a viral vector.

[00216] In one embodiment, the sequence encoding the B7 protein or its active fragment is located between the stop codon and the polyA recognition site of the L5 adenoviral gene and the stop codon and polyA recognition site of the E4 gene.

[00217] In one embodiment, the sequence encoding the B7 protein or its active fragment is located between approximately bp Petition 870170082359, dated 10 / 26 / 2017, pp. 525 / 684 32 / 119 29,356 and approximately 29,357 of the EnAd genome, for example, as shown in SEQ ID NO: 21, or an equivalent position. A knowledgeable individual will understand that the absolute numerical value of the location can change based on how the numbering is allocated. However, the relative position of the inserted gene remains the same regardless of the absolute numerical values ​​employed.

[00218] In one embodiment, the oncolytic adenovirus according to the present disclosure has a formula (I): 5'ITR-Bi-Ba-B2-Bx-Bb-By-B3-3'ITR (I)

[00219] where:

[00220] B1 is a link or comprises: E1A, E1B or E1A-E1B (in particular, E1A, E1B or E1A-E1B);

[00221] Ba is E2B-L1-L2-L3-E2A-L4;

[00222] B2 is a linkage or comprises E3 or a transgene, for example, under an exogenous or endogenous promoter;

[00223] Bx is a linkage or a DNA sequence comprising: a restriction site, one or more transgenes, or both;

[00224] Bb includes L5;

[00225] By comprises a transgene that encodes a B7 protein or an active fragment thereof; and

[00226] B3 is a link or comprises E4.

[00227] In one embodiment, the oncolytic virus has a formula (la): 5'ITR- Bi-Ba-B2-Bx-Bb-By-B3-3'ITR (la)

[00228] where:

[00229] B1 is a link or comprises: E1A, E1B or E1A-E1B (in particular, E1A, E1B or E1A-E1B);

[00230] Ba is E2B-Ll-L2-L3-E2A-L4;

[00231] B2 is a link or comprises E3;

[00232] Bb includes L5; Petition 870170082359, dated 10 / 26 / 2017, pp. 526 / 684 33 / 119

[00233] By comprises a transgene that encodes a B7 protein or an active fragment thereof; and

[00234] B3 is a link or comprises E4.

[00235] In one embodiment, the viral genome in constructs of formula (I) and / or (la) is Ad11 or EnAd, in particular, EnAd.

[00236] In one embodiment, the transgene encoding the B7 protein or its active fragment is under the control of an endogenous promoter, for example, the major posterior promoter. REGULATORY ELEMENTS

[00237] In one embodiment, By comprises a transgene cassette, wherein said cassette comprises a transgene encoding a B7 protein or fragment thereof and a regulatory element, such as a combination of regulatory elements.

[00238] In one embodiment, the regulating element is a join acceptor sequence.

[00239] In one embodiment, the regulating element is a Kozak sequence.

[00240] In one embodiment, for example, when the transgene encodes a polycistronic RNA molecule, the regulatory element is an IRES sequence.

[00241] In one embodiment, the regulatory sequence is a highly efficient self-cleaving peptide sequence such as P2A, T2A, F2A, E2A.

[00242] In one embodiment, the regulatory sequence is a pulley tail.

[00243] In one embodiment, there are at least two regulatory sequences, for example, a junction acceptor and a Kozak sequence, or a junction acceptor and a polyA tail, or a junction acceptor and an IRES sequence, or a junction acceptor and a P2A sequence. Petition 870170082359, dated 10 / 26 / 2017, pp. 527 / 684 34 / 119

[00244] In one embodiment, there are at least three regulatory sequences, for example, a junction acceptor sequence, a Kozak sequence and a polyA tail or a junction acceptor sequence, an IRES or 2A sequence and a polyA tail; or a junction acceptor sequence, a Kozak sequence and an IRES or 2A sequence.

[00245] In one embodiment, there are at least four regulatory sequences, for example, a junction acceptor sequence, a Kozak sequence, an IRES or 2A sequence and a polyA tail, in particular, located between L5 and E4 in the order junction acceptor sequence, Kozak sequence, IRES or 2A sequence and a polyA tail.

[00246] In one embodiment, the transgene encodes a polycistronic RNA molecule comprising both IRES and 2A regulatory sequences. VIRUS-ENCODED PROTEINS

[00247] In one embodiment, the virus of the present disclosure encodes multiple proteins for expression on the surface of the infected cancer cell wherein at least one is a B7 protein or an active fragment thereof, for example, two, three, four or more different proteins are encoded, in particular, two or three proteins are encoded by the virus for expression on the surface of a cancer cell or secretion into the extracellular space. The protein in this context includes a fusion protein. In another embodiment, the virus of the present disclosure encodes two different B7 proteins, active fragments thereof or combinations thereof, for example, both for expression on a cancer cell surface.

[00248] In one embodiment, the virus according to the present disclosure encodes one or two proteins for expression on the cell surface and one or two proteins that have the ability to be anchored on the cell surface, for example, if they are intended to act with the cancer cell or are for secretion / release from the cells.

[00249] In one embodiment, a B7 protein or Petition 870170082359, dated 10 / 26 / 2017, pp. 528 / 684 35 / 119 an active fragment of the same is encoded by the virus of the present disclosure for expression on the surface of the cancer cell and a soluble form, which is released or secreted from the cell, of the same B7 protein or a different B7 protein (which includes active fragments) is also encoded by the virus.

[00250] In one embodiment, at least two different B7 proteins or active fragments are encoded by a virus of the present disclosure.

[00251] In one embodiment, at least one protein expressed on the cell surface is a B7 protein and at least one protein not anchored to the cell (e.g., secreted) is a non-B7 protein.

[00252] In one embodiment, multiple proteins can be encoded to be expressed as separate proteins that are independently processed and expressed on the cancer cell membrane. The independence of proteins on the cancer cell surface may make a positive contribution to immune activation. While not wishing to dwell on the theory, lipid packing can influence the fluidity (i.e., viscosity) of the lipid bilayer in the cancer cell membrane. Membrane viscosity can affect the rotation and orientation of proteins and other biomolecules within the membrane, which affects the functions of these molecules.Thus, when the virus-encoded proteins are located as individual, separate proteins within the membrane of the infected cancer cell, the fluidity of the lipid bilayer allows independent movement of the molecules, which may have a particularly suitable shape, for example, similar to a natural shape that is conducive to biological function.

[00253] In one embodiment, independently processed and expressed proteins are located (anchored) in different locations, such as physically separate locations on the cancer cell membrane. Petition 870170082359, dated 10 / 26 / 2017, pp. 529 / 684 36 / 119

[00254] In one embodiment, one or more proteins (e.g., all proteins) encoded by the virus and expressed on the surface of the infected cancer cell are not fusion proteins.

[00255] As described above, in some embodiments, the proteins are expressed as a fusion protein.

[00256] In one embodiment, the virus of the present disclosure provides one or more separate independent proteins for expression on the cell surface and one or more fusion proteins for expression on the cell surface.

[00257] Thus, in one embodiment, the virus according to the present disclosure comprises DNA sequences encoding said multiple proteins for expression, for example, on the surface or infected cancer cell. Thus, in one embodiment, the virus according to the present disclosure comprises two or more transgenes, at the same or different locations in the viral genome. When located at the same position in the viral genome, the multiple proteins will still be expressed independently on the surface of the cancer cell.

[00258] In one embodiment, multiple proteins (including fusion proteins) are encoded at different locations in the viral genome, for example, at E3, Bx and / or By, and are expressed separately on the surface of the infected cancer cell.

[00259] In one embodiment, multiple proteins (including fusion proteins) are encoded at the same location in the viral genome and expressed together on the surface of an infected cancer cell, for example, when the encoded proteins are provided as a fusion protein, in particular, where the fusion protein comprises a B7 protein or an active fragment thereof.

[00260] In one embodiment, the B7 protein in the fusion protein is a full-length protein, in particular, a protein described herein, such as B7-1 and / or B7-2, fused or linked to Petition 870170082359, dated 10 / 26 / 2017, pp. 530 / 684 37 / 119 another protein of interest or an active fragment thereof. In one embodiment, the fusion protein comprises a transmembrane subunit of a B7 protein. In another embodiment, B7 is an active fragment that excludes the transmembrane domain. In the latter embodiment, a transmembrane subunit other than that derived from a B7 protein may be employed to ensure that the fusion protein is presented on the surface of the infected cancer cell.

[00261] In one embodiment, multiple proteins are encoded at the same location on the virus and are expressed as one or more fusion proteins that fuse together on the surface of the infected cancer cell.

[00262] When the location of the gene (or genes) encoding a protein (or proteins) of interest in the virus is the same, then the genes can, for example, be linked by an IRES sequence or a 2A peptide.

[00263] In one embodiment, the virus according to the present disclosure comprises a second transgene and, optionally, a third transgene (i.e., one or more of said multiple proteins, for example, encoding a polypeptide selected from the group comprising a cytokine, a chemokine, a ligand and an antibody molecule, such as an antagonistic antibody molecule, and an agonistic antibody molecule.

[00264] In one embodiment, the additional protein or proteins is / are independently selected from the group comprising an antibody, antibody fragment or protein ligand that binds CD3, CD28, CD80, CD86, 4-1 BB, GITR, 0X40, CD27, CD40 and combinations thereof, in forms suitable for expression on the surface of a cancer cell.

[00265] In one embodiment, the additional protein is an anti-CD3 antibody, for example, independently selected from a Petition 870170082359, dated 10 / 26 / 2017, pp. 531 / 684 38 / 119 Muromonab-CD3 (also known as OKT3), otelixizumab (also known as TRX4), teplizumab (also known as hOKT3yl(AlaAla)) or visilizumab.

[00266] In one embodiment, the anti-CD3 antibody is in the form of an antibody fragment, for example, an scFv that is part of a protein fusion with the transmembrane region of another protein, for example, the transmembrane domain of the PDGF receptor or the cell surface form of IgG. In one embodiment, an antibody molecule is an inhibitor (antagonistic antibody) that is independently selected from the group comprising an inhibitor of an angiogenesis factor, such as an anti-VEGF antibody molecule, and an inhibitor of T cell deactivating factors, such as an anti-CTLA-4, anti-PD1 or anti-PDL1 antibody molecule. In one embodiment, the antibody molecule is an agonist independently selected from the group comprising antibodies to CD40, GITR, OX40, CD27 and 4-1BB.

[00267] In one embodiment, an additional transgene encodes a cytokine, or soluble variant thereof, selected from the group comprising IL-2, IFNα, IFNβ, IFNγ, GM-CSF, IL-15, IL-12, and fms-related tyrosine kinase ligand 3 (FLT3L). Advantageously, one or more of this group of proteins expressed by the virus, in particular as a free protein secreted from the cancer cell, may be particularly suitable for stimulating an in vivo immune response to the cancer cell.

[00268] In one embodiment, an additional transgene encodes a chemokine selected from the group comprising MlPlalpha, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, and CCL21. Advantageously, one or more of this group of proteins is expressed by the virus as a free protein that can be secreted from the cancer cell and may be particularly suitable for attracting Petition 870170082359, dated 10 / 26 / 2017, pp. 532 / 684 39 / 119 immune cells and stimulate an immune response to the cancer cell in vivo.

[00269] In one embodiment, in addition to at least the B7 protein or an active fragment thereof expressed on the surface of the infected cancer cell, one or more molecules are also expressed on the surface and / or secreted.

[00270] Thus, in one embodiment, the virus encodes B7-1, B7-2, or an active fragment of either, or a combination thereof.

[00271] Thus, in one embodiment, the virus encodes B7-1, B7-2, or an active fragment of either or a combination thereof for expression on the surface of the infected cancer cell, and an anti-CD3 antibody (agonist) or binding antibody fragment (such as scFv) also for expression on the surface of the cancer cell, in particular, when the proteins are expressed as individual proteins on the cell surface.

[00272] Thus, in one embodiment, the virus encodes B7-1, B7-2, or an active fragment of either of them, or a combination thereof, for expression on the surface of the infected cancer cell, and an anti-VEGF antibody (antagonist) or a binding fragment thereof, also for expression on the surface of the cancer cell, or for release from the cancer cell, for example, by secretion or after lysis / death of the infected cancer cell.

[00273] Thus, in one embodiment, the virus encodes B7-1, B7-2, or an active fragment of either or a combination thereof for expression on the surface of the infected cancer cell and an antibody, antibody fragment, or protein ligand that binds CD3, CD28, CD80, CD86, 4-1BB, GITR, 0X40, CD27, CD40 also for expression on the surface of a cancer cell or for release from the cancer cell, for example, by secretion or release. Petition 870170082359, dated 10 / 26 / 2017, pp. 533 / 684 40 / 119 after lysis / death of the infected cancer cell.

[00274] Thus, in one embodiment, the virus encodes B7-1, B7-2, or an active fragment of either or a combination thereof for expression on the surface of the infected cancer cell and a cytokine selected from IL-2, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, IL-15, IL-12, and FLT3L, for example, for release from the cancer cell, in particular, by secretion or release after lysis / death of the infected cancer cell.

[00275] Thus, in one embodiment, the virus encodes B7-1, B7-2 or an active fragment of either or a combination thereof for expression on the surface of the infected cancer cell and a chemokine selected from MlPl-alpha, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21, for example, for release from the cancer cell, in particular, by secretion or release after lysis / cell death of the infected cancer cell.

[00276] Thus, in one embodiment, the virus encodes B7-1, B7-2, or an active fragment of either or a combination thereof for expression on the surface of the infected cancer cell and an anti-CD3 antibody (agonist) or binding antibody fragment (such as an scFv) also for expression on the surface of the cancer cell (in particular, when the proteins are expressed as individual proteins on the cell surface) and additionally encodes a cytokine or chemokine selected from IL-2, IFN-alpha, IFN-gamma, GM-CSF, IL-15, IL-12, FLT3L, MIP1-alpha, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21 for example, for release from the cancer cell, in Specifically, through secretion or after lysis / cell death of the infected cancerous cell.

[00277] Thus, in one form, the virus Petition 870170082359, dated 10 / 26 / 2017, pp. 534 / 684 41 / 119 encodes B7-1, B7-2 or an active fragment of either or a combination thereof for expression on the surface of the infected cancer cell and an anti-CD3 antibody (agonist) or antibody fragment (such as an scFv) also for expression on the surface of the cancer cell (in particular, when the proteins are expressed as individual proteins on the cell surface) and additionally encodes an antibody, antibody fragment or protein ligand that binds CD28, CD80, CD86, 4-1BB, GITR, 0X40, CD27, CD40 or an anti-VEGF antibody (antagonist) also for expression on the surface of the cancer cell or for release from the cancer cell, for example, by secretion or release after lysis / death of the infected cancer cell.

[00278] Thus, in one embodiment, the virus encodes B7-1, B7-2 or an active fragment of either or a combination thereof for expression on the surface of the infected cancer cell and two different cytokines or chemokines selected from IL-2, IFNα, IFNβ, IFNγ, GM-CSF, IL-15 and IL-12, FLT3L, MIα1α, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21, for example, for release from the cancer cell, in particular, by secretion after lysis / cell death of the infected cancer cell.

[00279] Thus, in one embodiment, the virus encodes B7-1, B7-2, or an active fragment of either or a combination thereof for expression on the surface of the infected cancer cell and an anti-CD3 antibody (agonist) or binding antibody fragment (such as an scFv) also for expression on the surface of the cancer cell (in particular, when the proteins are expressed as individual proteins on the cell surface) and additionally encodes a cytokine independently selected from IL-2, IFNα, IFNγ, GM-CSF, IL-15, and IL-12, and / or a chemokine selected from Petition 870170082359, dated 10 / 26 / 2017, pp. 535 / 684 42 / 119 from RANTES (CCL5), MI1α (isoforms LD78a (CCL3) or LD78P (CCL3L1)), MI1β which can be released from the cancer cell, in particular, by secretion before and release after lysis / cell death of the infected cancer cell. In one embodiment, in particular, it can be combined with any of the above embodiments, the virus additionally encodes an anti-PD-1 antibody (an antagonist).

[00280] In one embodiment, the protein or proteins encoded in the transgene cassette for expression on the cell membrane may also comprise a peptide linker or spacer between the 5 transmembrane domains and the extracellular ligand-binding domain. Such linkers or spacers may add flexibility to the cell surface-expressed protein, enhancing the protein's ability to interact with its target molecule, for example, in an adjacent cell. Such linkers or spacers may also be designed or selected to promote dimerization or trimerization of proteins on the cell surface through the formation of disulfide bonds or protein-protein interactions. For example, the binding regions of immunoglobulin or CD8 molecules may be employed to enhance both flexibility and dimerization.

[00281] In one embodiment, the protein or proteins encoded in the transgene cassette may also comprise a peptide tag. The peptide tag may include c-myc, polyhistidine, V5, or SIGNAL tags and may be located at the N- or C-terminus of the polypeptide, either intracellularly or extracellularly, or may be encoded within the protein, for example, in an extracellular loop or between the transmembrane domain and the extracellular domain. Peptide tags may be used as spacers or linkers between different protein domains, for example, the transmembrane and extracellular domains, and may be used for detection or purification of the protein, or cells expressing the protein.

[00282] In one embodiment, one or more additional transgenes (other than the gene encoding protein B7 or a fragment of Petition 870170082359, dated 10 / 26 / 2017, pp. 536 / 684 43 / 119 same) is under the control of an exogenous or endogenous promoter, for example, an endogenous promoter. In one embodiment, a transgene in the E3 (B2) region is under the control of an exogenous promoter.

[00283] In one embodiment, one or more additional transgenes are located between the E3 region and the L5 fiber in the adenovirus genome, for example, at position Bx in the construct of formula (I), in particular, under the control of an exogenous promoter, therefore, in one embodiment, a transgene e, Bx is under the control of an exogen.

[00284] In one embodiment, one or more additional transgenes are located between the E4 region and the L5 fiber in the adenovirus genome, for example, at a By position in the formula construct (I) or (la), in particular, under the control of an endogenous promoter, such as the major posterior promoter. This may additionally be the B7 protein or an active fragment thereof encoded in the By region.

[00285] In one embodiment, a composition is provided comprising an oncolytic adenovirus according to the present disclosure, for example, a pharmaceutical composition 35 comprising, in particular, a pharmaceutically acceptable excipient, such as a diluent or carrier.

[00286] In one embodiment, an oncolytic adenovirus according to the present disclosure or a composition comprising the same is provided for use in treatment, in particular, for use in the treatment of cancer. In another embodiment, a method of treating a patient in need thereof is provided, comprising administering a therapeutically effective amount of an oncolytic virus according to the present disclosure or a composition, such as a pharmaceutical composition comprising the same.

[00287] In one embodiment, use is provided of an oncolytic adenovirus according to the present disclosure or a composition comprising the same for the manufacture of a medicament for the Petition 870170082359, dated 10 / 26 / 2017, pp. 537 / 684 44 / 119 treatment of cancer, in particular carcinomas, for example, colorectal, lung, bladder, kidney, pancreatic, liver, head and neck, breast or ovarian cancer. In one embodiment, a polynucleotide is provided comprising a genomic sequence of at least 50% of a virus according to the present disclosure (for example, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) and comprising a sequence encoding a B7 protein or an active fragment thereof, for example, a B7 protein disclosed herein, such as B7-1 or an active fragment thereof. In one embodiment, the polynucleotide sequence is in the form of a plasmid.

