[go: up one dir, main page]

WO2018083259A1 - Adénovirus oncolytique codant pour des transgènes - Google Patents

Adénovirus oncolytique codant pour des transgènes Download PDF

Info

Publication number
WO2018083259A1
WO2018083259A1 PCT/EP2017/078218 EP2017078218W WO2018083259A1 WO 2018083259 A1 WO2018083259 A1 WO 2018083259A1 EP 2017078218 W EP2017078218 W EP 2017078218W WO 2018083259 A1 WO2018083259 A1 WO 2018083259A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
virus
replication
protein
transgene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2017/078218
Other languages
English (en)
Inventor
Alice Claire Noel BROMLEY
Brian Robert Champion
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Akamis Bio Ltd
Original Assignee
Psioxus Therapeutics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Psioxus Therapeutics Ltd filed Critical Psioxus Therapeutics Ltd
Publication of WO2018083259A1 publication Critical patent/WO2018083259A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to an oncolytic adenovirus encoding transgenes; compositions comprising the same; and use of the virus and compositions in treatment, particularly in the treatment of cancer.
  • Cancer is still a huge social burden to society in terms of the hardship and suffering of patients and their loved ones, and also in terms of the high financial cost of treating, caring for and supporting patients. It is now thought that the immune system of healthy individuals clears cancerous cells routinely. However, in those patients with cancer one or more of the defense mechanisms involved in this clearance is /are down regulated or turned off completely.
  • tumors change their microenvironment to make it more permissive to their growth. This occurs by the tumor releasing extracellular signals that, for example, promote tumor angiogenesis and/or induce local immune suppression or immune tolerance.
  • the present inventors have engineered certain oncolytic adenoviruses, to express a protein or proteins on the surface of a cancer cell it infects. Some early work they did three transgenes in an expression cassette was constructed with the transmembrane domain of the relevant protein at a terminal end of the encoding polynucleotide, i.e. in the format:
  • gene 1 and gene 2 are soluble proteins and gene 3 is a transmembrane anchored protein.
  • the proteins are expressed as one long polypeptide and require post-translational modification to release the individual proteins.
  • the soluble proteins encoded where small because it is known in the art at the viruses have limited "space" ability to accommodate transgenes.
  • all the transgenes are driven by one endogenous promoter and the said promoter may have reduced efficiency at the furthest extremity of the cassette.
  • this cassette format was designed to ensure the virus was not overloaded with genetic material and was able to express the proteins, without adversely affecting the virus life cycle, for example if the virus becomes inefficient the replication of the virus may be reduced or inhibited.
  • the life cycle of the virus is not adversely affected because the gene expression and oncolytic properties are closely linked to the virus life cycle. That is to the say the advantageous therapeutic properties of the virus may be lost, if virus replication is halted.
  • transgenes which encode proteins for cancer cell surface expression i.e. proteins comprising a transmembrane domain or GPI anchor
  • CD80 transgenes which encode proteins for cancer cell surface expression
  • the virus comprises at least one further transgenes (even where the further transgene encodes a soluble/secreted protein) in comparison to where the virus only comprises a transgene encoding a surface anchored protein.
  • the expression of the surface anchored protein is even improved when the surface expressed transgene is at the "front" of the expression cassette and is only released as an individual protein by a post-translational modification.
  • the expression is still better when four, five or six transgenes are co- located in the cassette. Surprisingly the virus can handle expressing all these transgenes.
  • a replication deficient or replication capable oncolytic adenovirus comprising:
  • a first transgene comprising a DNA sequence which encodes membrane anchored form of an immunomodulatory protein or an active fragment thereof
  • transgenes are located between the virus fibre gene L5 and the virus E4 gene and wherein the said transgenes are under the control of a promoter endogenous to the virus, such as the major late promoter.
  • a replication deficient or replication capable oncolytic adenovirus according to claim 1, where the first transgene encodes a membrane anchored form of CD80 or an active CD80 fragment.
  • a replication deficient or replication capable oncolytic adenovirus according to claim 1 or 2, wherein the virus comprises at least one further transgene gene encoding a protein between the virus fibre gene L5 and the virus E4 gene, wherein said further transgene is under the control of a promoter endogenous to the virus, such as the major late promoter.
  • a replication deficient or replication capable oncolytic adenovirus according to any one of claims 1 to 3, wherein two or more transgenes are located within a single transgene cassette, for example the first and second transgenes are in a single transgene cassette, or the first, second and further transgenes are in a single transgene cassette.
  • a replication deficient or replication capable oncolytic adenovirus according to any one of claims 1 to 4, wherein the cassette does not consists of:
  • a splice acceptor sequence Kozak sequence, start codon, INFa sequence, P2A sequence, MIP-la sequence, T2A sequence and hCD80 sequence, stop codon, and polyA tail sequence.
  • a replication deficient or replication capable oncolytic adenovirus according to any one of claims 1 to 5, wherein the first transgene adjoins the start codon.
  • a replication deficient or replication capable oncolytic adenovirus according to any one of claims 1 to 5, wherein the first transgene adjoins a transgene.
  • a replication deficient or replication capable oncolytic adenovirus according to any one of claims 1 to 9, wherein the second protein is an antibody or binding fragment thereof.
  • the antibody or binding fragment is human or humanised.
  • the antibody or antibody binding fragment is a cell membrane-anchored form, for example wherein the antibody or binding fragment comprises a transmembrane domain sequence or GPI anchor, such as a transmembrane domain from a PDGF receptor.
  • a replication deficient or replication capable oncolytic virus according to any one of claims 1 to 9, wherein the second protein is a cytokine or chemokine.