[00288] In one embodiment, a host cell is provided, for example, a mammalian cell, such as an HEK293 cell or a derivative thereof, comprising an oncolytic virus according to the present disclosure or a polynucleotide sequence according to the present disclosure.

[00289] In one embodiment, a process is provided for preparing an oncolytic adenovirus according to the present disclosure comprising a step of inserting a polynucleotide encoding protein B7 or an active fragment thereof into an oncolytic adenovirus.

[00290] In one embodiment, a process for replicating a virus according to the present disclosure is provided, comprising the step of culturing host cells in the presence of the virus under conditions suitable for replication. In general, the method will comprise a further step of harvesting the virus, for example, from the supernatant or after lysis of the host cells. DEFINITIONS

[00291] The oncolytic virus with selectivity for cancer cells, as used in this document, refers to a virus that preferentially exterminates cancer cells, for example, Petition 870170082359, dated 10 / 26 / 2017, pp. 538 / 684 45 / 119 due to the fact that it preferentially infects cancerous cells and / or the viral life cycle is dependent on a gene, such as p53, which is deregulated, for example, overexpressed in cancerous cells. In one embodiment, the oncolytic virus preferentially infects cancerous cells and continues to replicate its genome and produce capsid proteins to generate new virus particles, for example, as by EnAd.

[00292] Selectivity for cancer cells (therapeutic index) can be tested as described in document WO2005 / 118825, incorporated herein by reference.

[00293] The transgene, as used herein, refers to a gene that has been inserted into the adenovirus genome sequence, wherein the gene is not native to the virus (exogenous) or not normally found at that particular location in the virus. Examples of transgenes are given herein. The transgene, as used herein, also includes a functional gene fragment, which is a portion of the gene that, when inserted, is suitable to perform the function or most of the function of the full-length gene, for example, 50% of the function or more.

[00294] The transgene and coding sequence are used interchangeably in this document in the context of insertion into the viral genome, unless the context indicates otherwise. The coding sequence, as used herein, means, for example, a DNA sequence that codes for a functional RNA, peptide, polypeptide, or protein. Typically, the coding sequence is cDNA for the transgene encoding the functional RNA, peptide, polypeptide, or protein of interest. The functional RNA, peptides, polypeptide, and proteins of interest are described below.

[00295] In one embodiment, the transgene, as used herein, refers to a segment of DNA containing a gene or cDNA sequence that has been isolated from an organism. Petition 870170082359, dated 10 / 26 / 2017, pp. 539 / 684 46 / 119 and is introduced into a different organism, that is, the virus of the present disclosure. In one embodiment, this non-native segment of DNA will generally retain the ability to produce functional RNA, peptide, polypeptide, or protein. The transgenes employed may, for example, encode a single protein or active fragment thereof, a chimeric protein, or a fusion protein.

[00296] Evidently, the viral genome contains DNA coding sequences. Endogenous (naturally occurring genes) in the viral genomic sequence are not considered a transgene in the context of this descriptive report, except if they have been modified by recombinant techniques, such as those in which they are in an unnatural location or in an unnatural environment.

[00297] Thus, in one embodiment, the inserted transgene encodes a human or humanized protein, polypeptide, or peptide.

[00298] In one embodiment, the transgene comprises a DNA sequence encoding a B7 protein or an active fragment thereof. The present disclosure ensures that the B7 protein or active fragment thereof can be provided in one or more independently selected formats from a fusion protein, a simple B7 protein, or an active fragment thereof.

[00299] The B7 single protein or an active fragment thereof, as employed herein, refers to proteins that are essentially wild-type proteins, i.e., not part of a fusion protein, and that have an identical or similar sequence to the relevant known protein, in particular, the known human protein. The single gene also includes the fact that 10% of the amino acids are substituted or deleted along the entire length of the relevant protein.

[00300] The GPI anchor, as used in this document, refers to a glycolipid that can be attached to the C-terminus of a protein during post-translational modification. It is Petition 870170082359, dated 10 / 26 / 2017, pp. 540 / 684 47 / 119 is composed of a phosphatidylinositol group linked via a carbohydrate-containing linker (glucosamine and mannose glycosidically linked to the inositol residue) and via an ascorbyl ethanolamine (EtNP) phosphate bridge to the C-terminal amino acid of a mature protein. The two fatty acids within the hydrophobic inositol-phosphatidyl group anchor the protein to the cell membrane.

[00301] Glypiated (GPI-linked) proteins generally contain a signal peptide, which directs them to the endoplasmic reticulum (ER). The C-terminus is composed of hydrophobic amino acids that are inserted into the ER membrane. The hydrophobic end is then cleaved and replaced by the GPI anchor. As the protein progresses along the secretion pathway, it is transferred through vesicles to the Golgi apparatus and finally to the extracellular space where it remains attached to the outer leaflet of the cell membrane. Since glipiation is the only means of attaching such proteins to the membrane, cleavage of the group by phospholipases will result in the controlled release of the protein from the membrane. The latter mechanism is used in vitro; that is, the membrane proteins released from the membranes in the enzymatic assay are glipiated proteins.

[00302] Phospholipase C (PLC) is an enzyme known to cleave the phosphoglycerol bond found in GPI-anchored proteins. Treatment with PLC will cause the release of GPI-bound proteins from the outer cell membrane. The T-cell marker Thy-1 and acetylcholinesterase, as well as intestinal and placental alkaline phosphatases, are known to be GPI-bound and released by PLC treatment. GPI-bound proteins are considered to be preferentially located on a lipid base, suggesting a high level of organization within plasma membrane microdomains.

[00303] A review of GPI anchors written by Ferguson, Kinoshita, and Hart is available in Chapter 11 of Essentials of Glycobiology 2nd Edition. VIRUS Petition 870170082359, dated 10 / 26 / 2017, pp. 541 / 684 48 / 119

[00304] Replication competent in the context of this descriptive report refers to a virus that possesses all the necessary machinery to replicate in cells in vitro and in vivo, that is, without the assistance of a packaging cell line. A viral vector, for example, deleted in at least the E1A region, with the capacity to replicate in a complementary packaging cell line is not a replication competent virus in this context.

[00305] A viral vector is a replication-deficient virus that requires a packaging cell line (comprising a transgene) to replicate.

[00306] A virus capable of replication, as used herein, refers to a virus competent for replication or a virus whose replication is dependent on a factor in cancer cells, for example, an upregulation factor such as p53 or similar.

[00307] In one embodiment, adenovirus is a human adenovirus. Adenovirus, serotype, or “adenoviral serotype,” as used herein, refers to any adenovirus that can be assigned to any of the 50 currently known adenoviral serotypes, which are classified into subgroups A to F, and additionally extends to any, and even unidentified or unclassified, adenoviral serotypes. See, for example, Strauss, Adenovirus infections in humans, in The Adenoviruses, Ginsberg, et al., Plenum Press, New York, NY, pages 451 to 596 (1984) and Shenk, Adenoviridae: The Viruses and Their Replication, in Fields Virology, Volume 2, Fourth Edition, Knipe, 35th ed., Lippincott Williams & Wilkins, pages 2265 to 2267 (2001), as shown in Table 1. Subgroup Serotype Adenoviral A 12, 18, 31 B 3, 7, 11, 14, 16, 21, 34, 35, 51 Petition 870170082359, dated 10 / 26 / 2017, pp. 542 / 684 49 / 119 Subgroup Serotype Adenoviral C 1,2,5,6 D 8 to 10, 13, 15, 17, 19, 20, 22 to 30, 32, 33 to 36 E 4 F 40,41

[00308] Adenoviruses are grouped based on their capsid.

[00309] In one embodiment, the adenovirus is a subgroup B, for example, independently selected from the group comprising or consisting of: Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34 and Ad51, such as Ad11, in particular, Ad11p (the Slobitski strain). In one embodiment, the adenovirus of the invention has the capsid, such as the exon and / or fiber of a subgroup B adenovirus, such as Ad11, in particular, Ad11p. In one embodiment, the adenovirus is Ad11 or has the fiber and / or exon and / or penton of Ad11, such as Ad11p.

[00310] In one embodiment, the virus of the present disclosure is not a Group A virus.

[00311] In one embodiment, the virus of the present disclosure does not comprise an adenovirus death protein (ADP).

[00312] In one embodiment the virus of the present disclosure is not a group C virus.

[00313] In one embodiment, the virus of the present disclosure comprises no more than a fragment of part of an Ad5 virus.

[00314] Enadenotucirev (EnAd) is a chimeric oncolytic adenovirus, formerly known as ColoAdl (document no. WO2005 / 118825), with fiber, penton, and exon of Ad11p; therefore, it is a subgroup B virus. It has a chimeric E2B region comprising DNA from Ad11p and Ad3. Almost all of the E3 region and part of the E4 region is deleted in EnAd. Therefore, it has significant space in the genome to accommodate this. Petition 870170082359, dated 10 / 26 / 2017, pp. 543 / 684 50 / 119 additional genetic material while remaining viable. Furthermore, due to EnAd being a subgroup B adenovirus, pre-existing immunity in humans is less common than, for example, Ad5. Other examples of chimeric oncolytic viruses with Ad11 fiber, penton, and exon include OvAd1 and OvAd2 (see document WO2006 / 060314).

[00315] EnAd appears to preferentially infect tumor cells, replicate rapidly in these cells, and cause cell lysis. This, in turn, can generate inflammatory immune responses that also stimulate the body to fight cancer. Part of EnAd's success is hypothetically related to the virus's rapid replication in vivo.

[00316] It is important to note that EnAd has been clinically demonstrated to be able to be administered systemically (e.g., by intravenous injection or infusion) and then subsequently selectively infect and express proteins in tumor cells. This property of EnAd, which may be shared by Ad11p and other group B adenoviruses, in particular those expressing Ad11p capsid proteins (such as those described herein), makes it possible to express proteins on the surface of cancer cells without having to directly inject the transgenes into the tumor, which is not feasible for many cancers.

[00317] Although EnAd selectively lyses tumor cells, it may be possible to introduce additional beneficial properties, for example, by increasing the therapeutic activity of the virus or reducing side effects of the virus by arming it with transgenes, such as a transgene that encodes a cell signaling protein or an antibody, or a transgene that encodes an entity that stimulates a cell signaling protein (or proteins).

[00318] By advantageously arming a virus with DNA that encodes certain proteins that can be expressed within the cancer cell, it may be possible to enable the body's own defenses to be employed to fight tumor cells more effectively, for example, Petition 870170082359, dated 10 / 26 / 2017, pp. 544 / 684 51 / 119 making the cells more visible to the immune system or delivering a therapeutic gene / protein preferentially to target tumor cells.

[00319] In one embodiment, the oncolytic adenovirus of the present disclosure stimulates the patient's immune system to fight the tumor, for example, by reducing the cancer's ability to suppress immune responses.

[00320] In one embodiment, the oncolytic virus has fiber, exon and penton proteins of the same serotype, for example, Ad11, in particular, Ad11p, for example, found at positions 30,812 to 31,789, 18,254 to 21,100 and 13,682 to 15,367 of the genomic sequence of the latter where the nucleotide positions are relative to Genbank ID 217307399 (accession number: GC689208).

[00321] In one embodiment, the adenovirus is enadenotucirev (also known as EnAd and formerly as ColoAdl). Enadenotucirev, as used herein, refers to the chimeric adenovirus of SEQ ID NO: 21. It is a replication-competent oncolytic chimeric adenovirus that has enhanced therapeutic properties compared to wild-type adenovirus (see WO2005 / 118825). EnAd has a chimeric E2B region that features Ad11p and Ad3 DNA and E3 / E4 deletions. The structural changes in enadenotucirev result in a genome that is approximately 3.5 kb smaller than Ad11p, providing additional “space” for transgene insertion.

[00322] Antibody molecules as employed may comprise a complete antibody molecule having full-length heavy and light chains, a bispecific antibody form comprising full-length antibodies or a fragment thereof, including but not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, Petition 870170082359, dated 10 / 26 / 2017, pp. 545 / 684 52 / 119 Bis-scFv, diabodies, triabodies, tetrabodies, and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). The methods for creating and manufacturing these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in International Patent Applications No. WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies can have multiple specificities, for example, bispecific, or they can be monospecific (see, for example, n2WO 92 / 22853, WO05 / 113605, WO2009 / 040562 and WO2010 / 035012).

[00323] The antibody, as used herein, unless the context indicates otherwise, refers to a full-length antibody.

[00324] Antibody-binding fragments refer to a fragment comprising binding domains, such as VH and / or VL, that retain specificity for the target antigen to which it binds and, for example, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabodies, tribodies, tetrabodies and epitope-binding fragments of any of the same. CONNECTORS Suitable ligands for use in presently revealed fusion proteins include: TABLE 2 · CONNECTOR SEQUENCES OF ARTICULATION SEQ ID NO: SEQUENCE 26 DKTHTCAA Petition 870170082359, dated 10 / 26 / 2017, pp. 546 / 684 53 / 119 SEQID NO: SEQUENCE 27 DKTJITCPPCPA 28 DKTJITCPPCPATCPPCPA 29 DKTJITCPPCPATCPPCPATCPPCPA 30 DKTJITCPPCPAGKPTLYNSLVMSDTAGTCY 31 DKTJITCPPCPAGKPTIIVNVSVVMAEVDGTCY 32 DKTHTCCVECPPCPA 33 DKTHTCPRCPEPKSCDTPPPCPRCPA 34 DKTHTCPSCPA TABLE 3. FLEXIBLE CONNECTOR SEQUENCES SEQID NO: SEQUENCE 35 SGGGGSE 36 DKTHTS 37 fSIGGGGS 38 (S)GGGGSGGGGS 39 (S)GGGGSGGGSGGGGS 40 (S)GGGGSGGGGSGGGGSGGGGS 41 (5)GGGGSGGGSGGGGSGGGGSGGGGS 42 AAAGSG-GASAS 43 AAAGSG-XGGGS-GASAS 44 AAAGSG-XGGGSXGGGS -GASAS 45 AAAGSG-XGGGSXGGGSXGGGS-GASAS 46 AAAGSG-XGGGSXGGGSXGGGSXGGGS-GASAS 47 ÁAAGSG-XS-GASAS 48 PGGNRGTTTTRRPATTTGSSPGPTQSHY 49 ATTTGSSPGPT 50 ATTTGS - GS Petition 870170082359, dated 10 / 26 / 2017, pp. 547 / 684 54 / 119 SEQ ID NO: SEQUENCE 51 EPSGPISTJNSPPSKESHKSP 52 GTVAAPSVFJFPPSD 53 GGGGIAPSMVGGGGS 54 GGGGKVEGAGGGGGS 55 GGGGSMKSHDGGGGS 56 GGGGNLJTIVGGGGS 57 GGGGVVPSLPGGGGS 58 GGEKSIPGGGGS 59 60 68 NPSLNPPSYLHRAPSRIS 69 LPWRTSLLPSLPLRRRP 70 PPLFAKGPVGLLSRSFPP 71 VPPAPVVSLRSAHARPPY 72 LRPTPPRVRSYTCCPTP- 73 PNVAHVLPLLTVPWDNLR 74 CNPLLPLCARSPAVRTFP (S) is optional in sequences 37 to 41,

[00325] Examples of rigid linkers include the peptide sequences GAPAPAAPAPA (SEQ ID NO: 75), PPPP (SEQ ID NO: Petition 870170082359, dated 10 / 26 / 2017, pp. 548 / 684 55 / 119 76) and PPP.

[00326] Other connectors are shown in TABLE 4: SEQ ID NO: SEQUENCE 77 DLCLRDWGCLW 78 DICLPRWGCLW 79 MED1CLPRWGCLWGD 80 QRLMFrUGLPRWGGLWEDDE 81 QGI.IGDtCLPRWGCLWGRSV 82 OGI.IGDICLPRWGCLWGRSVK 83 EDIC1TRWGCI.WRDD 84 RI.MEDlGLPRWGCl.WEDD 85 MEDICLPRWGCI.WEDD 86 MEDJCLPRWGCl.WED 87 RLMEDICLARWCCLWEDD 88 EVRSFCTRWPAEKSCKPLRG 89 RAPESFVCYWETICFERSEO 90 EMCYFPGICWM RELEVANT DEFINITIONS FOR FORMULA (I) AND (Ia)

[00327] A linkage refers to a covalent bond that connects one DNA sequence to another DNA sequence, for example, connecting one section of a virus genome to another. Thus, when a variable in formula (I) and (Ia) in this document represents a linkage, the feature or element represented by the linkage is absent, i.e., excluded.

[00328] Since the structure of adenoviruses is generally similar, the elements below are discussed in terms of the structural elements and the nomenclature commonly used to refer to them, which are known to the versed individual. When an element is indicated in this document, reference is then made to the DNA sequence that encodes the element or a DNA sequence that encodes the same protein. Petition 870170082359, dated 10 / 26 / 2017, pp. 549 / 684 56 / 119 structural element in an adenovirus. The latter is relevant due to the redundancy of the DNA code. The virus's preference for codon usage may need to be considered for optimized results.

[00329] Any structural element of an adenovirus employed in the virus of the present disclosure may comprise or consist of the natural sequence or may have similarity throughout the specified length of at least 95%, such as 96%, 97%, 98%, 99% or 100%. The original sequence may be modified to omit 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the genetic material. The skilled individual is aware that when making changes, the reading frames of the virus must not be disturbed so that the expression of structural proteins is not disrupted.

[00330] In one embodiment, the element determined is a full-length sequence, that is, a full-length gene. The full-length gene, as used herein, refers to at least the entire coding sequence of a gene, but may include any associated non-coding regions, especially if these are relevant to the gene's function.

[00331] In one embodiment, the determined element is smaller than a full length and retains the same or corresponding functions as the full-length sequence.

[00332] In one embodiment, for a given element that is optional in the constructs of the present disclosure, the DNA sequence may be shorter than a full length and have no functionality, for example, the E3 region may be totally or partially deleted. However, it may be useful to essentially delete the entire E3 region as this optimizes the space available for inserting transgenes.

[00333] The structural genes encoding structural or functional adenovirus proteins are generally linked to non-coding DNA regions. Therefore, there is some flexibility regarding where to “cut” the genomic sequence of the structural element of Petition 870170082359, dated 10 / 26 / 2017, pp. 550 / 684 57 / 119 of interest (especially non-coding regions thereof) for the purpose of inserting a transgene into the virus of the present disclosure. Thus, for the purpose of this descriptive report, the element will be considered a reference structural element to the extent that it meets the purpose and does not encode extrinsic material. Thus, if appropriate, the gene will be associated with suitable non-coding regions, for example, as found in the natural structure of the virus.

[00334] Thus, in one embodiment, an insert, such as DNA encoding a restriction site and / or transgene, is inserted into a non-coding region of genomic viral DNA, such as an intron or intergenic sequence. Having done so, some non-coding regions of adenovirus may have a function, for example, in alternative junction, transcription regulation, or translation regulation, and this may need to be taken into consideration. The sites identified in this document, which are associated with the L5 region, are suitable for accommodating a variety of DNA sequences encoding complex entities, such as RNAi, cytokines, multimeric or single-stranded proteins, such as antibodies.

[00335] The gene, as used herein, refers to coding sequences and any associated non-coding sequences, for example, associated introns and exons. In one embodiment, a gene comprises or consists of only essential structural components, for example, a coding region.

[00336] Below is a discussion related to specific structural elements of adenovirus.

[00337] Terminal Replication Sequences Inverted trichrome replication (ITR) sequences are common to all known adenoviruses (so named because of their symmetry) and are the viral chromosomal origins of replication. Another property of these sequences is their ability to form a hairpin.

[00338] A 5'ITR, as employed herein Petition 870170082359, dated 10 / 26 / 2017, pp. 551 / 684 58 / 119 document, refers to part or all of an ITR from the 5' end of an adenovirus, which retains the function of the ITR when incorporated into an adenovirus at an appropriate location. In one embodiment, the 5'ITR comprises or consists of a sequence of about 1 bp to 138 bp of SEQ ID NO: 21 or a sequence 90, 95, 96, 97, 98 or 99% identical to the same along its entire length, in particular, the sequence consists of about 1 bp to 138 bp of SEQ ID NO: 21.

[00339] A 3'ITR, as used herein, refers to part or all of an ITR from the 3' end of an adenovirus that retains ITR function when incorporated into an adenovirus at an appropriate location. In one embodiment, the 3'ITR comprises or consists of the sequence from about 3.2189 bp to 32.326 bp of SEQ ID NO: 21 or a sequence 90, 95, 96, 97, 98 or 99% identical to the same along its entire length, in particular, the sequence consists of from about 32.189 bp to 32.326 bp of SEQ ID NO: 21.

[00340] B1, as used herein, refers to the DNA sequence that encodes: part or all of an adenovirus E1A, part or all of an adenovirus EIB region, and independently, part or all of the adenovirus E1A and E1B regions.

[00341] When B1 is a link, then the E1A and EIB sequences will be omitted from the virus. In one embodiment, B1 is a link and, in this way, the virus is a vector.

[00342] In one embodiment, B1 additionally comprises a transgene. It is known in the art that the E1 region can accommodate a transgene that can be inserted in a disruptive manner in the E1 region (i.e., in the middle of the sequence) or part or all of the E1 region can be deleted to provide more space to accommodate genetic material.

[00343] E1A, as used in this document, refers to the DNA sequence that encodes part or all of an adenovirus E1A region. The latter refers to the E1A polypeptide / protein. The same Petition 870170082359, dated 10 / 26 / 2017, pp. 552 / 684 59 / 119 can be modified so that the protein encoded by the E1A gene has conservative or non-conservative amino acid changes (e.g., 1, 2, 3, 4, or 5 amino acid changes, additions, and / or deletions along the entire length) so that it has: the same function as the wild-type protein (i.e., the corresponding unmodified protein); increased function compared to the wild-type protein; decreased function, such as no function compared to the wild-type protein; or a new function compared to the wild-type protein or an appropriate combination thereof.

[00344] E1B, as used herein, refers to the DNA sequence that encodes part or all of an adenovirus E1b region (i.e., polypeptide or protein), which may be modified so that the protein encoded by the E1B gene / region has conservative or non-conservative amino acid changes (e.g., 1, 2, 3, 4, or 5 amino acid changes, additions, and / or deletions along its length) such that it has: the same function as the wild type (i.e., the corresponding unmodified protein); increased function compared to the wild-type protein; decreased function, such as no function compared to the wild-type protein; or has a new function compared to the wild-type protein or a combination thereof as appropriate.

[00345] Thus, B1 may or may not be modified in relation to the wild-type EI region, just like wild-type E1A and / or E1B. A knowledgeable individual can easily identify whether E1A and / or E1B are present or excluded (partially) or modified.

[00346] Wild type, as used herein, refers to a known adenovirus or a sequence of a known adenovirus. A known adenovirus is one that has been identified and named, regardless of whether sequence information is available. Petition 870170082359, dated 10 / 26 / 2017, pp. 553 / 684 60 / 119

[00347] In one embodiment, B1 has the sequence from 139 bp to 3,932 bp of SEQ ID NO: 21.

[00348] Ba, as used herein, refers to the DNA sequence encoding E2B-L1-L2-L3

[00349] Regions E2A-L4 include any non-coding sequences, as appropriate (in particular, corresponding to the natural sequence of an adenovirus). Generally, this sequence will not comprise a transgene. In one embodiment, the sequence is substantially similar or identical to a contiguous sequence of a known adenovirus, for example, a serotype shown in Table 1, in particular, a group B virus, for example, Ad3, Ad7, Ad11, Adl4, Adl6, Ad21, Ad34, Ad35, Ad51 or a combination thereof, such as Ad3, Ad11 or a combination thereof. In one embodiment, E2B-L1-L2-L3-E2A-L4 refers to the understanding of these elements and other structural elements associated with the region, for example, BA generally includes the sequence that encodes the IV2a protein, for example, as follows: IV2A IV2a-E2B-L1-L2-L3-E2AL4.

[00350] In one embodiment, the E2B region is chimeric. That is, it comprises DNA sequences from two or more different adenoviral serotypes, for example, from Ad3 and Ad11, such as Ad11p. In one embodiment, the E2B region has the sequence from 5,068 bp to 10,355 bp of SEQ ID NO: 21 or a sequence 95%, 96%, 97%, 98% or 99% identical to it along its entire length.

[00351] In one embodiment, the E2B in component Ba comprises the sequences shown in SEQ ID NO: 22 (which corresponds to SEQ ID NO: 3 disclosed in the document at WO2005 / 118825).

[00352] In a Ba modality, the sequence is from 3.933 bp to 2.7184 bp with SEQ ID NO: 21.

[00353] E3, as used in this document, refers to the DNA sequence that encodes part or all of a Petition 870170082359, dated 10 / 26 / 2017, pp. 554 / 684 61 / 119 adenovirus E3 region (i.e., protein / polypeptide), it can be modified so that the protein encoded by the E3 gene has conservative or non-conservative amino acid changes (e.g., 1, 2, 3, 4, or 5 amino acid changes, additions and / or deletions along the entire length), so that it has the same function as the wild-type (the corresponding unmodified protein); increased function compared to the wild-type protein; decreased function, such as no function compared to the wild-type protein or has a new function compared to the wild-type protein or a combination thereof, as appropriate.

[00354] In one embodiment, the E3 region forms an adenovirus serotype specified in Table 1 or a combination thereof, in particular, a group B serotype, for example, Ad3, Ad7, Ad11 (in particular, Ad11p), Ad14, Ad16, Ad21, Ad34, Ad35, Ad51 or a combination thereof, such as Ad3, Ad11 (in particular, Ad11p) or a combination thereof, such as Ad3, Ad11 (in particular, Ad11p) or a combination thereof. In one embodiment, the E3 region has a sequence shown in SEQ ID NO: 23.

[00355] In one embodiment, the E3 region is partially excluded, for example, it is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% excluded.

[00356] In a B2 form, it is a linkage in which the DNA encoding the E3 region is absent.

[00357] In one embodiment, the DNA encoding the E3 region may be replaced or interrupted by a transgene. As used herein, “transgene-replaced E3 region” includes part or all of the E3 region that is replaced by a transgene.

[00358] In one embodiment, region B2 comprises the sequence from 27,185 bp to 28,165 bp of SEQ ID NO: 24.

[00359] In a B2 modality, it consists of the sequence Petition 870170082359, dated 10 / 26 / 2017, pp. 555 / 684 62 / 119 from 27,185bp to 28,165bp of SEQ ID NO: 24.

[00360] Bx, as used herein, refers to the DNA sequence in the vicinity of the 5' end of the L5 gene in BB. In the vicinity of the 5' end of the L5 gene, as used herein, refers to: adjacent (contiguous) to the 5' end of the L5 gene or a non-coding region inherently associated with it, i.e., contiguous to the 5' drive end of the L5 gene or a non-coding region inherently associated with it. Alternatively, in the vicinity may refer to being close to the L5 gene such that there are no coding sequences between the BX region and the 5' end of the L5 gene.

[00361] Thus, in one embodiment, Bx is directly joined to an L5 base that represents, for example, the beginning of an L5 gene coding sequence.

[00362] Thus, in one embodiment, Bx is directly joined to an L5 base that represents, for example, the beginning of a non-coding sequence, or directly joined to a non-coding region naturally associated with L5. The non-coding region naturally associated with L5, as used herein, refers to part of all non-coding regions that are part of or contiguous to the L5 gene, but not part of another gene.

[00363] In one embodiment, Bx comprises the sequence of SEQ ID NO: 24. This sequence is an artificial non-coding sequence into which a DNA sequence, for example, comprising a transgene (or transgene cassette), a restriction site, or a combination thereof, may be inserted. This sequence is advantageous because it acts as a buffer that allows some flexibility in the exact location of the transgene while minimizing disruptive effects on virus stability and viability.

[00364] The insert (or inserts) can occur in Petition 870170082359, dated 10 / 26 / 2017, pp. 556 / 684 63 / 119 anywhere within SEQ ID NO: 24 from the 5' end, from the 3' end, or anywhere between bp 1 and 201, for example, between base pairs 1 / 2, 2 / 3, 3 / 4, 4 / 5, 5 / 6, 6 / 7, 7 / 8, 8 / 9, 9 / 10, 10 / 11, 11 / 12, 12 / 13, 13 / 14, 14 / 15, 15 / 16, 16 / 17, 17 / 18, 18 / 19, 19 / 20, 20 / 21, 21 / 22, 22 / 23, 23 / 24, 24 / 25, 25 / 26, 26 / 27, 27 / 28, 28 / 29, 29 / 30, 30 / 31, 31 / 32, 32 / 33, 33 / 34, 34 / 35, 35 / 36, 36 / 37, 37 / 38, 38 / 39, 39 / 40, 40 / 41, 41 / 42, 42 / 43, 43 / 44, 44 / 45, 45 / 46, 46 / 47, 47 / 48, 48 / 49, 49 / 50, 50 / 51, 51 / 52, 52 / 53, 53 / 54, 54 / 55, 55 / 56, 56 / 57, 57 / 58, 58 / 59, 59 / 60, 60 / 61, 61 / 62, 62 / 63, 63 / 64, 64 / 65, 65 / 66, 66 / 67, 67 / 68, 68 / 69, 69 / 70, 70 / 71, 71 / 72, 72 / 73, 73 / 74, 74 / 75, 75 / 76, 76 / 77, 77 / 78, 78 / 79, 79 / 80, 80 / 81, 81 / 82, 82 / 83, 83 / 84, 84 / 85, 85 / 86, 86 / 87, 87 / 88, 88 / 89, 89 / 90, 90 / 91, 91 / 92, 92 / 93, 93 / 94, 94 / 95, 95 / 96, 96 / 97, 97 / 98, 98 / 99, 99 / 100, 100 / 101, 101 / 102, 102 / 103, 103 / 104, 104 / 105, 105 / 106, 106 / 107, 107 / 108, 108 / 109, 109 / 110, 110 / 111, 111 / 112, 112 / 113, 113 / 114, 114 / 115, 115 / 116, 116 / 117, 117 / 118, 118 / 119, 119 / 120, 120 / 121, 121 / 122, 122 / 123, 123 / 124, 124 / 125, 125 / 126, 126 / 127, 127 / 128, 128 / 129, 129 / 130, 130 / 131, 131 / 132, 132 / 133, 133 / 134, 134 / 135, 135 / 136, 136 / 137, 137 / 138, 138 / 139, 139 / 140, 140 / 141, 141 / 142, 142 / 143, 143 / 144, 144 / 145, 145 / 146, 146 / 147, 147 / 148, 148 / 149, 150 / 151, 151 / 152, 152 / 153, 153 / 154, 154 / 155, 155 / 156, 156 / 157, 157 / 158 158 / 159, 159 / 160, 160 / 161, 161 / 162, 162 / 163, 163 / 164, 164 / 165, 165 / 166, 166 / 167, 167 / 168, 168 / 169, 169 / 170, 170 / 171, 171 / 172, 172 / 173, 173 / 174, 174 / 175, 175 / 176, 176 / 177, 177 / 178, 178 / 179, 179 / 180, 180 / 181, 181 / 182, 182 / 183, 183 / 184, 184 / 185, 185 / 186, 186 / 187, 187 / 188, 189 / 190, 190 / 191, 191 / 192, 192 / 193, 193 / 194, 194 / 195, 195 / 196, 196 / 197, 197 / 198, 198 / 199, 199 / 200 or 200 / 201.

[00365] In one embodiment, Βχ comprises SEQ ID NO: 24 with a DNA sequence inserted between bp 27 and bp 28 or a corresponding location between positions 28,192 bp and 28,193 bp of SEQ ID NO: 24.

[00366] In one form, Βχ has the sequence of 28.166 bp to 28.366 bp of SEQ ID NO: 21. In a modality, Βχ is a link.

[00367] Bβ, as employed herein Petition 870170082359, dated 10 / 26 / 2017, pp. 557 / 684 64 / 119 document, refers to the DNA sequence that encodes the L5 region. As used herein, the L5 region refers to the DNA sequence containing the gene encoding the fiber protein / polypeptide, as appropriate in the context. The fiber gene / region encodes the fiber protein, which is a major capsid component of adenovirus. The fiber functions in receptor recognition and contributes to the adenovirus's ability to selectively bind to and infect cells.

[00368] In the viruses of the present disclosure, the fiber can be of any adenovirus serotype, and adenoviruses that are chimeric, resulting from the fiber being changed to a different serotype, are also disclosed in the present disclosure. In one embodiment, the fiber is a group B virus, in particular, Ad11, such as Ad11p.

[00369] In a Bb modality, the sequence is from 28,367 bp to 29,344 bp with SEQ ID NO: 21.

[00370] The DNA sequence in relation to By, as used herein, refers to the DNA sequence in the vicinity of the 3' end of the L5 gene of Ββ. In the vicinity of the 3' end of the L5 gene, as used herein, refers to: adjacent (contiguous) to the 3' end of the L5 gene or a non-coding region inherently associated with it, i.e., contiguous to the 3' drive end of the L5 gene or a non-coding region inherently associated with it (i.e., all or part of a non-coding sequence endogenous to L5). Alternatively, in the vicinity may refer to being close to the L5 gene such that there are no coding sequences between the By region and the 3' end of the L5 gene.

[00371] Thus, in one embodiment, By is directly joined to an L5 base that represents the end of an encoding sequence.

[00372] Thus, in one embodiment, By is directly joined to a basis of L5 that represents the endpoint of a Petition 870170082359, dated 10 / 26 / 2017, pp. 558 / 684 65 / 119 non-coding sequence, or joined directly to a non-coding region naturally associated with L5.

[00373] Inherent and naturally are used interchangeably in this document. In one embodiment, Βγ comprises the sequence of SEQ ID NO: 25. This sequence is a non-coding sequence into which a DNA sequence, for example, comprising a transgene (or transgene cassette), a restriction site, or a combination thereof, may be inserted. This sequence is advantageous because it acts as a buffer that allows some flexibility in the exact location of the transgene, while minimizing disruptive effects on viral stability and viability.

[00374] The insert (or inserts) can occur anywhere within SEQ ID NO: 22 from the 5' end to the 3' end, or at any point between 1 bp and 35 bp, for example, between base pairs 1 / 2, 2 / 3, 3 / 4, 4 / 5, 5 / 6, 6 / 7, 7 / 8, 8 / 9, 9 / 10, 10 / 11, 11 / 12, 12 / 13, 13 / 14, 14 / 15, 15 / 16, 16 / 17, 17 / 18, 18 / 19, 19 / 20, 20 / 21, 21 / 22, 22 / 23, 23 / 24, 24 / 25, 25 / 26, 26 / 27, 27 / 28, 28 / 29, 29 / 30, 30 / 31, 31 / 32, 32 / 33, 33 / 34 or 34 / 35.

[00375] In one embodiment, Βγ comprises SEQ ID NO: 25 with a DNA sequence inserted between positions 12 bp and 13 bp or a location corresponding to 29,356 bp and 29,357 bp in SEQ ID NO: 21. In one embodiment, the insert is a restriction site insert. In one embodiment, the restriction site insert comprises one or more restriction sites. In one embodiment, the restriction site is a 19 bp restriction site insert comprising 2 restriction sites. In one embodiment, the restriction site insert has a 9 bp restriction site insert comprising 1 restriction site. In one embodiment, the restriction site insert comprises one or two restriction sites and at least one transgene, for example, one or two or three transgenes, such as one or two transgenes. In one embodiment, the restriction site is a 19 bp restriction site insert comprising 2 restriction sites and at least one transgene, for example, one or two transgenes. Petition 870170082359, dated 10 / 26 / 2017, pp. 559 / 684 66 / 119 In one embodiment, the restriction site insert is a 9 bp restriction site insert comprising 1 restriction site and at least one transgene, for example, one or two transgenes. In another embodiment, two restriction sites sandwich one or more of these two transgenes (for example, in a transgene cassette). In another embodiment, when By comprises two restriction sites, said restriction sites are different from each other.

[00376] In one embodiment, said one or more restriction sites in By are of unnatural occurrence (such as unique) in the particular adenovirus genome into which they were inserted. In one embodiment, said one or more restriction sites in By are different from other restriction sites located elsewhere in the adenovirus genome, for example, different from naturally occurring restriction sites or restriction sites introduced elsewhere in the genome, such as Bx. Thus, in one embodiment, the restriction site or sites allow the DNA in the section to be specifically cut.

[00377] In one mode, By has the sequence from 29.345 bp to 29.379 bp of SEQ ID NO: 21. In one mode, By is a link.

[00378] In one embodiment, the insert is after bp 12 in SEQ ID NO: 25.

[00379] In one embodiment, the insert is approximately at position 29.356 bp of SEQ ID NO: 21.

[00380] In one embodiment, the insert is a transgene cassette comprising one or more transgenes, for example, 1, 2 or 3, such as 1 or 2.

[00381] E4, as used herein, refers to the DNA sequence that encodes part or all of an adenovirus E4 region (i.e., polypeptide / protein region), which may be modified so that the protein encoded by the E4 gene has conservative or non-conservative amino acid changes (e.g., 1, 2, 3, 4, or 5). Petition 870170082359, dated 10 / 26 / 2017, pp. 560 / 684 67 / 119 changes, additions and / or deletions of amino acids), and has the same function as the wild type (the corresponding unmodified protein); increased function compared to the wild type protein; decreased function, such as no function compared to the wild type protein or has a new function compared to the wild type protein or a combination thereof as appropriate.

[00382] In one embodiment, region E4 is partially excluded, for example, it is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% excluded. In one embodiment, region E4 has the sequence from 32,188 bp to 29,380 bp of SEQ ID NO: 21.

[00383] In one embodiment, E4 is present except for the E4orf4 region which is excluded. In another embodiment, B3 is a link, that is, where E4 is absent.

[00384] In one embodiment, B3 has the sequence consisting of 32,188 bp to 29,380 bp of SEQ ID NO: 21.

[00385] As used in this document, numerical ranges include endpoints.

[00386] The skilled individual will observe that the elements in the formulas in the present document, such as formula (I), (la), are contiguous and may incorporate non-coding DNA sequences as well as the genes and coding DNA sequences (structural features) mentioned in the present document. In one or more embodiments, the formulas of the present disclosure attempt to describe a naturally occurring sequence in the adenovirus genome. In this context, it will be evident to the skilled individual that the formula is referring to the main elements that characterize the relevant section of the genome and is not intended to be an exhaustive description of the genomic elongation of DNA.