  • a replication deficient or replication capable oncolytic virus according to any one of claims 1 to 12 and 15, wherein the third protein is a cytokine or chemokine.
  • a replication deficient or replication capable oncolytic virus according to any one of claims 1 to 16, wherein the virus is a group B adenovirus, such as Adll.
  • a replication deficient or replication competent oncolytic virus according to any one of claims 1 to 17, wherein the virus is a chimeric virus, for example wherein the virus comprises a modified E2B region.
  • 20. A replication competent oncolytic virus according to any one of claims 1 to 19, wherein the virus has a formula (I) :
  • Bi comprises: E1A, E1B or E1A-E1B;
  • B A is E2B-L1-L2-L3-E2A-L4;
  • B2 is a bond or comprises E3 or a transgene, for example under an endogenous or exogenous promoter
  • ⁇ is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both;
  • ⁇ comprises L5
  • ⁇ comprises the transgenes according to the present disclosure
  • B3 is a bond or comprises E4.
  • a replication deficient or replication competent oncolytic virus according to any one of claims 3 to 2-, wherein the transgene cassette comprises one or more high efficiency self- cleavable sequences, for example two high efficiency cleavable sequences, in particular selected from P2A, T2A and combinations thereof.
  • transgene cassette contains only two transgenes and one high efficiency self-cleavable sequence, for example selected from P2A and T2A.
  • a pharmaceutical composition comprising a replication deficient competent oncolytic adenovirus according to any one of claims 1 to 23, and a diluent or carrier.
  • a method of treating a patient comprising administering a therapeutically effective amount of a replication competent adenovirus according to any one of claims 1 to 22 or a composition according to claim 24.
  • the first transgene when the second transgene encodes a membrane anchored form of CD80 the first transgene does not encode a membrane anchored from of an antibody or binding fragment specific to CD3.
  • the virus of the present disclosure does not have a sequence shown in SEQ ID NO: 12 (i.e. the genomic sequence of NG-348).
  • the oncolytic virus does not encode CD80.
  • the oncolytic virus does not encode a B7 protein.
  • the oncolytic virus does not encode an anti-CD3 antibody or binding fragment thereof.
  • the virus is not a group B virus.
  • Figure 1A is a schematic of the transgene cassette employed in the plasmid pNG-500.
  • Figure IB is a graph showing the oncolytic potency of NG-500 compared to EnAd.
  • Figure 1C is a bar chart showing the viral genome replication capability of NG-500 compared to EnAd.
  • Figure 2A is a series of scatter-plots showing the cell surface expression of CD80 in A549 cells infected with NG-500 vs EnAd.
  • Figure 2B is a bar chart showing the production of secreted MlPla in NG-500 vs EnAd infected A549 cells.
  • Figure 3 shows the results of a Jurkat Dual reporter assay demonstrating the functional activity of the CD80 and anti-CD3 ScFv transgenes in the NG-500 virus vs NG-348 virus.
  • Figure 4 is a series of scatter-plots showing the cell surface expression of CD107a in
  • Figure 5 is a bar chart showing the secretion of IFNy in NG-500, NG-348 and EnAd infected A549 cells.
  • SEQ ID NO: 6 Poly-adenylation sequence (SV40 late polyA sequence)
  • SEQ ID NO: 11 Splice Acceptor Sequence
  • SEQ ID NO: 12 NG-348 virus genome sequence comprising the EnAd genome with a transgene cassette that encodes a membrane-anchored chimeric form of the single chain Fv anti-human CD3e and the T lymphocyte activation antigen, CD80.
  • SEQ ID NO: 14 Membrane anchored form of the anti-human CD3 scFv with C- terminal V5 tag
  • SEQ ID NO: 28 Insulin-like Growth Factor 1 amino acid sequence
  • the first transgene is located closer to L5 than the second transgene, in particular the relative orientation of the genes is as shown in as the simplified representation (wherein not all regulatory elements, etc are shown) :
  • first transgene is the transgene encoding a therapy entity located after L5 in the direction toward the E4 region, in particular the relative orientation of the genes is as shown in as the simplified representation (wherein not all regulatory elements, etc are shown):
  • the oncolytic virus encodes a third transgene, for example co- located the first and second transgene.
  • the oncolytic virus encodes a fourth transgene, for example co- located with the first and second transgene, such as co-located with the first, second and third transgene.
  • the oncolytic virus encodes a fifth transgene, for example co- located with first and second transgene, such as co-located with the first, second, third and fourth transgene.
  • the oncolytic virus encodes a sixth transgene, for example co- located with the first and second transgene, such as with the first, second, third, fourth and fifth transgene.
  • the first transgene is protein is independently selected from an antibody or binding fragment thereof (such as an antagonistic antibody or binding fragment), a chemokine, a cytokine, an immunomodulator and an enzyme.
  • the second transgene is protein is independently selected from an antibody or binding fragment thereof (such as an antagonistic antibody or binding fragment), a chemokine, a cytokine, an immunomodulator and an enzyme.
  • the third transgene is protein is independently selected from an antibody or binding fragment thereof (such as an antagonistic antibody or binding fragment), a chemokine, a cytokine, an immunomodulator and an enzyme.
  • the fourth transgene is protein is independently selected from an RNAi, an antibody or binding fragment thereof (such as an antagonistic antibody or binding fragment), a chemokine, a cytokine, an immunomodulator and an enzyme.
  • the fifth transgene is protein is independently selected from an RNAi, an antibody or binding fragment thereof (such as an antagonistic antibody or binding fragment), a chemokine, a cytokine, an immunomodulator and an enzyme.
  • the sixth transgene is protein is independently selected from an RNAi, an antibody or binding fragment thereof (such as an antagonistic antibody or binding fragment), a chemokine, a cytokine, an immunomodulator and an enzyme.