[00387] E1A, E1B, E3 and E4, as used in this document, each independently refers to the type Petition 870170082359, dated 10 / 26 / 2017, pp. 561 / 684 68 / 119 wild-type and equivalents thereof, modified or partially deleted forms of each region as described herein, in particular a wild-type sequence of a known adenovirus.

[00388] Insert, as used herein, refers to a DNA sequence that is incorporated at the 5' end, the 3' end, or within a given reference sequence of DNA sequence such that it interrupts the reference sequence. A reference sequence used as a reference point relative to that insert is located. In the context of this disclosure, inserts generally occur within SEQ ID NO: 24 or SEQ ID NO: 25. An insert may be a restriction site insert, a transgene cassette, or both. When the sequence is interrupted, the virus will still comprehend the original sequence, but it will generally be as two fragments sandwiching the insert.

[00389] In one embodiment, the transgene or transgene cassette does not comprise a non-polarized insertion transposon, such as a TN7 transposon or part thereof. TN7 transposon, as used herein, refers to a non-polarized insertion transposon as described in WO2008 / 080003.

[00390] In one embodiment, the transgene or transgene cassette additionally comprises a regulatory element or sequence. OTHER REGULATORY SEQUENCES

[00391] Gene expression regulator (or regulatory element), as used herein, refers to a genetic element, such as a promoter, enhancer, or splicing acceptor sequence, that plays a role in gene expression, typically initiating or enhancing transcription or translation.

[00392] Junction acceptor sequence, junction acceptor or junction site, as used in this document, refers to a regulatory sequence that determines when a molecule of Petition 870170082359, dated 10 / 26 / 2017, pp. 562 / 684 69 / 119 mRNA will be recognized by small nuclear ribonucleoproteins of the spliceosome complex. Once assembled, the spliceosome catalyzes the splicing between the acceptor splicing site of the mRNA molecule and an upstream donor splicing site, producing a mature mRNA molecule that can be translated to produce a single polypeptide or protein.

[00393] Joining acceptor sequences of different dimensions can be employed in the present invention and can be described as short (small) joining acceptor, (medium) joining acceptor and branched (large) joining acceptor.

[00394] SSA, as used herein, refers to a short-junction acceptor typically comprising only the junction site, for example, 4 bp. SA, as used herein, refers to a junction acceptor typically comprising the short-junction acceptor and the polypyrimidine tract, for example, 16 bp. bSA, as used herein, refers to a branched-junction acceptor typically comprising the short-junction acceptor, polypyrimidine tract and the branching point, for example, 26 bp.

[00395] In one embodiment, the join acceptor employed in the disclosure constructs is CAGG or SEQ ID NO: 3 or 4. In one embodiment, the SSA has the nucleotide sequence of SEQ ID NO: CAGG. In one embodiment, the SA has the nucleotide sequence of SEQ ID NO: 23. In one embodiment, the bSA has the nucleotide sequence of cagg. In one embodiment, the join acceptor sequence is independently selected from the group comprising: tgctaatctt cctttctctc ttcagg (SEQ ID NO: 4), tttctctctt cagg (SEQ ID NO: 3) and cagg.

[00396] In one embodiment, the junction site is immediately followed (i.e., followed in a 5' to 3' direction) by a consensus Kozak sequence comprising CCACC. In another embodiment, the junction site and the Kozak sequence are interleaved (separated) by up to Petition 870170082359, dated 10 / 26 / 2017, pp. 563 / 684 70 / 119 100 bp or less. In one embodiment, the Kozak sequence has the nucleotide sequence CCACC.

[00397] Typically, when under the control of an exogenous or endogenous promoter (such as an endogenous promoter), the coding sequence will be immediately preceded by a Kozak sequence. The start of the coding region is indicated by the start codon (AUG), for example, in the context of the sequence (gcc)gccRccAUGg [SEQ ID NO: 105] the start of the coding sequences is indicated by the bases in bold. A lowercase letter indicates common bases at that position (which may, however, vary) and uppercase letters indicate highly conserved bases, i.e., the sequence “AUGG” is constant or rarely changes, if it changes at all; “R” indicates that a purine (adenine or guanine) is usually observed at that position and the parenthetical sequence (gcc) is of uncertain significance. Thus, in one embodiment, the AUG start codon is incorporated into a Kozak sequence.

[00398] The Internal Ribosome Entry DNA Sequence, as used herein, refers to a DNA sequence that encodes an Internal Ribosome Entry Sequence (IRES). IRES, as used herein, means a nucleotide sequence that allows translational initiation of a messenger RNA (mRNA) sequence, which includes initiation that begins within an mRNA sequence. This is particularly useful when the cassette encodes polycistronic mRNA. The use of IRES results in a polycistronic mRNA that is translated into multiple individual proteins or polypeptides. In one embodiment, the Internal Ribosome Entry DNA sequence has the nucleotide sequence SEQ ID NO: 6. In one embodiment, a particular IRES is used only once in the genome. This may have benefits regarding genome stability.

[00399] Highly autocleatable peptide 2A or peptide 2A, as used herein, refers to a Petition 870170082359, dated 10 / 26 / 2017, pages 564 / 684 71 / 119 peptide that is efficiently cleaved following translation. Suitable 2A peptides include P2A, F2A, E2A, and T2A. The present inventors noted that once a specific DNA sequence encoding a given 2A peptide is used for the first time, the same specific DNA sequence cannot be used a second time. However, redundancy in the DNA code can be used to generate a DNA sequence that is translated into the same 2A peptide. The use of 2A peptides is particularly useful when the cassette encodes polycistronic mRNA. The use of 2A peptides results in a single polypeptide chain that is translated, which is modified post-translationally to generate multiple individual proteins or peptides.

[00400] In one embodiment, the encoded P2A peptide employed has the amino acid sequence of SEQ ID NO: 7. In one embodiment, the encoded F2A peptide employed has the amino acid sequence of SEQ ID NO: 8. In one embodiment, the encoded E2A peptide employed has the amino acid sequence of SEQ ID NO: 9. In one embodiment, the encoded T2A peptide employed has the amino acid sequence of SEQ ID NO: 10.

[00401] In one embodiment, an mRNA or each transgene-encoded mRNA comprises / comprise a polyadenylation signal sequence, such as, typically at the end of an mRNA sequence, for example, as shown in SEQ ID NO: 5. Thus, in one embodiment, the transgene or transgene cassette comprises at least one sequence encoding a polyadenylation signal sequence.

[00402] PolyA, Polyadenylation signal or polyadenylation sequence, as used in this document, means a DNA sequence, which generally contains an AATAAA site, which once transcribed can be recognized by a multiprotein complex that cleaves and polyadenylates the nascent mRNA molecule.

[00403] In one modality, the sequence of Petition 870170082359, dated 10 / 26 / 2017, pages 565 / 684 72 / 119 polyadenylation has the nucleotide sequence SEQ ID NO: 5.

[00404] In one embodiment, the construct does not include a polyadenylation sequence. In one embodiment, the gene expression regulator is a junction acceptor sequence.

[00405] In one embodiment, the sequence encoding a protein / polypeptide / peptide, such as an antibody or antibody-binding fragment, additionally comprises a polyadenylation signal.

[00406] In one embodiment, a virus or construct with a sequence disclosed in this document is provided, for example, a selected virus NG-330 (SEQ ID NO: 16); NG-334 (SEQ ID NO: 17); NG345 (SEQ ID NO: 18); NG-346 (SEQ ID NO: 19); NG-347 (SEQ ID NO: 20) and NG348 (SEQ ID NO: 96).

[00407] In one embodiment, the virus is NG-347 (SEQ ID NO: 20) or NG-348 (SEQ ID NO: 96). FORMULATIONS

[00408] The present disclosure also refers to the pharmaceutical formulation of a virus as described in this document.

[00409] In one embodiment, a liquid parenteral formulation is provided, for example, for infusion or injection, of a replicating oncolytic agent according to the present disclosure, wherein the formulation provides a dose in the range of 1x1010 to 1x1014 viral particles per dose volume.

[00410] Parenteral formulation means a formulation designed not to be delivered via the GI tract. Typical parenteral delivery routes include injection, implantation, or infusion. In one embodiment, the formulation is provided in a form for bolus delivery.

[00411] In one embodiment, the parenteral formulation is in the form of an injection. Injection includes intravenous, subcutaneous, intratumoral, or intramuscular injection. Injection, as used herein, means the insertion of fluid into the body via a syringe. In Petition 870170082359, dated 10 / 26 / 2017, pp. 566 / 684 73 / 119 one modality, the method of the present disclosure does not involve intratumoral injection.

[00412] In one embodiment, the parenteral formulation is in the form of an infusion.

[00413] Infusion, as used in this document, means the administration of fluids at a slower rate by drip, infusion pump, syringe delivery device or equivalent device. In one embodiment, the infusion is administered over a period in the range of 1.5 minutes to 120 minutes, such as approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 65, 80, 85, 90, 95, 100, 105, 110 or 115 minutes.

[00414] In one embodiment, a dose of the formulation less than 100 ml, for example, 30 ml, as administered by a syringe dispenser.

[00415] In one embodiment, the injection is administered as a slow injection, for example, over a period of 1.5 to 30 minutes.

[00416] In one embodiment, the formulation is for intravenous (IV) administration. This route is particularly effective for delivering oncolytic viruses because it allows rapid access to most organs and tissues and is particularly useful for treating metastases, for example, established metastases especially located in highly vascularized regions such as the liver and lungs.

[00417] Therapeutic formulations will typically be sterilized under manufacturing and storage conditions. The composition may be formulated as a solution, microemulsion, liposome, or other parenteral formulation suitable for administration to a human and may be formulated as a pre-filled device such as a syringe or vial, particularly as a single dose.

[00418] The formulation will generally comprise a pharmaceutically acceptable diluent or carrier, for example, a carrier Petition 870170082359, dated 10 / 26 / 2017, pp. 567 / 684 74 / 119 is non-toxic, isotonic, compatible with the virus, and stable against the virus for the required period of time.

[00419] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Appropriate fluidity may be maintained, for example, by the use of a dispersant or surfactant, such as lecithin or a non-ionic surfactant such as polysorbate 80 or 40. In dispersions, the maintenance of the required particle size may be assisted by the presence of a surfactant. Examples of isotonic agents include sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[00420] In one embodiment, parenteral formulations employed may comprise one or more of the following: a buffer, for example, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, a phosphate buffer and / or a Tris buffer, a sugar, for example, dextrose, mannose, sucrose or similar, a salt such as sodium chloride, magnesium chloride or potassium chloride, a detergent such as a non-ionic surfactant such as Brij, PS-80, PS-40 or similar. The formulation may also comprise a preservative such as EDTA or ethanol or a combination of EDTA and ethanol, which are considered to prevent one or more pathways of possible degradation.

[00421] In one embodiment, the formulation will comprise purified oncolytic virus according to the present disclosure, for example, 1x10¹⁰ to 1x10¹⁴ viral particles per dose, such as 1x10¹⁰ to 1x10¹² viral particles per dose. In one embodiment, the virus concentration in the formulation is in the range of 2x10⁸ to 2x10¹⁴ vp / ml, such as 2x10¹² vp / ml.

[00422] In one embodiment, the parenteral formulation comprises glycerol.

[00423] In one embodiment, the formulation comprises oncolytic adenovirus as described herein, HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), glycerol, and buffer. Petition 870170082359, dated 10 / 26 / 2017, pages 568 / 684 75 / 119

[00424] In one embodiment, the parenteral formulation consists of a detection virus, HEPES for example, 5 mM, glycerol for example, 5 to 20% (v / v), hydrochloric acid for example, to adjust the pH to the range of 7 to 8 and water for injection.

[00425] In one embodiment, 0.7 ml of detection virus at a concentration of 2 x 1012 vp / ml is formulated in 5 mM eHEPES, 20% glycerol with a final pH of 7.8.

[00426] A complete discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ 1991).

[00427] In one embodiment, the formulation is provided as a formulation for topical administration, which includes inhalation. Suitable inhalable preparations include inhalable powders, metered aerosols containing propellant gases, or inhalable solutions free of propellant gases. Inhalable powders according to the disclosure will generally contain a virus, as described herein, with a physiologically acceptable excipient.

[00428] These inhalable powders may include monosaccharides (e.g., glucose or arabinose), disaccharides (e.g., lactose, sucrose, maltose), oligo- and polysaccharides (e.g., dextrans), polyalcohols (e.g., sorbitol, mannitol, xylitol), salts (e.g., sodium chloride, calcium carbonate) or mixtures of these with others. Mono- or disaccharides are appropriately used, such as lactose or glucose, particularly, but not exclusively, in the form of their hydrates.

[00429] Particles for deposition in the lung require a particle size of less than 10 microns, such as 1 to 9 microns, for example, 0.1 to 5 μm, in particular, 1 to 5 μm. The size of the particle carrying the virus is of great importance and, therefore, in one embodiment, the virus according to the present disclosure can be adsorbed or absorbed onto a particle, such as a lactose particle of a determined size. Petition 870170082359, dated 10 / 26 / 2017, pages 569 / 684 76 / 119

[00430] The propellant gases that can be used to prepare inhalable aerosols are known in the art. Suitable propellant gases are selected from hydrocarbons, such as n-propane, n-butane or isobutane, and halohydrocarbons, such as chlorinated and / or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The propellant gases mentioned above can be used alone or in mixtures thereof.

[00431] Particularly suitable propellant gases are derived from halogenated alkanes selected from TG 11, TG 12, TG 134a and TG227. Of the halogenated hydrocarbons mentioned above, TG134a (1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable.

[00432] Inhalable aerosols containing propellant gas may also contain other ingredients, such as cosolvents, stabilizers, surface-active agents (surfactants), antioxidants, lubricants, and pH adjusters. All of these ingredients are known in the art.

[00433] Inhalable aerosols containing propellant gas according to the invention may contain up to 5% by weight of active substance. The aerosols according to the invention contain, for example, 0.002 to 5% by weight, 0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to 2% by weight or 0.5 to 1% by weight of active ingredient.

[00434] Alternatively, topical administration to the lung may also be by administering a solution or liquid suspension formulation, for example, employing a device such as a nebulizer, for example, a nebulizer connected to a compressor (for example, the Pari LC-Jet Plus(R) nebulizer connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).

[00435] The virus of the invention can be delivered dispersed in a solvent, for example, in the form of a solution or a Petition 870170082359, dated 10 / 26 / 2017, pp. 570 / 684 77 / 119 suspension, for example, as described above in parenteral formulations. It may be suspended in an appropriate physiological solution, for example, saline or another pharmacologically acceptable solvent, or a buffered solution. Buffer solutions known in the art may contain 0.05 mg to 0.15 mg of sodium edetate, 8.0 mg to 9.0 mg of NaCl, 0.15 mg to 0.25 mg of polysorbate, 0.25 mg to 0.30 mg of anhydrous citric acid, and 0.45 mg to 0.55 mg of sodium citrate per 1 ml of water, so as to achieve a pH of about 4.0 to 5.0.

[00436] Therapeutic solution or suspension formulations may also contain one or more excipients. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions may be encapsulated in biodegradable liposomes or microspheres. The formulation will generally be supplied in a substantially sterile form employing sterile manufacturing processes.

[00437] This may include production and sterilization by filtering the solvent / buffered solution used for the formulation, aseptic suspension of the antibody in the buffered solvent solution, and dispensing the formulation into sterilized receptacles using methods familiar to those skilled in the art.

[00438] The nebulizable formulation according to the present disclosure may be supplied, for example, as single-dose units (e.g., sealed plastic containers or vials) packaged in foil envelopes. Each vial contains a unit dose in a volume, for example, 2 ml of solvent buffer / solution.

[00439] The present disclosure also extends to intranasally delivered liquid solutions or suspensions, for example, Petition 870170082359, dated 10 / 26 / 2017, pp. 571 / 684 78 / 119 employing a device as disclosed in documents WO2009 / 068877 and US2004 / 0153033, both incorporated herein by reference. TREATMENT

[00440] In a further aspect, the present disclosure extends to a virus or a formulation thereof, as described herein, for use in treatment, in particular, for the treatment of cancer.

[00441] In one modality, the treatment method is for use in the treatment of a tumor.

[00442] Tumor, as used in this document, refers to an abnormal mass of tissue resulting from excessive, uncontrolled, and progressive cell division, also called a neoplasm. Tumors can be benign (non-cancerous) or malignant. Tumor encompasses all forms of cancer and metastasis. In one modality, the tumor is cancerous.

[00443] In one embodiment, the tumor is a solid tumor. The solid tumor may be localized or metastasized.

[00444] In one type, the tumor is of epithelial origin.

[00445] In one modality, the tumor is a malignancy, such as colorectal cancer, hepatoma, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, kidney cancer, bladder cancer, head and neck cancer, or lung cancer.

[00446] In one form, the tumor is a colorectal malignancy.

[00447] Malignancy, as used in this document, refers to cancerous cells.

[00448] In one embodiment, oncolytic adenovirus is used in the treatment or prevention of metastasis. Petition 870170082359, dated 10 / 26 / 2017, pp. 572 / 684 79 / 119

[00449] In one embodiment, the method or formulation in this document is employed in the treatment of drug-resistant cancers. In another embodiment, the virus is administered in combination with the administration of an additional cancer treatment or therapy.

[00450] In one embodiment, a virus or formulation in accordance with the present disclosure is provided for use in the manufacture of a medicament for the treatment of cancer, for example, a cancer described above.

[00451] In a further aspect, a method of treating cancer is provided comprising administering a therapeutically effective amount of a virus or formulation according to the present disclosure to a patient in need thereof, for example, a human patient.

[00452] In one embodiment, the oncolytic virus or formulation in this document is administered in combination with another therapy.

[00453] In combination, as used in this document, it is intended to encompass when the oncolytic virus is administered before, concurrently with, and / or after cancer treatment or therapy. However, treatment regimens are generally combination therapy.

[00454] Cancer therapy includes surgery, radiotherapy, targeted therapy and / or chemotherapy.

[00455] Cancer treatment, as used in this document, refers to treatment with a therapeutic compound or biological agent, for example, an antibody intended to treat cancer and / or maintenance therapy for the same.

[00456] In one embodiment, cancer treatment is selected from any other anticancer therapy which includes a chemotherapeutic agent; a targeted anticancer agent, such as a conjugate of Petition 870170082359, dated 10 / 26 / 2017, pp. 573 / 684 80 / 119 antibody drug; radiotherapy, radioisotope therapy or any combination thereof.

[00457] In one embodiment, the virus of the present disclosure, such as an oncolytic adenovirus, can be used as a pretreatment to therapy, such as surgery (neoadjuvant therapy), to shrink the tumor, to treat metastasis and / or prevent metastasis or further metastasis. The oncolytic adenovirus can be used after therapy, such as surgery (adjuvant therapy), to treat metastasis and / or prevent metastasis or further metastasis.

[00458] In one embodiment, a virus or formulation of the present disclosure is employed in maintenance therapy.

[00459] Consequently, as employed in this document, it is the administration of additional cancer treatment at the same time, or approximately the same time, as the oncolytic adenovirus formulation. The treatment may be contained within the same formulation or administered as a separate formulation.

[00460] In one embodiment, the virus is administered in combination with the administration of a chemotherapeutic agent.

[00461] The chemotherapeutic agent, as used in this document, refers to specific antineoplastic chemical agents or drugs that are selectively destructive to malignant cells and tissues. For example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. Examples of specific chemotherapeutic agents include doxorubicin, 5-fluorouracil (5-FU), paclitaxel, capecitabine, irinotecan, and platinums such as cisplatin and oxaliplatin. The dose may be chosen by the practitioner based on the nature of the cancer being treated. In one embodiment, the therapeutic agent is ganciclovir, which may assist in controlling immune responses and / or tumor vascularization.

[00462] In a modality, one or more therapies Petition 870170082359, dated 10 / 26 / 2017, pp. 574 / 684 81 / 119 employed in the method in this document are metronomic, which is a continuous or frequent treatment with low doses of anticancer drugs, often given concomitantly with other methods of therapy.

[00463] Oncolytic adenovirus of subgroup B, in particular Ad11 and those derived from it such as EnAd, may be particularly synergistic with chemotherapies due to the fact that they appear to have a mechanism of action that is highly independent of apoptosis, exterminating cancerous cells by a predominantly necrolytic mechanism. Furthermore, the immunosuppression that occurs during chemotherapy may allow the oncolytic virus to function more efficiently.

[00464] The therapeutic dose, as used in this document, refers to the amount of virus, such as oncolytic adenovirus, that is adequate to achieve the intended therapeutic effect when used in an appropriate treatment regimen, for example, improving the symptoms or conditions of a particular disease without eliciting dose limits due to side effects. A dose may be considered a therapeutic dose in the treatment of cancer or metastases when the number of viral particles may be sufficient to result in the following: tumor or metastatic growth becomes slower or is stopped, or the tumor or metastasis shrinks in size and / or the patient's life is extended. Adequate therapeutic doses are generally a balance between therapeutic effect and tolerable toxicity, for example, when the side effect and toxicity are tolerable due to the benefit achieved by the therapy.

[00465] In one embodiment, systematic administration of multiple doses of a parenteral formulation of an oncolytic adenovirus according to the present disclosure is provided in a single treatment cycle, for example, wherein the total dose determined in each administration is in the range of 1x1010 to 1x1014 viral particles per dose.

[00466] In one embodiment, one or more doses (for example, each dose) of virus or composition comprising the same is Petition 870170082359, dated 10 / 26 / 2017, pages 575 / 684 82 / 119 administered so that the viral particle delivery rate is in the range of 2x1010 particles per minute to 2x1012 particles per minute.

[00467] In one embodiment, a virus or therapeutic construct according to the present disclosure (including a formulation comprising the same) is administered weekly, for example, week 1 the dose is administered on days 1, 3, 5, followed by a dose each subsequent week.

[00468] In one embodiment, a virus or therapeutic construct according to the present disclosure (including a formulation comprising the same) is administered bi- or tri-weekly, for example, it is administered in week 1 on days 1, 3 and 5, and in week 2 or 3 it is also administered on days 1, 3 and 5 of the same. This dosing regimen may be repeated as often as appropriate.

[00469] In one embodiment, a virus or therapeutic construct according to the present disclosure (including a formulation comprising the same) is administered monthly, for example, in a treatment cycle or as maintenance therapy.

[00470] In one embodiment, the virus and constructs of the present disclosure are prepared by recombinant techniques. The skilled individual will observe that the armed adenovirus genome can be manufactured by other means of technique, which includes synthesizing the entire genome or a plasmid comprising part of the entire genome. The skilled individual will observe that in the case where synthesizing the genome, the insertion region may not comprise the restriction site nucleotides as the latter are artifacts following gene insertion using cloning methods.

[00471] In one embodiment, the armed adenovirus genome is entirely synthetically manufactured, for example, in SEQ ID NO: 109, which was employed with transgene cassettes in SEQ ID Nos: 18, 20, 96, 101, 102, 103.

[00472] The revelation in this document Petition 870170082359, dated 10 / 26 / 2017, pp. 576 / 684 83 / 119 additionally extends to an adenovirus of formula (I) or a subformula thereof, obtained or obtainable by insertion of a transgene or transgene cassette.

[00473] “É conforme, used in this document, means that it includes.

[00474] In the context of this descriptive report, "comprising" should be interpreted as "including".

[00475] The embodiments of the invention comprising certain features / elements are also intended for extension to alternative embodiments that consist of, or essentially consist of, the relevant elements / features.

[00476] Where technically appropriate, embodiments of the invention may be combined. Technical references, such as patents and applications, are incorporated herein by reference.

[00477] Any specific and explicitly cited modalities in this document may form the basis for a refusal of opinion alone or in combination with one or more additional modalities.

[00478] Headings in this document are used to divide the document into sections and are not intended to be used to construct the meaning of the disclosure provided in this document.

[00479] The present invention is further described by way of illustration only in the following examples. EXAMPLES EXAMPLE 1: PRODUCTION OF EnAD VIRUS EXPRESSING THE CD80 T-CELL ACTIVATING ANTIGEN

[00480] The pEnAd2.4 plasmid was used to generate the pNG-330 plasmid by direct insertion of a cassette encoding CD80. Petition 870170082359, dated 10 / 26 / 2017, pp. 577 / 684 84 / 119 T cell activator (SEQ ID NO: 11). The pNG-330 cassette contains a short 5' CAGG join acceptor sequence, a human CD80 CAGG cDNA sequence, and a 3' polyadenylation sequence (SEQ ID NO: 5). A schematic of the inserted transgene cassette is shown in Figure 3A. The plasmid construct was confirmed by DNA sequencing. VIRAL PRODUCTION AND CHARACTERIZATION

[00481] References in this document to viruses such as NG-330-00 are simply references to the “00” processed in a particular batch of the NG-330 virus. Similar nomenclature may be used for other viruses. The pNG-330 plasmid was linearized by restriction digestion with the enzyme Ascl to produce the genome of the NG-330 virus (SEQ ID NO: 16). The digested DNA was purified by phenol / chloroform extraction and precipitated for 16 h at 20 °C in 300 μl > 95% molecular biology grade ethanol and 10 μl of 3M sodium acetate. The precipitated DNA was pelletized by centrifugation at 14,000 rpm for 5 minutes and washed in 500 μl of 170% ethanol before centrifuging again at 14,000 rpm for 5 minutes. minutes. The cleaned DNA pellet was air-dried, resuspended in 500 μl of OptiMEM containing 15 μl of lipofectamine transfection reagent, and incubated for 30 minutes, RT.After incubating the cells with the transfection mixture for 2 hours at 37°C, 5% CO2, 4 ml of cell medium (high DMEM glucose with glutamine supplemented with 2% FBS) were added to the cells and the flasks were incubated at 37°C, 5% CO2.

[00482] The 293 transfected cells were monitored every 24 hours and supplemented with additional medium every 48 to 72 hours. Virus production was monitored by observing a significant cytopathic effect (CPE) in the cell monolayer. Once extensive CPE was observed, virus was harvested from 293 cells by three freeze-thaw cycles. The harvested viruses were used to reinfect 293 cells in order to amplify the virus stocks. The production of viable virus during the Petition 870170082359, dated 10 / 26 / 2017, pp. 578 / 684 85 / 119 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 stock was used for further amplification before the virus was purified by double-pair cesium chloride banding to produce an NG-330 virus stock. EXAMPLE 2: CHARACTERIZATION OF NG-330 VIRUS ACTIVITY IN COMPARISON WITH EnAD CARCINOMA CELL LINES

[00483] The replication of NG-330 or EnAd virus (evaluated by qPCR), and CD80 membrane expression (evaluated by flow cytometry (Figures 4 and 5)) was compared in the HT-29 colon carcinoma cell line and the A549 lung carcinoma cell line. NG-330 is an EnAd-derived virus containing a transgene cassette encoding the human T-cell activator antigen, CD80, after the late EnAd gene, L5 (Fiber). A schematic of the inserted cassette is shown in Figure 3A. NG-330 virus production is detailed in Example 1. HT-29 or A549 carcinoma cell lines were seeded in 6-well plates at cell wells of 7.5e5 cells / well for A549 cells or 2e6 cells / well for HT-29 cells. The plates were incubated for 18 h at 37 °C. 5% CO2, before cells were infected with 100 EnAd or NG-330 virus particles per cell (ppc) or were allowed to become infected. Assays were performed 24, 48, or 72 hours post-infection. Virus replication assessed by qPCR

[00484] HT-29 and A549 cell lines infected for 24, 48, or 72 h with 100 ppc EnAd or NG-330, or left uninfected, were used for viral DNA quantification by qPCR. Cell supernatants were collected and clarified by centrifugation for 5 minutes at 1200 rpm. DNA was extracted from 10 μL of supernatant using a Sigma Genelute DNA extraction kit, according to the manufacturer's protocol. A standard curve was created. Petition 870170082359, dated 10 / 26 / 2017, pp. 579 / 684 86 / 119 with the use of EnAd virus particles (25e10-2.5e5vp) was also prepared and extracted using the Sigma Genelute Kit. Each extracted sample or standard was analyzed by qPCR using an EnAd E3 gene-specific primer probe set.

[00485] Quantification of the number of viral genomes detected per cell demonstrated that the replication of NG-330 virus and EnAd virus was comparable in both HT-29 (Figure 4A) and A549 (Figure 4B) cell lines. No viral genomes could be detected in uninfected cells (data not shown). CD80 CELL SURFACE EXPRESSION ASSESSED BY FLOW CYTOMETRY

[00486] HT-29 and A549 cell lines, both infected for 24, 48, or 72 h with 100 ppc EnAd or NG-330, or left uninfected, were used for analysis of CD80 transgene expression on the cell surface. Tumor cells were removed from the plate surface by trypsin treatment, centrifuged, and then resuspended in 1% BSA / PBS. Samples were then incubated at 5 °C for 1 h with buffer, Cy5-conjugated mouse isotype control antibody, or Cy5-conjugated anti-human CD80 antibody (clone 2D10). All samples were also stacked with live / killed Zombie Aqua for differentiated viable cells. The samples were washed 3 times with 1% BSA / PBS before analysis by flow cytometry (FACS, Attune) for cell viability and CD80 protein expression on the cell surface.Analysis showed that CD80 could be detected on the cell surface in both A549 (Figure 5A) and HT-29 (Figure 5B) cells treated with NG-330, but not in those treated with EnAd or left untreated. COMPARISON OF ONCOLYTIC POTENCY OF VIRUSES

[00487] HT-29 colon carcinoma cells were seeded in 96-well plates at a cell density of 2.5e4 cells / well. The plates were incubated for 4 h at 37 °C, 5% CO2, before Petition 870170082359, dated 10 / 26 / 2017, pages 580 / 684 87 / 119 cells were infected with EnAd or NG-330 virus particles in an infection density range of 100-0.39 particles per cell (ppc). HT-29 cell viability was assessed using MTS 96 Cell Titration Reagent (Promega: G3581) 72 h post-infection. Quantification of % cell survival at each infection density demonstrated that the oncolytic potency of NG-330 was comparable to that of EnAd in HT29 cells (Figure 6). EXAMPLE 3: PRODUCTION OF ENAD VIRUSES EXPRESSING THE CD80 TEA CELL ACTIVATING ANTIGEN CYTOKINE IFNα

[00488] The pEnAd2.4 plasmid was used to generate the pNG-343 plasmid by direct insertion of a cassette encoding human T-cell activating antigen CD80 (SEQ ID NO: 11) and human cytokine interferon α (IFNa, SEQ ID NO: 12). The pNG-343 cassette contains: a short 5' CAGG join acceptor sequence; human IFN α cDNA; a high-efficiency autocleavable P2A peptide sequence (SEQ ID NO: 7); a human CD80 cDNA sequence and a 3' polyadenylation sequence (SEQ ID NO: 5). A schematic of the inserted transgene cassette is shown in Figure 3B. The plasmid construct was confirmed by DNA sequencing. VIRAL PRODUCTION AND CHARACTERIZATION

[00489] The pNG-343 plasmid was linearized by restriction digestion with the enzyme Ascl to produce the NG343 virus genome (SEQ ID NO: 17)). The NG-343 virus is amplified and purified according to the methods detailed in Example 1. EXAMPLE 4: PRODUCTION OF ENAD VIRUSES EXPRESSING THE EXTRACELLULAR DOMAIN OF THE FMS-TYPE TYROSINE KINASE-3 LIGAND, CHEMOKINAM1α AND THE CYTOKINE IFNα

[00490] The pEnAd2.4 plasmid is used to generate the pNG-345 plasmid by direct insertion of a cassette encoding a variant Petition 870170082359, dated 10 / 26 / 2017, pages 581 / 684 The pNG-345 cassette contains: a soluble 88 / 119 ligand of FMS-type tyrosine kinase-3 (Flt3L, SEQ ID NO: 13), MI1α (LD78p isoform, SEQ ID NO: 14), and IFNα (SEQ ID NO: 12). The pNG-345 cassette contains: a 5' short splicing acceptor sequence CAGG; human Flt3L cDNA; a high-efficiency autocleavable P2A peptide sequence (SEQ ID NO: 7); human MI1α cDNA; a high-efficiency autocleavable T2A peptide sequence (SEQ ID NO: 10); human IFNα cDNA; and a 3' polyadenylation sequence (SEQ ID NO: 5). A schematic of the inserted transgene cassette is shown in Figure 3D. The plasmid construct is confirmed by DNA sequencing. VIRAL PRODUCTION AND CHARACTERIZATION

[00491] The pNG-345 plasmid is linearized by restriction digestion with the enzyme Ascl to produce the NG345 virus genome (SEQ ID NO: 18)). The NG-345 virus is amplified and purified according to the methods detailed in Example 1. EXAMPLE 5: PRODUCTION OF ENAD VIRUSES EXPRESSING THE CD80 T-CELL ACTIVATING ANTIGEN, THE CHEMOKIN MI1α AND THE FLT3 LIGAND

[00492] The pEnAd2.4 plasmid was used to generate the pNG-346 plasmids by direct insertion of a cassette encoding the human T-cell activating CD80 antigen (SEQ ID NO 11), the human macrophage inflammatory protein 1α (MlP1a, SEQ ID NO. 14) and the human Flt3 ligand (SEQ ID NO: 13). The pNG-346 cassette contains: a 5' short splicing acceptor sequence CAGG; human IFNα cDNA; a high-efficiency autocleaving P2A peptide sequence (SEQ ID NO: 7); human MlP1α cDNA (LD78P isoform); a high-efficiency autocleaving T2A peptide sequence (SEQ ID NO: 10); Human Flt3 ligand cDNA sequence and a 3' polyadenylation sequence (SEQ ID NO: 5). A schematic of the inserted transgene cassette is shown in Figure 3E. The plasmid construct is confirmed by DNA sequencing. Petition 870170082359, dated 10 / 26 / 2017, pages 582 / 684 89 / 119 VIRAL PRODUCTION AND CHARACTERIZATION

[00493] The pNG-346 plasmid is linearized by restriction digestion with the enzyme Ascl to produce the NG346 virus genome (SEQ ID NO: 19). The NG-346 virus is amplified and purified according to the methods detailed in Example 1. EXAMPLE 6: PRODUCTION OF ENAD VIRUSES EXPRESSING THE CD80 T-CELL ACTIVATING ANTIGEN, THE CHEMOKIN MLP1a, AND THE CYTOKIN IFNa

[00494] The pEnAd2.4 plasmid was used to generate the pNG-347 plasmids by direct insertion of a cassette encoding human T-cell activating CD80 antigen (SEQ ID NO: 11), human macrophage inflammatory protein 1α (MIPIα, SEQ ID NO. 14), and human cytokine interferon (IFNa, SEQ ID NO: 12). The pNG-347 cassette contains: a 5' short splicing acceptor sequence CAGG; human IFNNa cDNA; a high-efficiency autocleaveable P2A peptide sequence (SEQ ID NO: 7); human MlP1 α cDNA (LD78P isoform); a high-efficiency autocleaveable T2A peptide sequence (SEQ ID NO: 10); Human CD80 cDNA sequence and a 3' polyadenylation sequence (SEQ ID NO: 5). A schematic of the inserted transgene cassette is shown in Figure 3F. The plasmid construct is confirmed by DNA sequencing. VIRAL PRODUCTION AND CHARACTERIZATION

[00495] The pNG-347 plasmid is linearized by restriction digestion with the enzyme Ascl to produce the NG347 virus genome (SEQ ID NO: 20). The NG-347 virus is amplified and purified according to the methods detailed in Example 1. EXAMPLE 7: PRODUCTION OF ENAD VIRUSES EXPRESSING THE CD80 CELL-ACTIVATING ANTIGEN TE, A MEMBRANE-ANCHORED SINGLE-CHAIN ​​FV FRAGMENT ANTIBODY FOR THE ε CHAIN ​​OF THE HUMAN CD3 COMPLEX (CD3e) Petition 870170082359, dated 10 / 26 / 2017, pages 583 / 684 90 / 119

[00496] The pEnAd2.4 plasmid was used to generate the pNG-348 plasmids by direct insertion of a cassette encoding the human T-cell activating CD80 antigen (SEQ ID NO: 11) and a membrane-anchored chimeric form of the single-stranded anti-human CD3e Fv (SEQ ID NO: 15). The pNG-348 cassette contains: a short 5' CAGG join acceptor sequence; membrane-anchored anti-human CD3e scFv cDNA; a high-efficiency autocleavable P2A peptide sequence (SEQ ID NO: 7); a human CD80 cDNA sequence and a 3' polyadenylation sequence (SEQ ID NO: 5). A schematic of the inserted transgene cassette is shown in Figure 3C. The plasmid construct is confirmed by DNA sequencing. VIRAL PRODUCTION AND CHARACTERIZATION

[00497] The pNG-348 plasmid is linearized by restriction digestion with the enzyme Ascl to produce the NG348 virus genome (SEQ ID NO: 96). The NG-348 virus is amplified and purified according to the methods detailed in Example 1. EXAMPLE 8: ACTIVITY OF EnAd VIRUS, NG-343, WHICH EXPRESSES TWO TRANSGENES; THE CD80 TEA CELL ACTIVATING ANTIGEN AND THE CYTOKINE IFNα

[00498] Characterization of NG-343 virus activity compared to NG-343 carcinoma cell lines or EnAd virus replication (evaluated by qPCR), CD80 transgene expression (evaluated by flow cytometry), or IFNoc transgene expression (evaluated by ELISA) was compared in the colon carcinoma cell line, HT-29, or the lung carcinoma cell line, A549. NG-343 is an EnAd-derived virus containing a transgene cassette encoding the human T-cell activator antigen, CD80, as well as the human cytokine interferon alpha 2b located after the late EnAd gene, L5 (Fiber). A schematic of the embedded cassette is shown in Figure 3B. NG-343 virus production is detailed in Example 3. Petition 870170082359, dated 10 / 26 / 2017, pages 584 / 684 91 / 119 A549 or HT-29 carcinoma cell lines were seeded in 12-well plates at cell densities of 7.5 x 10⁵ cells / well for A549 cells or 1.4 x 10⁶ cells / well for HT-29 cells. The plates were incubated for 18 h at 37 °C, 5% eCO₂, before the cells were infected with EnAd or NG-343 at 100 virus particles per cell (ppc) or left uninfected. Assays were performed 24, 48, or 72 hours post-infection. Virus replication assessed by qPCR

[00499] HT-29 cells infected for 24, 48, or 72 h with 100 ppc of EnAd or NG-343, or left uninfected, were used for viral DNA quantification by PCR. Cell supernatants were collected and clarified by centrifugation for 5 minutes at 1200 rpm. DNA was extracted from 10 μL of supernatant using the Sigma Genelute DNA extraction kit according to the manufacturer's protocol. A standard curve using EnAd virus particles (2.5 x 10¹⁰ to 2.5 x 10⁵ vp) was also prepared and extracted using the Sigma Genelute kit. Each extracted sample or standard was analyzed by qPCR using an EnAd gene-specific primer probe set, E3.