  • a B7-1 protein encoded in an oncolytic viruses of the present disclosure can be useful because the extracellular domain of the protein family member generally modulates a biological function, for example the B7-1 (CD80) extracellular domain may be employed to prime or stimulate T cells.
  • B7-1 may include the ability to bind CD28 and/or CTLA-4, and in particular to signal or activate the relevant signaling cascade or cascades.
  • the transmembrane domain of B7-1 protein can be employed to direct 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 B7-1.
  • B7-1 may be secreted from the oncolytic virus by omitting a transmembrane domain.
  • B7-1 protein or CD80 as employed herein, unless the context indicates otherwise, refers to the full-length sequence of the B7-1 protein or a sequence at least 95% similar or identical thereto (such as 96%, 97%, 98%, 99% or 100% similar or identical thereto along the entirety of the relevant sequence). When the full-length protein is employed then at least one normal biological function of the protein will generally be present.
  • Full-length protein refers to at least the extracellular domain of the protein in question.
  • chimeric B7-1 proteins wherein the sequence of the chimaera has the structure and a function of a B7-1 protein and wherein the sequences that make up the chimaera are selected from B7-1 in combination with other proteins from the B7 family.
  • the B7 family includes B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7- H5, B7-H6 and B7-H7.
  • the elements in a fragment or full length B7-1 protein may be from the same or different B7 proteins.
  • the B7 fragment or protein is chimeric.
  • chimeric B7-1 proteins as employed herein refer to where substantially all the sequences making up the chimaera are from a B7-1 protein, for example at least 98% of the sequence of the chimaera is fragments of B7-1 proteins fused together.
  • a chimeric fragment as employed herein refers a fragment comprising a sequence from two or more different B7 proteins.
  • Chimeric B7-1 proteins may comprise B7-1 fragments from human and non-human species (such as mouse CD80 or similar).
  • the full-length B7-1 protein comprises the extracellular domain and the transmembrane domain, for example from the same B7-1 protein or alternatively the extracellular domain from another B7 protein (such as B7-2) and a transmembrane domain or equivalent, such as lipid membrane anchor, from a completely different protein.
  • a full-length chimeric B7-1 protein may comprise an extracellular domain of B7-1 protein and the transmembrane from a different B7 protein.
  • the full-length B7-1 protein comprises the extracellular domain, the transmembrane domain and intracellular domain, for example all from the same B7-1 protein or from two or more different B7 proteins, such as B7-1 and B7-2.
  • Active fragment refers to a fragment of a relevant full-length protein that has at least one function of the full-length protein (such as all the function of the full-length protein).
  • a B7-1 active fragment may for example bind CD28 or perform some other biological function of the full-length B7-1 protein.
  • 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.
  • the active fragment comprises or consists of a B7-1 extracellular domain or a sequence at least 95% similar or identical thereto, such as 96, 97, 98, 99 or 100% similar or identical.
  • the B7-1 fragment comprises or consists of a transmembrane domain from a B7 protein in particular one described herein, such as B7-1. Employing the latter is thought to contribute expression on the cell surface.
  • the active B7-1 fragment may be part of an extracellular domain.
  • An active fragment for example a transmembrane fragment or a larger fragment comprising B7-1 and other B7 domains may be employed in a fusion protein with an additional protein, for example to facilitate expression of the additional protein on the cancer cell surface.
  • fragment as employed herein 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 B7-1 and another B7 domains) which in turn may provide more functionality.
  • the larger fragment comprises some biological activity of the relevant B7 protein.
  • an active B7-1 fragment is a fragment that retains the essential biological activity of the full-length protein, for example the ability to prime or activate T cells.
  • the activity of a given protein fragment may be analysed in a relevant in vitro assay, for example using full-length protein as a comparator, for example employing an assay described in the Examples herein.
  • the active fragment is a transmembrane domain
  • the activity can be assessed by analysing the surface expression on cells of the relevant protein to which the transmembrane domain is attached, for example using an assay described in the Examples herein.
  • the B7-1 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.
  • a full length B7-1 protein comprising a transmembrane domain can be employed to present the extracellular domain of the B7-1 protein and the protein or fragment fused or linked thereto on the surface of the infected cancer cell.
  • the B7-1 protein will be attached 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.
  • the proteins can be arrange as desired, for example with the B7-1 extracellular domain at the N-terminal, fused or linked at its C-terminal to the next protein or fragment, which in turn is fused or linked at the C-terminal to the transmembrane domain, for example a transmembrane domain from a B7 protein.
  • Fusion protein refers to at least two proteins or fragments or a combination of at least one protein and at least one fragment fused directly or connected to each other, for example by a linker.
  • Fused as employed herein 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).
  • Linker refers to wherein two entities, such as two polypeptide sequences are connected via a linker.
  • a linker is a sequence which is not naturally present in either polypeptide or a sequence, which is not present in that particular position relative to both polypeptides.
  • the fusion protein comprises a B7-1 protein or an active fragment thereof. Fusion proteins comprising B7-1 fragments or protein and additional proteins are not referred to as chimeric proteins herein. Generally fusion protein as employed herein refers to a combination of a B7-1 protein or fragment thereof, optionally other B7 proteins/fragment and another non-B7-protein/fragment.
  • viruses of the present disclosure may encode entities in addition to the B7-1 protein or active fragment thereof, such entities include further proteins.
  • the B7 family includes B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7- H6, B7-H7, active fragments of the same, and combinations thereof.
  • 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.
  • CD80 has the following sequence shown in SEQ ID NO: 1.
  • 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 these proteins bind PD-1.