[00500] Quantification of the number of viral genomes detected per cell demonstrated that the replication of NG-343 and EnAd viruses was comparable at all time points analyzed (Figure 7A). No viral genomes could be detected in uninfected cells (data not shown). IFNa EXPRESSION ANALYSIS BY ELISA

[00501] Supernatants from HT-29 or A549 cell lines infected for 24, 48, or 72 h with 10 ppc of EnAd or NG-343, or left uninfected, were analyzed for secreted IFNα expression by ELISA. Culture supernatants were removed from each well and centrifuged for 5 minutes at 1200 rpm to remove residues. Petition 870170082359, dated 10 / 26 / 2017, pages 585 / 684 92 / 119 cells. The supernatants were diluted in 5% BSA / PBS assay buffer (1:2 or 1:50 or 1:100) and ELISA was performed using the Verikine human alpha IFN kit (Pbl assay science) according to the manufacturer's protocol.

[00502] The concentrations of secreted IFNa were determined by interpolating from the standard curves. Increased IFNa expression in cell supernatants during the course of infection was detected in both HT-29 and A549 cell lines (Figure 7B) CD80 CELL SURFACE EXPRESSION ASSESSED BY FLOW CYTOMETRY

[00503] A549 cell lines infected for 48 or 72 h with 10 ppc EnAd or NG-343 or left uninfected were used for analysis of CD80 transgene expression on the cell surface. At the appropriate time post-infection, A549 cells were removed from the plate surface by trypsin treatment, centrifuged, and then resuspended in 1% BSA / PBS. Samples were then incubated at 5°C for 1 h with buffer, Cy5-conjugated mouse isotype control antibody, or Cy5-conjugated anti-human CD80 antibody (clone 2D10). All samples were also stacked with live / killed Zombie Aqua for viable differentiated cells. The samples were washed 3 times with 1% BSA / PBS before analysis by flow cytometry (FACS, Attune) for cell viability and CD80 protein expression on the cell surface.Analysis of CD80 expression versus live / dead staining showed that at both 48 and 72 h post-infection, CD80 could be detected on the cell surface of NG-343-treated cells, but not on EnAd or uninfected control cells (UIC) (Figure 8). Cell viability at 72 h post-virus treatment was not sufficient to perform comprehensive CD80 expression analysis; however, high levels of CD80 could be detected in both live and dying NG-343-treated cells at this time point (Figure 8D, bottom panel). Petition 870170082359, dated 10 / 26 / 2017, pages 586 / 684 93 / 119

[00504] CD80 protein expression was then compared in HT-29 and A549 cells 48 h after infection with 100 ppc. Samples were harvested and stained as described above prior to analysis of cell viability and CD80 protein expression on the cell surface. Analysis of CD80 expression at this time point only in cells stained as live cells showed that CD80 could be detected on the surface of -91% of NG-343-treated HT-29 cells and -98% of NG-343-treated A549 cells, but not in EnAd-treated controls. EXAMPLE 9: SELECTIVITY OF NG-343 VIRUS ACTIVITY AND TRANSGENE EXPRESSION IN CARCINOMA, STROMAL FIBROBLASTS, AND EPITHELIAL CELL LINES.

[00505] To show that the IFNa and CD80 transgenes encoded in the NG-343 virus are selectively expressed only in cells permissive to infection with NG-343 or EnAd, virus replication (assessed by qPCR), IFNa expression (assessed by ELISA), and CD80 expression (assessed by flow cytometry) were measured in cancer cells (HT-29) known to be permissive to infection with EnAd, fibroblast cell lines (WI-38 and MRC-5) previously characterized as non-permissive, and a bronchial epithelial cell line (BE) that shows only limited permissiveness to infection with EnAd. In summary, cells were seeded in 12-well plates and infected 18 h after seeding with 100 ppc of NG-343 virus or EnAd for WI38, MRC5, or BE cells, or 10 ppc of NG-343 virus or EnAd for HT-29 cells. Cells were incubated with virus particles for 4 h before the infection medium was removed from the cells and replaced with fresh culture medium.1 h or 72 h after the 4 h infection period, cell supernatants were collected for qPCR or ELISA analysis, and cells were treated with trypsin to remove them from the plates for flow cytometry analysis. NG-343 Selective Virus Replication and ENAD Petition 870170082359, dated 10 / 26 / 2017, pp. 587 / 684 94 / 119

[00506] For qPCR, cell supernatants were collected and clarified by centrifugation for 5 minutes at 1200 rpm. DNA was extracted from 10 μL of supernatant using a Sigma Genelute DNA extraction kit, according to the manufacturer's protocol. A standard curve was obtained using EnAd virus particles (2.5 x 10¹⁰ to 2.5 x 10¹⁰). <5> (vp) was also prepared and extracted using the Sigma Genelute kit. Each extracted sample or standard was analyzed by qPCR using an EnAd E3 gene-specific primer probe set.

[00507] Quantification of the number of viral genomes detected per cell demonstrated that the replication of NG-343 and EnAd viruses was comparable in all cell lines analyzed (Figure 9A). NG-343 Selective Transgene Expression

[00508] For detection of IFNa expression, cell supernatants were collected and clarified by centrifugation for 5 minutes at 1,200 rpm. The supernatants were diluted in 5% BSA / PBS assay buffer (1:2 or 1:50 or 1:100) and ELISA was performed using the Verikine human alpha IFN kit (Pbl assay science) according to the manufacturer's protocol.

[00509] The concentrations of secreted IFNa were determined by interpolation of the standard curve. IFNa expression could only be detected in the supernatants of HT-29 cells infected with NG-343 and was not detectable (less than the lower limit of quantification [ <LLOQ]) em qualquer uma das linhagens de célula de fibroblasto ou linhagem de célula epitelial bronquial (Figura 9B).

[00510] For CD80 expression on the cell surface, cells were then incubated at 5 °C for 1 h with buffer, Cy5-conjugated mouse isotype control antibody, or Cy5-conjugated anti-human CD80 antibody (clone 2D10). All samples were also stacked with live / killed Zombie Aqua for differentiated viable cells. Petition 870170082359, dated 10 / 26 / 2017, pages 588 / 684 95 / 119 The samples were washed 3 times with 1% BSA / PBS before analysis by flow cytometry (FACS, Attune) for cell viability and CD80 protein expression on the cell surface. Regarding IFNα expression data, CD80 expression could only be detected in HT-29 cells, with no detectable expression in bronchial epithelial cell lines or fibroblasts (Figure 9C).

[00511] Together these data demonstrated that both IFNa and CD80 transgenes are selectively expressed in cells permissive to infection by EnAd virus, i.e., carcinoma cells, and transgene coding does not alter the selectivity of the NG-343 virus when compared to the parental EnAd virus. EXAMPLE 10: NG-343 TRANSGENE EXPRESSION ACTIVITY IN IMMUNE CELL ACTIVATION

[00512] To determine whether treatment of tumor cells with NG-343 virus could lead to enhanced innate immune cell responses compared to no treatment or treatment with EnAd, freshly isolated peripheral blood monocells (PBMCs) were co-cultured with tumor cells infected with NG-343 or EnAd, or left uninfected. Immune cell activation was assessed by flow cytometry analysis of innate immune cell populations or ELISA analysis of co-culture supernatants. Briefly, A549 lung carcinoma cells were seeded in 12-well plates at a density of 4 x 10 <5> cells / cavity. After 20 h, cells were infected with 10 ppc of EnAd or NG-343 virus or left uninfected and then incubated for 24 h, 37 degrees, 5% CO2.PBMCs isolated from a healthy human donor by density gradient centrifugation were then added to A549 culture wells at a ratio of 5 PBMCs to 1 A549 cell. 48 h after the addition, co-culture supernatants of PBMCs were harvested from the plates. To analyze dendritic cell activation at this point, the cells were... Petition 870170082359, dated 10 / 26 / 2017, pages 589 / 684 96 / 119 were incubated at 5 °C for 1 h with buffer, mouse isotype control antibodies conjugated to Alexa Fluor 488, PE, PerCP / Cy5.5, BV605 or BV412, anti-CD14 antibody conjugated to Alexa Fluor 488, anti-CD80 antibody conjugated to PE, anti-HLA-DR conjugated to PerCP.Cy5.5, anti-CD3 conjugated to BV605 or anti-PD-L1 antibody conjugated to BV421. All samples were also stained with live / killed Zombie Aqua for differentiated viable cells. Samples were washed 3 times with 1% BSA / PBS before flow cytometry analysis (FACS, Attune). Viable cells with negative staining for CD14 and CD3, but positive for HLA-DR, were defined as dendritic cell population. The expression of the DC activation marker, CD80 and PD-L1, was compared in this population (Figure 10).These analyses revealed that tumor cells infected with NG-343 were able to induce increased surface levels of CD80 and PD-L1 on the surface of DCs when compared to uninfected or EnAd-infected tumor cell culture. EXAMPLE 11: ACTIVITY OF ENAD VIRUS, NG-347, WHICH EXPRESSES THREE TRANSGENES; THE T-CELL ACTIVATOR CD80, THE CHEMOKINE MI1α AND THE CYTOKINE IFNα Characterization of viral activity in NG347 compared to ENAD in carcinoma cell lines.

[00513] CD80 transgene expression (evaluated by flow cytometry) and IFNa or MlP1a (CCL3) transgene expression (evaluated by ELISA) were compared in colon carcinoma cell lines treated with EnAd and NG-347, lung carcinoma cell lines or HT-29, A549. NG-347 is an EnAd-derived virus containing a transgene cassette encoding the human T-cell activating antigen, CD80, the human cytokine Interferon alfa 2b, and the human chemokine MlP1a (LD78P isoform). Transgene expression is under the control of the upstream promoter. Petition 870170082359, dated 10 / 26 / 2017, pp. 590 / 684 97 / 119 main endogenous virus. The schematic of the inserted cassette is shown in Figure 3F. Production of NG-347 virus is detailed in Example 6. A549 or HT-29 carcinoma cell lines were seeded in 12-well plates at cell densities of 7.5 x 10⁵ cells / well for A549 cells or 1.4 x 10⁶ cells / well for HT-29 cells. The plates were incubated for 18 h at 37 °C, 5% eCO₂, before the cells were infected with 100 particles of EnAd or NG-347 virus per cell (ppc) or left uninfected. Assays were performed 24, 48, or 72 hours post-infection. LFNa or MIP1a expression analysis by ELISA

[00514] Supernatants from HT-29 or A549 cell lines infected for 24, 48, or 72 h with 100 ppc of EnAd or NG-347 or left uninfected were analyzed for expression of secreted IFNα or secreted MlP1a by ELISA.

[00515] Culture supernatants were prepared according to the methods detailed in Example 9. IFN α ELISA was performed using the Verikine Human IFN alpha kit (Pbl assay science) and MlP1a ELISA was performed using the Quantikine CCL3 Human ELISA kit (R&D systems). Both assays were performed according to the manufacturer's protocol.

[00516] The concentrations of secreted IFNa or MlP1a were determined by interpolating the standard curves. The expression of IFNa and MlP1a increased in the cell supernatants during the course of infection, and was detected for both HT-29 and A549 cell lines (Figure 11A and Figure 11B). CD80 Expression Analysis by Flow Cytometry

[00517] CD80 protein expression was compared on the surface of HT-29 and A549 cells 48 h post-infection. As Petition 870170082359, dated 10 / 26 / 2017, pp. 591 / 684 98 / 119 cells were harvested and stained according to methods detailed in Example 9. Cells were analyzed for cell viability and CD80 protein expression on the cell surface by flow cytometry. Analysis of CD80 expression at this point in time in live cells showed that CD80 could be detected on the surface of -96% of NG-347-treated HT-29 cells and -99% of NG-347-treated A549 cells, but no staining was detected in EnAd-treated controls (Figure 11C). EXAMPLE 12: ACTIVITY OF ENAD VIRUS, NG-345, WHICH EXPRESSES THREE TRANSGENES; THE CYTOKINE LIGAND FLT3, THE CHEMOKIN MIP1A AND THE CYTOKINE IFNa. Characterization of viral activity in NG345 compared to ENAD in carcinoma cell lines.

[00518] Flt3 ligand, IFN α and MlP1a transgene expression (evaluated by ELISA) were compared in colon carcinoma cell lines treated with EnAd and NG-345, HT-29 or lung carcinoma cell lines, A549. NG-345 is an EnAd-derived virus containing a transgene cassette encoding a soluble variant of human Flt-3 ligand, the human cytokine Interferon alfa 2b, and the human chemokine MlP1 α (LD78P isoform). Transgene expression is under the control of the endogenous virus's main posterior promoter. Schematic of the inserted cassette is shown in Figure 3D. The production of NG-345 virus is detailed in Example 4. A549 or HT-29 carcinoma cell lines were seeded in 12-well plates at cell densities of 7.5x10⁵ cells / well for A549 cells or 1.4x10⁶ cells / well for HT-29 cells.The plates were incubated for 18 h at 37 °C, 5% eCO2, before the cells were infected with 100 EnAd or NG-345 virus particles per cell (ppc) or left uninfected. Assays were performed 24, 48, or 72 hours post-infection. Petition 870170082359, dated 10 / 26 / 2017, pages 592 / 684 99 / 119 FLT-3 Ligand Analysis, IFNa or MLP1a Expression by ELISA

[00519] Supernatants from HT-29 or A549 cell lines infected for 24, 48, or 72 h with 100 ppc of EnAd or NG-345 or left uninfected were analyzed for expression of secreted Fl3-ligand, secreted IFNa, or secreted MlP1a by ELISA.

[00520] Culture supernatants were prepared according to the methods detailed in Example 9. IFN α ELISA was performed using the Verikine Human IFN alpha Kit (Pbl Assay Science), MlP1 α ELISA was performed using the Quantikine Human CCL3 ELISA Kit (R&D Systems), and Flt3L ELISA was performed using the Flt3L Human ELISA Kit (Abeam). All assays were performed according to the manufacturer's protocol.

[00521] The concentrations of secreted IFNa, MlPa or FLt3L were determined by interpolation of standard curves. Expression of IFNa, MlP1 and FLt3L increased in cell supernatants during the course of infection and was detected in HT-29 and A549 cell lines (Figure 12A to C). EXAMPLE 13: ONCOLOGICAL ACTIVITY AND NG-347 and NG-348 Virus Infectivity in Colon Carcinoma Cells ONCOLYTIC POTENCY OF VIRUSES

[00522] HT-29 colon carcinoma cells were seeded in 96-well plates at a cell density of 2.5 and 4 cells / well. Plates were incubated for 4 h at 37 °C, 5% CO2, before cells were infected with EnAd, NG-347, or NG-348 virus particles in an infection density range of 100–0.39 particles per cell (ppc). HT-29 cell viability was assessed using MTS 96 Cell Titration Reagent (Promega: G3581) 72 h post-infection. Quantification of the % cell survival at each infection density demonstrated that the Petition 870170082359, dated 10 / 26 / 2017, pp. 593 / 684 The oncolytic potency of NG-347 and NG-348 was comparable to EnAd (Figures 13A and 13B). VIRAL PARTICLE INFECTIVITY

[00523] HT-29 colon carcinoma cells were seeded in 12-well plates at a cell density of 4e5 cells / well. Plates were incubated for 24 h at 37 °C, 5% CO2, before cells were infected with EnAd, NG-347, or NG-348 virus particles at an infection density range of 1.6e7 to 2e6 vp / ml. HT-29 cell infection was detected by antibody staining of the viral protein exon. Stained cells were quantified by manual counting of 6 fields of view per well, for 6 replication wells for each dilution tested. The particle-to-infectivity ratio (P:I) was calculated for each virus from the viral titer and demonstrated that NG-347 and NG-348 have similar infectivity ratios to EnAd reference controls (Figure 13C). EXAMPLE 14: CELL SURFACE EXPRESSION OF THE T-CELL ACTIVATING ANTIGEN, CD80, IN CARCINOMA CELL LINES INFECTED BY NG-348 AND NG-347

[00524] CD80 transgene expression (evaluated by flow cytometry) was compared in colon carcinoma cell lines treated with NG-347, NG-348 and EnAd, DLD-1 or lung carcinoma cell lines, A549. A549 or DLD-1 carcinoma cell lines were seeded in 12-well plates at cell densities of 7.5 and 5 cells / well. Plates were incubated for 18 h, 37 °C, 5% CO2, before cells were infected with 10 EnAd, NG-348 or NG347 virus particles per cell (ppc) or were left uninfected. CD80 protein expression was compared on the surface of A549 or DLD-1 cells at 24, 48, 72 or 96 h post-infection. At each time point, cells were harvested and stained according to methods detailed in Example 9. Cells were analyzed for cell viability and CD80 protein expression. Petition 870170082359, dated 10 / 26 / 2017, pp. 594 / 684 101 / 119 on the cell surface by flow cytometry. Analysis of CD80 expression 72 h post-infection in A549 cells showed CD80 could be detected on the surface of >95% of cells treated with NG-347 or NG-348 (Figures 14A and 14B). At 96 h post-infection, virus treatments lysed most A549 cells; therefore, FAC analysis was not performed. For DLD-1, cell expression could be detected in >50% of cells by 96 h post-treatment with NG-348 and >70% of cells following treatment with NG347 (Figures 15A and 15B). Staining was not detected in EnAd or untreated controls. EXAMPLE 15: T CELL ACTIVATION AND DEGRANULATION MEDIATED BY NG-348 INFECTED CARCINOMA CELL LINES

[00525] A549 lung carcinoma cells infected with NG-348 or EnAd virus particles or left uninfected were co-cultured with T cells isolated from human PBMC donors. The expression selectivity of NG-348-encoded CD80 was evaluated on the surface of A549 and T cells by flow cytometry. T cell activation was assessed by analyzing cell surface activation markers (by flow cytometry), cell surface CD107a expression as a marker for degranulation (by flow cytometry), and secretion of stimulating cytokines, IL-2 and IFNγ (by ELISA).

[00526] A549 cells were seeded in 12-well plates at a density of 5e5 cells / well. Plates were incubated for 18 h at 37 °C, 5% CO2, before cells were infected with 10 EnAd or NG-348 virus particles per cell (ppc) or left uninfected. At 48 h post-infection, CD3+ T cells, isolated by negative selection of PBMCs (MACs), were added to the A549 cell monolayers at a ratio of 8 T cells:1 tumor cell. Co-culture was performed for 16 h, after which cell supernatants were collected for ELISA analysis and cells Petition 870170082359, dated 10 / 26 / 2017, pages 595 / 684 102 / 119 tumor cells and T cells collected for flow cytometry analysis.