  • B7 proteins include B7-H2 (also known as ICOSLG, B7RP1, CD275: Uniprot number 075144) which binds ICOS, B7-H3 (also known as CD276: Uniprot number Q5ZPR3), B7-H4 (also known as VTCN1: Uniprot number Q727D3), B7-H5 (also known as VISTA, Platelet receptor Gi24, SISP1), B7-H6 (also known as NCR3LG1, NR3L1) which binds NKp30, B7-H7 (also known as HHLA2) which binds CD28H.
  • B7-H2 also known as ICOSLG, B7RP1, CD275: Uniprot number 075144
  • B7-H3 also known as CD276: Uniprot number Q5ZPR3
  • B7-H4 also known as VTCN1: Uniprot number Q727D3
  • B7-H5 also known as VISTA, Platelet receptor Gi
  • Individual proteins include single proteins, that is proteins or active fragments thereof that are not part of a fusion protein (including chimeric proteins), and also fusion proteins. In one embodiment the individual proteins are single proteins (including active fragments thereof).
  • the cytoplasmic domain of the B7-1 protein is present. In one 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 embodiments discussed below.
  • a transmembrane domain other than one derived from a B7-1 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 can be employed to present an active B7 protein fragment or another protein of interest on the surface of the infected cancer cell. Alternatively, it can be employed to present a fusion protein, for example comprising a B7 protein or active fragment thereof on said surface. In one embodiment the transmembrane domain from a PDGF receptor or fragment thereof is employed to express a B7 and/or another protein on the cancer cell surface.
  • a transmembrane tether or anchor sequence employed comprises a PDGFR TM domain (e.g. ala513-arg561), such as shown in SEQ ID NO: 9.
  • a tether or anchor sequence employed in the present disclosure comprises a tag attached, for example to a PDGF receptor or fragment thereof, such as PDGFR TM domain, in particular SEQ ID NO: 9.
  • Suitable tags include His-tags, Flag-tags, c-myc tag and the like. More specifically the tether or anchor may comprise a c-myc tag eg. of SEQ ID NO: 22 followed by a PDGFR TM domain is employed, (for example ala513-arg561), such as shown in SEQ ID NO: 9.
  • the c-myc tag comprises a spacer or spacer amino acids at the 3' and/or 5' end, for example gsEQKLISEEDLn (SEQ ID NO: 23 wherein the lowercase letters represent the amino acids which are added to the tag as spacers).
  • the tether or anchor sequence employed is gsEQKLISEEDLnAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 24) wherein the lowercase letters represent amino acid spacers).
  • the protein/polypeptide to which the tether or anchor is attached does not comprise a stop codon.
  • An exogenous protein or proteins encoded by the virus according to the present disclosure will generally comprise a leader sequence (also referred to as a signal peptide).
  • a leader sequence is, for example a sequence about 5 to 30 amino acids long located at the N-terminal of the protein or polypeptide.
  • the leader sequence for the protein to be expressed on the cancer cell surface is human, for example HuVHSS (SEQ ID NO: 104).
  • the structure of the ORF cassette comprising a first membrane anchored transgene comprises the following construct:
  • LS is a leader sequence, for example a human leader sequence
  • POLY is a polynucleotide encoding polypeptide or proteins of interest, in particular one disclosed herein;
  • TAG is a tag for example one disclosed herein, such as c-myc, in particular SEQ ID NO: 22 or 23;
  • TM_D is a TM domain for example a PDGFR TM domain, for example SEQ ID NO: 2.
  • the ORF may comprise the following construct:
  • LS is a leader sequence, for example a human leader sequence
  • VARi is a polynucleotide encoding a variable region such as VH region;
  • LINK is a linker, for example as disclosed herein, such as a linker based on the units of G 4 S, in particular SEQ ID NO: 25 GGGGSGGGGSGGGGS;
  • VAR 2 is a polynucleotide encoding a variable region, such as a VL region;
  • TAG is a tag, for example one disclosed herein, such as c-myc, in particular SEQ ID NO: 22 or 23;
  • TM_D is a TM domain for example a PDGFR TM domain, for example SEQ ID NO: 9.
  • transgenes in the construct may each be separated by a polynucleotide encoding a high efficiency self- cleavage peptide, for example independently selected from P2A, E2A, F2A and T2A.
  • the same peptide can be used in a construct more than once.
  • the DNA encoding the two peptides must be different because if the same DNA sequence is employed twice, it can result in stability in virus, for example it may result in homologous recombination and elimination of important section of DNA.
  • the disclosure also extends to embodiments, in particular those described specifically herein, which comprise a tag at the N- or C-termini of the polypeptide chains, such that it resides inside or on the outside of the membrane.
  • a C-termini tag located inside the membrane is advantageous because it is not likely to interfere with the binding or function of the polypeptide.
  • a combination of a transmembrane domain and a secretory signal sequence is employed to express a protein encoded by the virus (for example as described herein) on the surface of an infected cancer cell.
  • the present inventors have shown that the proteins encoded are expressed only on cells which are permissive to infection by the oncolytic virus, i.e. cancer cells.
  • the oncolytic adenovirus of the present disclosure comprises a fragment employed to express the protein on the surface of the infected cancer cell (such as the transmembrane fragment) is selected from about 20 to 25 hydrophobic amino acids which form a transmembrane alpha helix, for example from the proteins including PDGF receptor, insulin-like growth factor receptor, IL-6 receptor, CD28, glycophorin, LDL receptor, influenza HA protein, insulin receptor, Asialoglycoprotein receptor, Transferrin receptor.