[00527] The culture medium containing non-adherent cells was removed from the co-culture wells and centrifuged (300 x g). The supernatant was carefully removed, diluted 1 in 2 with 5% PBS BSA, and stored for ELISA analysis. The adherent cell monolayers were washed once with PBS and then detached using trypsin. The trypsin was inactivated using complete media, and the cells were added to cell pellets that were collected from the culture supernatants. The cells were centrifuged (300 x g), the supernatant discarded, and the cell pellet washed in 200 μL of PBS. The cells were centrifuged again, then resuspended in 50 μL of FACs buffer (5% BSA PBS) containing Aqua Viva / Morta (Life tech) for 15 minutes at room temperature.Cells were washed once in FACs buffer before staining with directly conjugated antibody panels: anti-CD3 conjugated to BV605; anti-CD25 conjugated to BV421; anti-CD107a conjugated to FITC; anti-EpCam conjugated to PE or anti-CD4 conjugated to PE; and anti-CD80 conjugated to PE / Cy5 or anti-HLADR conjugated to PE / Cy5. A cell sample from each co-culture condition was also stained with relevant isotype control antibodies. All staining was performed in FACs buffer in a total volume of 50 μL / well for 15 minutes at 4 °C. Cells were then washed with FACs buffer (200 μL) before resuspension in 200 μL of FACs buffer and analysis by flow cytometry (Attune). Selective expression of CD80

[00528] Similar to the results shown in example 14, CD80 expression was detectable on the surface of >80% of EpCam+A549 cells infected with NG-348 but not infected with EnAd, or uninfected control cells (Figure 16). In contrast, CD3+ T cells did not show CD80 expression on the cell surface, indicating, at least under these experimental conditions, that transgene expression is selective for tumor cells in co-culture. Petition 870170082359, dated 10 / 26 / 2017, pp. 596 / 684 103 / 119 Upregulation of T Cell Activation Markers

[00529] Flow cytometry analysis of T cell activation was evaluated by expression of the activation of the T cell markers CD25 and HLA-DR in live single CD3+ cells. These data showed that both the number of T cells expressing CD25 (Figure 17A and 17B) and the mean level of CD25 expression on the T cell surface (Figure 17C) were significantly higher for T cells cultured with NG-348-infected A549 cells than EnAd or uninfected controls. Specifically, there was no difference in T cell activation status when comparing untreated controls to EnAd (26.9% ± 3.4% and 25.3% ± 3.5% of CD25-expressing T cells, respectively), while CD25 was upregulated in most NG-348 co-cultured cells (83.2% ± 1.5%). CD25 expression was also analyzed in CD4 and CD8 T cell subsets by monitoring CD3+ T cells based on their CD4 expression.These analyses showed that CD25 expression is significantly upregulated in both CD4+ and CD4- T cell subsets in co-cultures treated with NG-348 compared to EnAd and uninfected controls (Figure 18).

[00530] In contrast to CD25, the number of cells expressing HLA-DR was low, < 5%, for all conditions tested (Figure 19A). This is likely due to the early time point after co-culture at which flow cytometry analysis was performed. However, there was a small but significant increase in the mean HLA-DR staining fluorescence intensity of CD3+HLA-DR+ cells from NG-348-treated co-cultures compared to controls (Figure 19B). T-cell degranulation stimulation

[00531] Analysis of CD107a expression on the surface of live CD3+ T cells showed a significant increase in the number of T cells that degranulated and were therefore stained with CD107a, Petition 870170082359, dated 10 / 26 / 2017, pp. 597 / 684 104 / 119 when A549 cells were infected with NG-348 (8.3% ± 1.7% of cells) compared with EnAd (0.6% ± 0.2% of cells) or untreated controls (0.1% ± 0.02% of cells) (Figure 20). Similar to the upregulation of CD25, both the CD4+ and CD4- T cell subsets showed a significant increase in CD107a expression compared with EnAd or A549 controls (Figure 21). SECRETION OF THE STIMULATING CYTOKINES IL-2 AND IFNγ

[00532] For detection of IL-2 or IFNγ expression, co-culture supernatants were diluted in 5% BSA / PBS assay buffer (in a range of 1:100 to 1:1000) and ELISA was performed using the Human IL-2 Ready Set Go Kit (Affymetrix) or Human IFNγ Ready Set Go Kit (Affymetrix) according to the manufacturer's protocol. The concentrations of secreted IL-2 or IFNγ were determined by interpolation of the standard curves. IL-2 expression could only be detected in co-culture supernatants using NG-348-infected A549 cells and was not detectable in EnAd, or untreated controls (Figure 22A). IFNγ expression could also be detected at very high levels (> 300 ng / ml) in supernatants of co-cultures of A549 cells infected with NG-348, which was significantly higher than EnAd or untreated controls (Figure 22B). EXAMPLE 16: ACTIVATION OF CD4 AND CD8 T CELLS CAN BE INDEPENDENTLY MEDIATED BY CARCINOMA CELL LINES INFECTED WITH NG-348

[00533] A549 lung cells infected with NG-348 or EnAd virus particles, or left uninfected, were co-cultured with CD4+ T cells or CD8+ T cells isolated from human PBMC donors. T cell activation was assessed by secretion of the stimulating cytokine IFNγ in the culture supernatants.

[00534] A549 cells were seeded and infected Petition 870170082359, dated 10 / 26 / 2017, pages 598 / 684 105 / 119 with NG-348 or EnAd virus particles or left uninfected according to the methods detailed in Example 14. 48 h after infection, CD4+ T cells or CD8+ T cells isolated by negative selection from a PBMC donor were added to the A549 cell monolayer at a ratio of 8 T cells to 1 tumor cell. After 16 h, co-culture supernatants were collected and evaluated for IFNγ according to the methods detailed in Example 14.

[00535] For CD4+ T cells, IFNγ expression was only detected in supernatants of co-cultures of A549 cells infected with NG-348 and was not detectable in EnAd or untreated controls (Figure 23A). For CD8+ T cells, IFNγ expression was detected at significantly higher levels for A549 cells infected with NG-348 than for EnAd or untreated controls (Figure 23B), demonstrating that both CD8 and CD4 cells can be activated to secrete IFNγ by NG-348 virus activity in tumor cell lines. EXAMPLE 17: T CELL ACTIVATION MEDIATED BY NG-347 INFECTED CELL LINES

[00536] A549 lung carcinoma cells infected with NG-347 or EnAd virus particles or left uninfected were co-cultured with T cells isolated from human PBMC donors. T cell activation was assessed by analyzing surface cell activation markers (by flow cytometry) and secretion of the stimulating cytokine, IFN γ (by ELISA analysis of cell supernatants).

[00537] A549 cells were seeded in 12-well plates at a density of 5e5 cells / well. Plates were incubated for 18 h at 37 °C, 5% CO2, before cells were infected with 10 EnAd or NG-347 virus particles per cell (ppc) or left uninfected. 24 h after infection, CD3+ T cells isolated by negative selection of PBMCs (MACs) were added to the A549 cell monolayers at a Petition 870170082359, dated 10 / 26 / 2017, pp. 599 / 684 106 / 119 ratio of 5 T cells: 1 tumor cell. Co-culture was performed for 48 h before cell supernatants were collected for ELISA analysis and tumor cells and T cells harvested for flow cytometry analysis according to the methods detailed in EG 15. Harvested cells were stained directly with conjugated antibodies: anti-CD3 conjugated to BV605 and anti-CD69 conjugated to BV421. A cell sample from each co-culture condition was also stained with relevant isotype control antibodies. All staining was performed in FACs buffer in a total volume of 5 CAL / well for 15 minutes at 4 °C. Cells were then washed with FACs buffer (200 μL) before resuspension in 200 μL of FACs buffer and flow cytometry analysis (Attune). Upregulation of the T-cell activation marker, CD69

[00538] Flow cytometry analysis of T cell activation was evaluated by the expression of the T cell activation marker CD69 in live single CD3+ cells. These data showed that the number of T cells expressing CD69 was significantly higher for T cells cultured with NG-347-infected A549 cells than EnAd or uninfected controls (Figure 24). SECRETION OF THE STIMULATING CYTOKINE IFNγ

[00539] For detection of IL-2 or IFNγ expression, co-culture supernatants were diluted in 5% BSA / PBS assay buffer (in a range of 1:100 to 1:1000) and ELISA was performed using the Human IFN gamma Ready Set Go kit (Affymetrix) according to the manufacturer's protocol.

[00540] The concentration of secreted IFNγ was determined by interpolation of the standard curve. IFNγ expression could only be detected in the supernatants of co-cultures using A549 cells infected with NG-347 and was not detectable in EnAd, or untreated controls. Petition 870170082359, dated 10 / 26 / 2017, pp. 600 / 684 107 / 119 (Figure 25). EXAMPLE 18: PRODUCTION OF ENAD VIRUSES EXPRESSING THE CD80 CELL-ACTIVATING ANTIGEN TE, A MEMBRANE-ANCHORED SINGLE-CHAIN ​​Fv FRAGMENT ANTIBODY FOR THE ε CHAIN ​​OF THE HUMAN CD3 COMPLEX (CD3ε)

[00541] The pEnAd2.4 plasmid was used to generate the pNG-348A plasmids by direct insertion of a cassette encoding the human T-cell activating CD80 antigen (SEQ ID NO 11) and a membrane-anchored chimeric form of single-stranded anti-human CD3e Fv with a C-terminal V5 tag (SEQ ID NO: 99). The pNG-348 cassette contains: a 5' short junction acceptor sequence (SEQ ID NO. 2); membrane-anchored anti-human CD3e ScFv cDNA; a C-terminal V5 tag (SEQ ID NO: 100); a high-efficiency autocleavable P2A peptide sequence (SEQ ID NO: 7); human CD80 cDNA sequence and a 3' polyadenylation sequence (SEQ ID NO: 5). A schematic of the NG-348A transgene cassettes is shown in Figure 26A. The plasmid construct is confirmed by DNA sequencing. VIRAL PRODUCTION AND CHARACTERIZATION

[00542] The pNG-348A plasmid is linearized by restriction digestion with the enzyme AscI to produce the NG348A virus genome (SEQ ID NO: 101). The NG-348A virus is amplified and purified according to the methods detailed in Example 1. EXAMPLE 19: PRODUCTION OF ENAD VIRUS AND A MEMBRANE-ANCHORED SINGLE-CHAIN ​​Fv FRAGMENT ANTIBODY FOR THE ε CHAIN ​​OF THE HUMAN CD3 COMPLEX (CD3ε)

[00543] The pEnAd2.4 plasmid was used to generate the pNG-420 and pNG-420A plasmids by direct insertion of coding cassettes of a single-stranded, membrane-anchored chimeric form of the anti-human Fv CD3e with or without a C-terminal V5 tag (SEQ ID NO: 99). Petition 870170082359, dated 10 / 26 / 2017, pages 601 / 684 108 / 119 a V5 tag (SEQ ID NO: 15). The pNG-420 cassette contains: a 5' short junction acceptor sequence CAGG; membrane-anchored anti-human scFv CD3e cDNA and a 3' polyadenylation sequence (SEQ ID NO: 5). The pNG-420A cassette contains: a 5' short junction acceptor sequence cagg; membrane-anchored anti-human ScFv CD3e cDNA; a C-terminal V5 tag (SEQ ID NO: 100) and a 3' polyadenylation sequence (SEQ ID NO: 5). Schematics of the NG-420 and NG-420A transgene cassettes are shown in Figures 26B and 26C. The construction of each plasmid is confirmed by DNA sequencing. VIRAL PRODUCTION AND CHARACTERIZATION

[00544] The pNG-420 and pNG-420A plasmids are linearized by restriction digestion with the enzyme Ascl to produce the genomes of NG-420 (SEQ ID NO: 102) and NG-420A (SEQ ID NO: 103) viruses. The NG-420 and NG-420A viruses are amplified and purified according to methods detailed in Example 1. EXAMPLE 20

[00545] Human lung carcinoma cells A549 and human fibroblast cells MRC5 were cultured with EnAd, NG-347, or NG-348 viruses (at 10 ppc) to compare virus genome replication, virus exon, and transgene expression by these cell types. After 72 hours of culture, cells were stained for FACS analyses of surface markers or supernatants, and cell lysates were prepared for virus genome replication (qPCR) or mRNA analyses (RT-qPCR) of exon or transgene expression.

[00546] Viral genome replication and exon mRNA expression for the two transgene-carrying viruses, NG-347 and NG348, were equivalent to those of the parental virus, EnAd (Figure 27). For NG348 (Figure 28), CD80 levels and anti-human CD3scFv transgene mRNA expression were high in A549 tumor cells, with only a low-level signal for non-tumor MRC5 cells. Protein expression of Petition 870170082359, dated 10 / 26 / 2017, pp. 602 / 684 109 / 119 CD80 on the surface of cells assessed by FACS was detected in most cells treated with NG-348, but was not detectable in MRC5 cells, with no CD80 detected in any cell type left untreated or treated with EnAd. Similarly, CD80 transgene mRNA and protein expression following treatment with NG-347 were selectively detected in A549 tumor cells, but not in MRC5 cells (Figure 29).

[00547] For cell cultures treated with EnAd and NG-347, mRNA levels of MIPI1α and IFNα in cell lysates and secreted proteins in supernatants were measured by RT-qPCR and specific ELISAs, respectively. The data (Figure 30) show selective expression of both transgenes by A549 tumor cells without detectable MIPI1α chemokine or IFNα cytokine in MRC5 supernatants. EXAMPLE 21

[00548] The selectivity / activity of EnAd, NG347, and NG-348 viruses with human T cells was evaluated by culturing isolated CD3+ T cells for 3 days with 500 ppc or 5,000 ppc of each virus. Selectivity / activity was evaluated by a) flow cytometry analysis of T cells stained with CD69, CD4, CD80, CD25, and CD3 staining antibodies, b) ELISA analysis of human MIPI1α, IFNα, and IFNY protein secretion, c) qPCR analysis of virus replication, and d) RT-qPCR analysis of gene expression.

[00549] As shown in Figure 31, T cells were not favorable to viral genome replication for any of the viruses tested with only antecedent signals in the viral exon RT-qPCR assay. A549 tumor cells supported high levels of exon mRNA expression. RT-qPCR analyses for transgene mRNA expression by T cells showed only antecedent signals (<1 copy / cell) for CD80 by both NG-347 and NG-348, and a similar absence of significant ScFv anti-CD3 mRNA expression by NG-348, despite high virus exposure (5,000 Petition 870170082359, dated 10 / 26 / 2017, pp. 603 / 684 110 / 119 ppc). High levels of expression of both transgenes were detected in treated A549 tumor cells (10 ppc) (Figures 32 and 33). mRNA expression of the IFna and MIP1a transgenes was also selectively detected by A549 tumor cells treated with NG-347 (non-EnAd) (at 10 ppc) and not by T cells treated with 5,000 ppc (Figure 34). Additionally, CD80 cell surface protein expression was only detectable in A549 cells, not T cells, for both NG-347 and NG-348 (Figures 32 and 33). Treatment with EnAd did not lead to CD80 expression by any cell type, and A549 cell death (as assessed by dye uptake) was similarly high for all three viruses; A low level of non-specific T-cell death was induced by all viruses due to the very high levels of virus particles used in the experiment (Figures 32 and 33).Transgene-like mRNA and protein expression data were obtained when viruses were used at 500 ppc (data not shown).

[00550] In the absence of tumor cells, purified human T cells were not activated to upregulate CD25 or CD69 activation markers when cultured with either virus (Table 5). TABLE 5 · ABSENCE OF EXPRESSION OF CD25 and CD69 activation markers by CD3+ T cells HUMANS PURIFIED WITH 5,000 PPC OF DIFFERENT VIRUSES Untreated EnAd NG-347 NG-348 CD25+ CD4 T cells 30.7 24.6 23.4 23.3 CD69+ CD4 T cells 0.1 0.4 0.3 0.7 CD25+ CD8 T cells 5.9 4.7 4.1 4.1 CD69+ CD8 T cells 0.5 1.0 0.9 1.3 EXAMPLE 22

[00551] A virus selectivity experiment Petition 870170082359, dated 10 / 26 / 2017, pages 604 / 684 111 / 119 similar to that described in Example 21 was performed using non-separated human PBMCs instead of purified T cells, which includes performing the same assessments. As with human T cells in Example 21, the data from this study collectively demonstrate the absence of virus replication and transgene expression by human PBMCs. Figures 35 to 37 show data using 5,000 ppc of EnAd, NG-347, or NG-348, but similar data were generated using 500 ppc (not shown). Figure 35 shows virus genome replication and exon mRNA expression, and Figures 36 and 37 show transgene mRNA expression. Assay backgrounds were established based on signals generated in the assay with the respective virus mixed in the culture media and then processed in the same manner as cell lysate samples.No detectable CD80 transgene expression was observed in CD3+ T cells or CD40+ cells (mostly B cells) in these PBMC cultures with either virus (not shown).

[00552] The activation mediated by NG347 and NG-348 virus particles of innate immune cells (monocytes, DCs) in PBMC cultures was similar to that of EnAd, as shown in Figures 38 and 39 for downregulation of CD14 expression and upregulation of endogenous cell surface HLA-DR and CD80, as well as secretion of MIPI1α and IFNa (it is noted that although NG-347 encodes these two molecules in its genome, there was no increase in production levels compared to those of Ad and NG-348 which do not encode the transgenes). EXAMPLE 23

[00553] This example is similar in design to experiments in Examples 15 to 17, 21 and 22, but in those studies, purified human PBMCs or T cells were co-cultured with virus-pretreated (48 hours) A549 tumor cells or MRC5 fibroblasts. The A549 or MRC5 cells were treated with 10 ppc of EnAd, NG-347, NG-348 or left untreated (UTC) and cultured for 48 hours to allow sufficient time for Petition 870170082359, dated 10 / 26 / 2017, pp. 605 / 684 112 / 119 virus replication and any transgene expression. PBMCs or T cells were then added to the cultures and left for 24 or 48 hours to assess the ability of virus-treated cells to activate T cells.

[00554] Figure 40 shows viral genome replication data that demonstrate comparable replication of the three viruses in PBMC or T cell cocultures with both cell types, replication levels with high A549 tumor cells and low MRC5 fibroblasts.

[00555] T cell activation as measured by upregulation of CD25 surface expression and CD8 effector T cell degranulation as measured by upregulation of CD107a on the cell surface and IFNγ production measured by intracellular cytokine staining were all selectively stimulated by NG-348-treated A549 cells compared to EnAd-unstimulated MRC co-cultures (Table 6). TABLE 6 · FLOW CYTOMETRY ANALYSES OF Activation of human CD3+ T cells in co-cultures of PBMC T cells with viruses Cells Treatment % of CD25+ % of CD8+ CD107a+ +% of IFNy+ A549+ T cells Untreated 373 0.1 0.1 A549+ T cells EnAd 38.4 0.1 0.2 A549+ T cells NG-348 88.2 17.9 12.0 MRCS+ T cells Untreated 38.8 0.3 0.4 MRCS+ T cells EnAd 38.9 0.2 0.4 MRCS+ T cells NG-348 39.1 0.3 0.3 A549+ PBMCs Untreated 28.3 ND ND A549+ PBMCs EnAd 29.4 ND ND A549+ PBMCs NG-348 73.7 ND ND MRCS+ PBMCs Not treated 23.0 ND ND MRCS + PBMCs EnAd 23.3 ND ND Petition 870170082359, dated 10 / 26 / 2017, pages 606 / 684 113 / 119 Cells Treatment % of CD25+ % of CD8+ CD107a+ +% of IFNy+ MRC5 + PBMCs NG-348 21.7 ND ND ND = Not determined

[00556] IFNγ secretion in co-culture supernatants was also quantified by ELISA. The data (Figure 41) similarly demonstrate the selective activation of T cells co-cultured with NG-348-treated A549 tumor cells, non-MRC5 fibroblasts, with purified T cells or PBMCs used in the assays.