  • the fragment employed to express the protein on the surface of the infected cancer cell is selected from the group comprising TM domain sequences (minimal portions) given in SEQ ID NO: 26, 27, 28, 29 or 30:
  • the transmembrane domain employed is derived from a G protein-coupled receptor or S antigen from hepatitis B.
  • a fusion protein comprising a full-length extracellular domain of a B7-1 protein or fragment and also a transmembrane domain derived from a protein other than B7-1 is arranged such that the B7-1 protein is located at the terminal end of the fusion protein distal from the cancer cell surface, that is on the outside of the cancer cell facing the extracellular space.
  • glycophospholipid anchor also referred to as a GPI anchor
  • Suitable glycophospholipid anchors include those from Thy-1, N-CAM and DAF.
  • transgenes according to the present disclosure under the control of an endogenous promoter is advantageous because the proteins are expressed in accordance with the virus life cycle as opposed to being constitutively expressed.
  • continuous expression under an exogenous promoter for example a strong promoter like the CMV promoter, may produce more protein than is necessary for a therapeutic effect and may result in off-target effects.
  • using an endogenous promoter reduces the size of the transgene or transgene cassette since there is no need to include an exogenous promoter.
  • Transgene(s) or transgene cassette in locations other than between L5 and the E4 region may be under the control of an exogenous promoter such as CMV, which may be advantageous because it can strongly and constitutively express the transgene which may be particularly useful in some situations, for example wherein the patient has a very pervasive cancer.
  • CMV exogenous promoter
  • the oncolytic virus according to present disclosure is an adenovirus, for example a group B adenovirus.
  • virus according to the present disclosure is a chimeric virus, for example EnAd.
  • the adenovirus is replication competent.
  • the virus is replication deficient and provided as a viral vector.
  • transgenes according to the present disclosure are located between the stop codon and polyA recognition site of the adenoviral gene L5 and the stop codon and polyA recognition site of the gene E4.
  • the transgenes according to the present disclosure are located between about bp 29356 and about 29357 of the EnAd genome, for example as shown in SEQ ID NO: 17, or a position equivalent thereto.
  • the skilled person 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 irrespective of the absolute numerical values employed.
  • the oncolytic adenovirus according to the present disclosure has a formula (I) :
  • Bi is a bond or comprises: E1A, E1B or E1A-E1B (in particular E1A, E1B or E1A-E1B); B A is E2B-L1-L2-L3-E2A-L4;
  • B2 is a bond or comprises E3 or a transgene, for example under an endogenous or exogenous promoter
  • ⁇ is a bond or a DNA sequence comprising: a restriction site, one or more transgenes or both;
  • ⁇ comprises L5
  • ⁇ comprises the transgenes according to the present disclosure.
  • B3 is a bond or comprises E4.
  • the oncolytic virus has a formula (la) : 5'ITR-B I-B A -B 2 -B B -BY-B3-3'ITR (la)
  • Bi is a bond or comprises: E1A, E1B or E1A-E1B (in particular E1A, E1B or E1A-E1B); B A is E2B-L1-L2-L3-E2A-L4;
  • B2 is a bond or comprises E3;
  • ⁇ comprises L5
  • ⁇ comprises the transgenes according to the present disclosure.
  • B3 is a bond or comprises E4.
  • the virus genome in constructs of formula (I) and/or (la) is from Adll or EnAd, in particular EnAd.
  • the transgene cassette is under the control of an endogenous promoter, for example the major late promoter.
  • ⁇ comprises a transgene cassette, said cassette comprising a transgene encoding a protein, such as a B7-1 protein or fragment thereof, and a regulatory element, such as combination of regulatory elements.
  • the regulatory element is splice acceptor sequence.
  • the regulatory element is a Kozak sequence.
  • the regulatory element is an IRES sequence.
  • the regulatory sequence is a high efficiency self-cleavable peptide sequence, such as a sequence independently selected from P2A, T2A, F2A, and E2A.
  • the regulatory sequence is a polyA tail.
  • splice acceptor and a Kozak sequence for example a splice acceptor and a polyA tail, or a splice acceptor and an IRES sequence, or a splice acceptor and a P2A sequence.
  • regulator sequences for example a splice acceptor sequence, a Kozak sequence and polyA tail, or a splice acceptor sequence an IRES or 2A sequence and a polyA tail; or a splice acceptor sequence, Kozak sequence and an IRES or 2A sequence.
  • regulatory sequences for example a splice acceptor sequence, a Kozak sequence, an IRES or 2A sequence and a polyA tail, in particular located between L5 and E4 in the order splice acceptor sequence, Kozak sequence, IRES or 2A sequence and a polyA tail.
  • the transgene encodes a polycistronic RNA molecule comprising both an IRES and a 2A regulatory sequence.
  • the virus of the present disclosure encodes multiple proteins, for example 3 proteins independently for expression on the surface of the infected cancer cell or secretion into the extracellular space, such as wherein at least one is a B7-1 protein or an active fragment thereof, one is an anti-human CD3 antibody or antibody fragment (such as a CD3s antibody or antibody fragment) and one is ⁇ - ⁇ or an active fragment thereof.
  • the virus may encode additional proteins, such a fourth, fifth or sixth protein.
  • 3 or more proteins are encoded by the virus for expression on the cancer cell surface or secretion into the extracellular space.
  • Protein in this context includes a fusion protein.
  • the virus of the present disclosure encodes two different B7 proteins, active fragments thereof or combinations of the same, for example both for expression on a cancer cell surface.
  • the virus according to the present disclosure encodes one or two protein(s) for cell surface expression and one or two proteins which are not capable of being anchored on the cell surface, for example are intended to act within the cancer cell or are for secretion/release from the cells.
  • a B7-1 protein or active fragment 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 B7-1 protein or a different B7 protein (including active fragments) is also encoded by the virus.
  • At least two different B7 proteins or active fragments are encoded by a virus of the present disclosure.
  • At least one protein expressed on the cell surface is a B7-1 protein and at least one non-cell-anchored (e.g. secreted) protein is a non-B7-l protein, for example ⁇ - ⁇ .
  • the non-B7-l protein, such as ⁇ - ⁇ may be anchored. This can be achieved for example by fusing ⁇ - ⁇ with a transmembrane domain.
  • the ⁇ - ⁇ (for example having an amino acid sequence given in SEQ ID NO: 3 or a sequence at least 95% identical thereto) is non-cell anchored and is secreted.
  • the ⁇ - ⁇ is cell anchored and expressed on the surface of the cancer cell.
  • the anti-CD3 antibody such as an anti-CD3s antibody having an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence at least 95% identical thereto is cell anchored and expressed on the surface of the cancer.
  • the anti-CD3 antibody is secreted and non-cell anchored.
  • the anti-CD3 antibody is fully human or humanised.
  • the multiple proteins may be encoded to be expressed as separate proteins which are independently processed and expressed in the cancer cell membrane.
  • the independence of the proteins on the surface of the cancer cell may make a positive contribution to the immune activation.
  • lipid packing can influence the fluidity (i.e. the viscosity) of the lipid bilayer in the membrane of the cancer cell. Viscosity of the membrane can affect the rotation and orientation of proteins and other bio-molecules within the membrane, thereby affecting the functions of these molecules.
  • the fluidity of the lipid bilayer allows independent movement of the molecules which may be a particularly suitable format, for example similar to a natural format that is conducive to biological function.
  • the independently processed and expressed proteins are located (anchored) in different locations, such as physically separate locations, in the cancer cell membrane.
  • one or more proteins (for example all the proteins) encoded by the virus and expressed on the surface of the infected cancer cell are not fusion proteins.
  • proteins are expressed as a fusion protein.
  • the virus of the present disclosure provides one or more separate independent proteins for cell surface expression and one or more fusion proteins for cell surface expression.
  • the virus according to the present disclosure comprises DNA sequences encoding said multiple proteins for expression, for example on the surface or the infected cancer cell, for example 1, 2, 3, 4, 5 or 6 proteins.
  • the virus according to the present disclosure comprises two or more transgenes, in the same or different locations in the virus genome.
  • the multiple proteins When located at the same position in the virus genome the multiple proteins will still be expressed independently at the surface of the cancer cell.
  • the multiple proteins are encoded in different locations in the virus genome, for example in E3, ⁇ and/or ⁇ and are expressed separately on the surface of the infected cancer cell.
  • the multiple proteins are encoded in the same location in the virus genome and expressed together on the infected cancer cell surface, for example where the proteins encoded are provided as a fusion protein, in particular wherein the fusion protein comprises a B7-1 protein or an active fragment thereof.
  • the oncolytic virus encodes 1, 2 or 3 surface expressed proteins and 5, 4, 3, 2 or 1 soluble proteins, wherein there are upto 6 transgenes encoded.
  • the B7-1 protein in the fusion protein is a full-length protein, fused or linked to another protein of interest or an active fragment thereof.
  • the fusion protein comprises a transmembrane domain from a B7-1 protein.
  • the B7-1 protein is an active fragment excluding the transmembrane domain.
  • a transmembrane other than one derived from a B7-1 protein may be employed to ensure the fusion protein is presented on the surface of the infected cancer cell.
  • the multiple proteins are encoded in the same location in the virus and are expressed as one or more fusion proteins together on the surface of the infected cancer cell.
  • genes may, for example be linked by an IRES sequence or a 2A peptide.
  • the virus according to the present disclosure comprises a fourth transgene and optionally a fifth 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.
  • 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, 4-1BB, GITR, 0X40, CD27, CD40 and combinations, in particular CD80, for example in forms suitable for expression on the surface of a cancer cell.
  • the protein is an anti-CD3 antibody, for example independently selected from a Muromonab-CD3 (also known as 0KT3), otelixizumab (also known as TRX4), teplizumab (also known as hOKT3yl (Ala-Ala) ), or visilizumab.
  • a Muromonab-CD3 also known as 0KT3
  • otelixizumab also known as TRX4
  • TRX4 teplizumab
  • teplizumab also known as hOKT3yl (Ala-Ala)
  • visilizumab for example independently selected from a Muromonab-CD3 (also known as 0KT3), otelixizumab (also known as TRX4), teplizumab (also known as hOKT3yl (Ala-Ala) ), or visilizumab.
  • the anti-CD3 antibody such as an anti-CD3s is in the form of an antibody fragment, for example an scFv that is part of a fusion protein with the transmembrane region of another protein, for example the transmembrane domain from the PDGF receptor or from the cell surface form of IgG
  • the protein is an anti-human CD3s scFv such as one having the amino acid sequence as set forth in SEQ ID NO: 2 or a sequence at least 95% identical thereto.
  • the antibody molecule is an inhibitor (antagonistic antibody) is independently selected from the group comprising an inhibitor of an angiogenesis factor, such as an anti-VEGF antibody molecule, and inhibitor of T cell deactivation factors, such as an anti-CTLA-4, anti-PDl or anti-PDLl antibody molecule.
  • the antibody molecule is an agonist independently selected from the group comprising antibodies to CD40, GITR, 0X40, CD27 and 4-1BB.
  • an additional transgene encodes a chemokine, selected from the group comprising MIP-la, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19 and CCL21.
  • a chemokine selected from the group comprising MIP-la, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19 and CCL21.
  • the transgene encodes MIP-la, for example having the amino acid sequence as set forth in SEQ ID NO: 3 or a sequence at least 95% identical thereto.