[00557] The ability of NG-347 to activate T cells was also evaluated by measuring CD69 levels in T cell cocultures of purified T cells or PBMCs with A549 tumor cells or MRC5 fibroblasts. As shown in Table Z, a small enhancement in CD69-positive T cells was seen with NG-347 treatment of A549 tumor cells compared to EnAd, which itself leads to upregulation of this earlier activation marker. These effects were not seen in MRC5 cocultures. No CD80 expression was detected on T cells (not shown). TABLE 7 · CD69 EXPRESSION IN NG-347 T CELLS OR CO-CULTURES TREATED WITH ENAD Cells Treatment % of CD69+ A549+ T cells Untreated 2.1 A549+ T cells EnAd 18.7 A549+ T cells NG-348 35.0 MRCS+ T cells Untreated 3.8 MRC5+ T cells EnAd 3.6 MRC5+ T cells NG-348 4.4 A549+ PBMCs Untreated 1.2 A549+ PBMCs EnAd 19.1 Petition 870170082359, dated 10 / 26 / 2017, pp. 607 / 684 114 / 119 Cells Treatment % of CD69+ A549 + PBMCs NG-348 28.7 MRCS + PBMCs Untreated 2.6 MRCS + PBMCs EnAd 2.7 MRCS + PBMCs NG-348 3.9

[00558] In a separate experiment, A549 cells treated with NG-347 and co-cultured with human CD3+ T cells led to upregulation of the activation marker CD69 in T cells and secretion of IFNγ (see Figures 24 and 25). EXAMPLE 24

[00559] CD14+ monolithic cells were isolated from PBMCs by antibody-coated magnetic bead separation and cultured with human IL-4 and GM-CSF to differentiate them into dendritic cells. After 3 days of culture, cells were treated with EnAd, NG-347, or NG-348 at 5,000 ppc or left untreated. As a positive activation control, some cells were stimulated with LPS. Two days later, the supernatants were taken for cytokine ELISAs, and cells were stained for surface activation marker expression and analyzed by flow cytometry. As shown in Table 8, all viruses induced upregulation of the co-stimulatory molecules CD80 and CD86, indicating that this previously identified particle-mediated innate immune cell activation effect was not altered by transgene incorporation into the NG-347 and NG-348 genomes.All viruses also stimulated secretion of similar levels of MIP1a and IFNa (Figure 42). TABLE 8 • PARTICLE-MEDIATED ACTIVATION OF HUMAN DENDRITIC CELLS BY ENAD, NG-347 AND NG-348 Treatment of CD % of CD80+ % of CD86+ Untreated 3.0 10.4 Petition 870170082359, dated 10 / 26 / 2017, pages 608 / 684 115 / 119 Treatment of CD % of CD80+ % of eCD86+ EnAd 81.6 99.3 NG-347 82.1 99.4 NG-348 62.5 99.5 Positive control of LPS 97.5 98.5 EXAMPLE 25

[00560] In a set of experiments, JurkatDual cells were used in co-cultures with tumor cells as a T-cell activation reporter assay to evaluate transgene expression functionality by NG-347, NG-348, and NG-420 viruses with EnAd serving as a negative control. JurkatDual cells stably express two different reporter genes: an NFKB reporter gene that produces a secreted form of luciferase that is responsive to signaling through the T-cell receptor complex and an IFNα-responsive secreted alkaline phosphatase (SEAP) reporter gene. A549 cells were pre-cultured with viruses at 10 ppc for two days, and then JurkatDual cells were added to an overnight culture (18–24 h) and then supernatants were collected for the luciferase assay and SEAP activities.As shown in Figure 43, A549 cells infected with NG-347 selectively induced SEAP production, which aligns with their IFNα production (see Figure 11), but did not induce luciferase activity. In contrast, NG-348 cells expressing the membrane anti-CD3 ScFv activated T cell receptor complex-induced luciferase, but not SEAP.

[00561] In another experiment, A549 lung carcinoma cells and HCT-116, HT-29, and DLD colon carcinoma cells were pre-cultured for 48 hours with 10 ppc of EnAd, NG-347, NG-348, or NG420 virus before co-culture with JurkatDual cells overnight, with supernatants tested for luciferase levels to indicate the level of activation induced. As shown in Figure 44, all four types of Petition 870170082359, dated 10 / 26 / 2017, pp. 609 / 684 116 / 119 tumor cells with NG-348 or NG-420 viruses, which encode cell surface anti-CD3 ScFv, activated JurkatDual cells while EnAd and NG-347 did not, with luciferase levels similar to those of uninfected tumor cell (UIC) controls.

[00562] In another experiment, A549 or HT-29 tumor cells were pre-cultured with different amounts of NG-348 or NG420 before adding JurkatDual cells and measuring their luciferase secretion. The data in Figure 45 show that the activation of NFKB activity in JurkatDual cells is dependent on the dose of virus used to treat the tumor cells. EXAMPLE 26

[00563] The in vivo pharmacokinetic biodistribution and particle-mediated systemic cytokine induction activities of EnAd and NG-348 following IV dosing in immunocompetent CD1 mice were compared. Mice were intravenously dosed with 5x10⁹ particles of EnAd or NG-348 and bled 2, 10, 30, 60, and 120 minutes post-dosing. Whole blood was DNA-extracted and analyzed by qPCR for viral genome levels (Figure 46). The elimination of both viruses from the blood followed similar kinetics. Similarly, the induction of MCP-1 cytokine response (a measure of particle-mediated activation of innate immunology such as liver Kupffer cells) was also similar for both viruses, as were the tissue biodistribution patterns (Figure 46). EXAMPLE 27

[00564] CB17 SCID mice were subcutaneously implanted with HCT116 cells and injected intratumorally (IT) or intravenously (IV) with EnAd, NG-347, or NG-348 virus (5x109 virus particles), or control once tumors were larger than 70 mm3. For IV-dosed mice, blood samples were taken from three mice from each group 3, 15, and 30 minutes after IV dosing, DNA was extracted, and the level of viral genomes in the blood was assessed by qPCR (analysis). Petition 870170082359, dated 10 / 26 / 2017, pp. 610 / 684 117 / 119 of pharmacokinetics [PK]). The data (Figure 47) show that NG-347 and NG-348 have similar PK to EnAd (and to each other). After 6 hours, tumors, livers, lungs, and spleens were cut from 3 mice from each group. The homogenized tissues were extracted for extracted DNA and analyzed for viral genome level by qPCR (biodistribution analysis). The data (Figure 48A) show similar tissue biodistribution for the three viruses. After 7 days or 14 to 21 days, tumors were excised from three mice from each group and homogenized to produce a tumor lysate that was used to prepare both DNA and RNA. The viral genome level in the tumors at the two time points was measured by qPCR analysis of the extracted DNA.The data (Figure 48B) show that mouse tumors dosed IV and IT have higher levels of viral genomes than the amount of virus dosed, indicating that viral replication in the tissue, with dosing IT producing higher genome levels than IV on day 7, but both being similarly high in the 14–21 day range. All three viruses replicated to similar levels.

[00565] Similarly, viral exon mRNA levels in tumor lysates detected by RT-qPCR were comparable between EnAd, NG-347, and NG-348 at both time points tested (Figures 49 and 50). Similar levels of anti-CD3 and CD80 ScFv mRNA were detected at both time points for both dosage routes for NG-348 treatment, with only antecedent assay reads using EnAd dosage (Figures 50 and 51). MIPI1α and IFNα mRNA levels were also selectively detected following NG-347, IT, or IV dosage (Figure 52).

[00566] The levels of CD80 protein encoded by NG347 and NG-348, and MlP1a protein encoded by NG-347 were measured in tumor lysates using specific ELISAs. The data in Figure 53 show that following a single IV virus dose, both proteins could also be selectively detected in tumor extracts. No protein Petition 870170082359, dated 10 / 26 / 2017, pp. 611 / 684 118 / 119 was detected in blood samples from the same mice. EXAMPLE 28 To evaluate the activity and tumor cell dependence of NG-348 virus in vivo, different combinations of human PBMCs (5x10⁷ cells), human A549 tumor cells (5x10⁶), and EnAd or NG-348 (at 5x10⁹ ppc) were injected into the peritoneum of beige SCID immunodeficient mice, with virus or control (saline) dosed within 15 minutes after cell injection. After 3 days, the peritoneal cavity was washed with 5 ml of saline, and recovered cells were analyzed by flow cytometry with a panel of T cell activation markers (CD25, CD69, and HLA-DR) to assess T cell activation levels, followed by monitoring of the CD3+ T cell population. Data from two separate experiments (Table 9) demonstrated that NG-348 selectively led to human T cell activation in vivo in a tumor cell-dependent manner. TABLE 9. IN VIVO ACTIVATION OF T CELLS IN Mice bearing tumor A549 B NG-348 Tumor Virus Group N % CD25+ % CD69+ % DR+ % CD25+, CD69+ % CO25+, DR+ Experiment 1 1 EnAd Saline 2 1.9; 2.3 1.6; 3.0 7.7; 9.1 0.2; 0.5 0.3; 0.6 2 EnAd 5x106 cells A549 2 4.2; 2.9 6.2; 5.5 8.4; 8.4 0.8; 0.3 1.4; 0.4 3 NG348 Saline 1 3.4 2.6 9.2 0.5 0.8 4 NG348 5x106 cells A549 2 35.8, 36.6 50.4, 42.2 26.3, 19.2 22.4, 18.0 16.4, 12.2 Experiment 2 Petition 870170082359, dated 10 / 26 / 2017, pp. 612 / 684 119 / 119 1 Saline Saline 1 25.6 37.3 14.8 14.1 7.08 2 EnAd Saline 2 6.5; 7.3 17.8; 18.2 5.50; 6.1 3.58, 3.46 1.01, 1.49 3 NG- 348 Saline 2 10.2; 6.5 26.7,18.3 7.7,6.0 6.73, 3.61 2.16, 1.44 4 Saline 5x106 cells A549 2 28.4, 22.7 54.4; 51.1 133 15.0 22.3 17.5 8.54, 7.72 5 EnAd 5x106 cells A549 1 13.2 29.4 5.1 7.84 1.62 6 NG348 5x106 cells A549 3 34.4; 29.6; 56.4 58.9; 59.2; 85.0 12.5 9.8; 17.0 27.2; 23.3; 52.7 9.07 7.5; 14.2 Petition 870170082359, dated 10 / 26 / 2017, pp. 613 / 684

Claims

1 / 6 CLAIMS 1. Oncolytic adenovirus of group B competent for replication with selectivity for cancer cells, wherein the adenovirus is characterized in that it comprises a transgene under the control of an endogenous viral promoter, wherein the transgene comprises a DNA sequence encoding a B7 protein, and said transgene is located between the stop codon polyA recognition site of the L5 adenovirus gene and the stop codon polyA recognition site of the E4 adenovirus gene, wherein said transgene is under the control of the endogenous late major promoter.

2. Replication-competent oncolytic virus according to claim 1, characterized in that the virus is a chimeric virus, optionally in which the main structure of the virus is enadenotucirev, optionally in which the virus has a formula (I): 5'ITR-Bi-Ba-B2-Bx-Bb-By-B3-3'ITR (I), wherein: B1 comprises: E1A, E1B or E1A-E1B; Ba is E2B-L1-L2-L3-E2A-L4; B2 is a linkage or comprises: E3 or a transgene, for example, under an exogenous or endogenous promoter; Bx is a linkage or a DNA sequence comprising: a restriction site, one or more transgenes or both; Bb comprises: L5; By comprises: a transgene encoding a B7 protein thereof; and B3 is a linkage or comprises: E4.

3. Replication-competent oncolytic virus according to claim 1 or 2, characterized in that the B7 protein is independently selected from the group comprising B7-1, B7-2, B7-DC, Petition 870260060809, dated 06 / 22 / 2026, page 32 / 39 2 / 6 B7-H1, B7-H2, B7-H3, B7-H4, B7-H5 and B7-H6; and / or the B7 protein comprises a transmembrane sequence, optionally wherein the transmembrane sequence comprises a PDGF receptor or a GPI anchor suitable for anchoring the protein or fragment to a cell membrane.

4. A replication-competent oncolytic virus according to any one of claims 1 to 3, characterized in that it additionally comprises a second transgene, optionally wherein the second transgene encodes a polypeptide selected from the group comprising a cytokine, a chemokine, an antagonist antibody molecule or fragment thereof, and an agonist antibody molecule or fragment thereof, optionally further, wherein the replication-competent oncolytic virus also comprises a third transgene, optionally, wherein the third transgene encodes a polypeptide selected from the group comprising a cytokine, a chemokine, a ligand, an antagonist antibody molecule and an agonist antibody molecule.

5. Replication-competent oncolytic virus according to claim 4, characterized in that: a) the second and third transgenes are located between the stop codon polyA recognition site of the L5 adenovirus gene and the stop codon polyA recognition site of the E4 adenovirus gene, wherein the second and third transgenes are under the control of the endogenous major late promoter; b) the second or third transgenes encode a cytokine, selected from the group comprising IL-2, IFN-alpha, IFN-beta, IFN-gamma, Flt3 ligand, GM-CSF, IL-15 and IL-12; c) the second or third transgenes encode a chemokine, selected from the group comprising MIP-1 alpha, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, Petition 870260060809, dated 06 / 22 / 2026, p.33 / 39 3 / 6 CCL2, CCL19 and CCL21; and / or d) the second and third transgenes encode a combination of a cytokine and a chemokine selected from the group comprising Mipl1a and Flt3 ligand and Mipl1a and IFNa.

6. A replication-competent oncolytic virus according to any one of claims 1 to 5, characterized in that the virus encodes an antibody molecule or fragment thereof, optionally, wherein the antibody molecule or antibody fragment comprises a transmembrane sequence or GPI anchor, such that it is a cell membrane-anchored form; the antibody molecule or antibody fragment comprises an anti-human CD3 antigen-binding domain; the antibody molecule is optionally an inhibitor, wherein the inhibitor is selected from the group comprising an inhibitor of an angiogenesis factor and an inhibitor of a T-cell deactivating factor; and / or the antibody molecule is optionally an agonist, wherein the agonist is an agonist of one or more selected from the group comprising CD40, GITR, OX40, CD27 and 4-1BB.

7. Pharmaceutical composition characterized in that it comprises an oncolytic adenovirus competent for replication as defined in any one of claims 1 to 6, and a diluent or carrier.

8. A replication-competent adenovirus according to any one of claims 1 to 6, or a composition according to claim 7, characterized in that it is for use in treatment, optionally for use in the treatment of cancer.

9. Replication-competent oncolytic group B adenovirus EnAd, characterized in that it comprises: two or more transgenes and a gene expression regulator; a first transgene comprising a DNA sequence encoding a B7-1 protein; and an additional transgene encoding an anti-CD3 antibody or a CD3-binding fragment thereof; wherein said transgenes are located between the polyA stop codon recognition site of the L5 adenovirus gene and the polyA stop codon recognition site of the E4 adenovirus gene; wherein said transgenes are under the control of the endogenous major late promoter for the virus.

10. Replication-competent oncolytic adenovirus according to claim 9, characterized in that the virus has a formula (I): 5'ITR-Bi-Ba-B2-Bx-Bb-By-B3-3'ITR (I), wherein: Bi comprises: E1A, E1B or E1A-E1B; Ba is E2B-L1-L2-L3-E2A-L4; B2 is a linkage or comprises E3 or a transgene; Bx is a linkage or a DNA sequence comprising: a restriction site, one or more transgenes or both; Bb comprises: L5; By comprises: a transgene cassette comprising a transgene encoding said protein B7-1, a transgene encoding said anti-CD3 antibody or anti-CD3 binding fragment thereof, and a regulatory element or a combination of regulatory elements; and B3 is a linkage or comprises: E4.

11. Replication-competent adenovirus, according to any one of claims 1 to 10, characterized in that the transgene comprises a regulatory element or regulatory sequence, optionally, wherein the regulatory element is a highly efficient self-cleaving peptide, optionally, wherein the peptide is selected from P2A, T2A, F2A and E2A.

12. A replication-competent oncolytic adenovirus according to any one of claims 1 to 11, characterized in that the virus encodes one or more proteins for expression on the surface of an infected cancer cell, optionally, wherein the virus encodes multiple proteins for expression on the surface of an infected cancer cell, and / or the proteins are independently processed and expressed at physically separate locations on the cancer cell membrane.

13. Replication-competent oncolytic adenovirus according to any one of claims 6 to 12, characterized in that: a) the anti-CD3 antibody or anti-CD3 binding fragment thereof further comprises a transmembrane domain or GPI anchor, such that it is a cell membrane-anchored form; and / or b) the anti-CD3 antibody or anti-CD3 binding fragment thereof is an scFv antibody fragment.

14. Replication-competent oncolytic adenovirus according to any one of claims 3 to 13, characterized in that the B7-1 protein: a) is a full-length B7-1 protein; b) comprises a GPI transmembrane or anchor domain; c) comprises a transmembrane domain of a B7 protein; d) is a human B7-1 protein; and / or e) has the sequence as defined in SEQ ID NO:

11.

15. Replication-competent oncolytic adenovirus, from Petition 870260060809, dated 06 / 22 / 2026, pp. 36 / 39 6 / 6 according to any of claims 9 to 14, characterized in that the anti-CD3 antibody molecule or the anti-CD3 binding fragment thereof comprises an anti-human CD3 antigen-binding domain.

16. Replication-competent oncolytic adenovirus according to any one of claims 6 to 15, characterized in that the anti-CD3 binding fragment has a sequence as defined in SEQ ID NO: 15 or 99.

17. Replication-competent oncolytic adenovirus according to claim 1, characterized in that the virus comprises a selected sequence of SEQ ID NO: 16, 19, 20, 96, 101.

18. Replication-competent oncolytic adenovirus according to claim 1 or 9, characterized in that the adenovirus comprises the sequence defined in SEQ ID NO: 96 or 101.

19. Pharmaceutical composition, characterized in that it comprises a replication-competent oncolytic adenovirus as defined in any one of claims 9 to 18 and a pharmaceutically acceptable excipient.

20. A replication-competent oncolytic adenovirus as defined in any one of claims 9 to 18, or a pharmaceutical composition as defined in claim 19, characterized in that it is for use in the treatment of, optionally, cancer, wherein the cancer is selected from colorectal, lung, bladder, renal, pancreatic, hepatic, head and neck, breast and ovarian cancer.

21. Method for generating a replication-competent oncolytic adenovirus as defined in any one of claims 1 to 18, characterized by being replication-capable in a host cell. Petition 870260060809, dated 06 / 22 / 2026, pp. 37 / 39

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