  • an additional transgene encodes a cytokine, or soluble variant thereof selected from the group comprising IL-2, IFNa, IFNp, IFNy, GM-CSF, IL-15, IL-12 and fms-related tyrosine kinase 3 ligand (FLT3L).
  • a cytokine or soluble variant thereof selected from the group comprising IL-2, IFNa, IFNp, IFNy, GM-CSF, IL-15, IL-12 and fms-related tyrosine kinase 3 ligand (FLT3L).
  • FLT3L fms-related tyrosine kinase 3 ligand
  • one or more molecules are also expressed on the surface and/or secreted.
  • the virus encodes B7-1 and B7-2 or an active fragment of any one of the same or a combination thereof.
  • the virus encodes the B7-1 or an active fragment thereof for expression on the surface of the infected cancer cell and an anti-CD3 (agonist), such as anti-CD 3 ⁇ antibody or antibody binding fragment (for example as a scFv, for example having the amino acid sequence set forth in SEQ ID NO: 2 or a sequence at least 95% identical thereto) also for expression on the cancer cell surface, in particular where the proteins are expressed as individual proteins on the cell surface.
  • an anti-CD3 agonist
  • anti-CD 3 ⁇ antibody or antibody binding fragment for example as a scFv, for example having the amino acid sequence set forth in SEQ ID NO: 2 or a sequence at least 95% identical thereto
  • the virus encodes B7-1 or an active fragment thereof for expression on the surface of the infected cancer cell and an anti-CD3e, and an anti-VEGF (antagonist) antibody or a binding fragment thereof also for expression on the cancer cell surface or for release from the cancer cell, for example by secretion or after lysis/death of the infected cancer cell.
  • the virus encodes B7-1 or an active fragment 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, in particular CD80, also for expression on the cancer cell surface or for release from the cancer cell, for example by secretion or release after lysis/death of the infected cancer cell.
  • the virus encodes B7-1 or an active fragment of 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 cell lysis/death of the infected cancer cell.
  • a cytokine selected from IL-2, IFN- alpha, IFN-beta, IFN-gamma, GM-CSF, IL-15, IL-12, and FLT3L
  • the virus encodes B7-1 or an active fragment thereof for expression on the surface of the infected cancer cell and a chemokine selected from MIP-la, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21, in particular MIP-la, for example for release from the cancer cell, in particular by secretion or release after cell lysis/death of the infected cancer cell.
  • a chemokine selected from MIP-la, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21, in particular MIP-la, for example for release from the cancer cell, in particular by secretion or release after cell lysis/death of the infected cancer cell.
  • the virus encodes B7-1 or an active fragment of any one of the same or a combination thereof for expression on the surface of the infected cancer cell and an anti-CD3, such as an anti-CD3s (agonist) antibody or antibody binding fragment (such as a scFv, for example having the amino acid sequence set forth in SEQ ID NO: 2 or a sequence at least 95% identical thereto) also for expression on the cancer cell surface (in particular where the proteins are expressed as individual proteins on the cell surface) and further encodes a cytokine or chemokine selected from IL-2, IFN-alpha, IFN-gamma, GM- CSF, IL-15, IL-12, FLT3L, MIP-la, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21, in particular MIP-la, for example for release from the cancer cell, in particular by an anti-
  • the virus encodes B7-1 or an active fragment thereof for expression on the surface of the infected cancer cell and an anti-CD3 (agonist) antibody or antibody fragment (such as a scFv, for example having the amino acid sequence set forth in SEQ ID NO: 2 or a sequence at least 95% identical thereto) also for expression on the cancer cell surface (in particular where the proteins are expressed as individual proteins on the cell surface) and further encodes an antibody, antibody fragment or protein ligand that binds CD28, CD80, CD86, 4-1BB, GITR, 0X40, CD27, CD40 or an anti-VEGF (antagonist) antibody, in particular CD80, also for expression on the cancer cell surface or for release from the cancer cell, for example by secretion or release after lysis/death of the infected cancer cell.
  • an anti-CD3 (agonist) antibody or antibody fragment such as a scFv, for example having the amino acid sequence set forth in SEQ ID NO: 2 or a sequence at least 95% identical thereto
  • the virus encodes B7-1 or an active fragment thereof for expression on the surface of the infected cancer cell and two different cytokines or chemokines selected from IL-2, IFNa, IFNp, IFNy, GM-CSF, IL-15, and IL-12, FLT3L, ⁇ , IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21, in particular MIP-la, for example for release from the cancer cell, in particular by secretion of after cell lysis/death of the infected cancer cell.
  • two different cytokines or chemokines selected from IL-2, IFNa, IFNp, IFNy, GM-CSF, IL-15, and IL-12, FLT3L, ⁇ , IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10,
  • the virus encodes B7-1 or an active fragment thereof for expression on the surface of the infected cancer cell and an anti-CD3 (agonist) antibody or antibody binding fragment (such as a scFv, for example having the amino acid sequence set forth in SEQ ID NO: 2 or a sequence at least 95% identical thereto) also for expression on the cancer cell surface (in particular where the proteins are expressed as individual proteins on the cell surface) and further encodes a cytokine independently selected from IL-2, IFNa, IFNy, GM-CSF, IL-15, and IL-12, and or a chemokine selected from RANTES (CCL5), MIP-la (LD78a (CCL3), LD78P (CCL3L1) isoforms)or ⁇ , in particular MIP-la, which can be released from the cancer cell, in particular by secretion before and release after cell lysis/death of the infected cancer cell.
  • an anti-CD3 (agonist) antibody or antibody binding fragment such as a sc
  • the virus further encodes an anti-PD-1 antibody (an antagonist).
  • the protein or proteins encoded in the transgene cassette for cell membrane expression may also comprise a peptide linker or spacer between the transmembrane domain and the extracellular ligand binding domain.
  • linkers or spacers may add flexibility to the cell surface expressed protein that enhances the ability of the protein to interact with its target molecule, for example on an adjacent cell.
  • linkers or spacers may also be designed or selected to promote dimerisation or trimerisation of the proteins at the cell surface, via disulphide bond formation or protein-protein interactions.
  • the hinge regions of immunoglobulin molecules or CD8 may be employed to enhance both flexibility and dimerisation.
  • the protein or proteins encoded in the transgene cassette may also comprise a peptide tag.
  • the peptide tag may include c-myc, poly-histidine, V5 or FLAG tags and can be located on the N-terminus 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 can be used as spacers or linkers between different protein domains, for example the transmembrane and the extracellular domain, and can be used for detection or purification or detection of the protein, or cells expressing the protein.
  • the one or more additional transgenes is/are under the control of an exogenous or endogenous promoter, for example an endogenous promoter.
  • a transgene in the E3 region (B2) is under control of an exogenous promoter.
  • the one or more additional transgenes genes are between the E3 region and the fibre L5 in the adenovirus genome, for example at a position ⁇ in the construct of formula (I), in particular under the control of an exogenous promoter.
  • a transgene in ⁇ is under the control of an exogenous promoter.
  • the one or more additional transgenes genes are between the E4 region and the fibre L5 in the adenovirus genome, for example at a position ⁇ in the construct of formula (I) or (la), in particular under the control of an endogenous promoter, such as the major late promoter. This may be in addition to the B7-1 protein or active fragment thereof encoded in the region ⁇ .
  • compositions comprising an oncolytic adenovirus according to the present disclosure, for example a pharmaceutical composition, in particular comprising a pharmaceutically acceptable excipient, such as a diluent or carrier.
  • a pharmaceutically acceptable excipient such as a diluent or carrier.
  • an oncolytic adenovirus according to the present disclosure or a composition comprising the same for use in treatment, in particular for use in the treatment of cancer.
  • a method of treating a patient in need thereof 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.
  • an oncolytic adenovirus according to the present disclosure or a composition comprising the same for the manufacture of a medicament for the treatment of cancer, in particular carcinomas, for example colorectal, lung, bladder, renal, pancreatic, hepatic, head and neck, breast or ovarian cancer.
  • a polynucleotide 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 B7-1 protein or an active fragment thereof.
  • the polynucleotide sequence is in the form of a plasmid.
  • a host cell for example a mammalian cell, such as a HEK293 cell or a derivative thereof, comprising an oncolytic virus according to the present disclosure or a polynucleotide sequence according to the present disclosure.
  • a process for preparing an oncolytic adenovirus according to the present disclosure comprising a step of inserting a polynucleotide encoding the transgenes according to the present disclosure into an oncolytic adenovirus.
  • a process of replicating a virus according to the present disclosure comprising the step of culture host cells in the presence of the virus under conditions suitable for replication.
  • the method will comprise a further step of harvesting the virus, for example from the supernatant or after lysis of the host cells.
  • Oncolytic virus with selectivity for cancer cells refers to a virus that preferentially kills cancer cells, for example because it preferentially infects cancer cells and/or the virus life cycle is dependent on a gene, such as p53 that is disregulated, for example over-expressed in cancer cells.
  • the oncolytic virus preferentially infects cancer cells and goes on to replicate its genome and produce capsid proteins to generate new virus particles, for example as per EnAd.
  • the selectivity for cancer cells can be tested as described in WO2005/118825 incorporated herein by reference.
  • Transgene as employed herein refers to a gene that has been inserted into the genome sequence of the adenovirus, wherein the gene is unnatural to the virus (exogenous) or not normally found in that particular location in the virus. Examples of transgenes are given herein. Transgene as employed herein also includes a functional fragment of the gene that is a portion of the gene which 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.
  • Transgene and coding sequence are used interchangeably herein in the context of inserts into the viral genome, unless the context indicates otherwise.
  • Coding sequence as employed herein means, for example a DNA sequence encoding a functional RNA, peptide, polypeptide or protein.
  • the coding sequence is cDNA for the transgene that encodes the functional RNA, peptide, polypeptide or protein of interest.
  • Functional RNA, peptides, polypeptide and proteins of interest are described below.
  • transgene as employed herein refers to a segment of DNA containing a gene or cDNA sequence that has been isolated from one organism and is introduced into a different organism i.e. 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. Transgenes employed may for example encode a single proteins or active fragment thereof, chimeric protein or a fusion protein.
  • virus genome contains coding sequences of DNA. Endogenous (naturally occurring genes) in the genomic sequence of the virus are not considered a transgene, within the context of the present specification unless then have been modified by recombinant techniques such as that they are in a non-natural location or in a non-natural environment.
  • the transgene (s) inserted encodes a human or humanised protein, polypeptide or peptide.
  • the transgene (s) may be located within a transgene cassette for example.
  • the transgene comprises a DNA sequence encoding a B7-1 protein or an active fragment thereof.
  • the present disclosure provides that the B7-1 protein or activate fragment thereof may be provided in one or more formats independently selected from a fusion protein, a simple B7 protein or an active fragment thereof.
  • Simple B7-1 protein or an active fragment thereof as employed herein refers to proteins which are essentially wild-type proteins, for example which are not part of a fusion protein and which has a sequence identical or similar to the relevant known protein, in particular the known human protein. Simple gene also includes wherein 10% of the amino acids are substituted or deleted over the whole length of the relevant protein.
  • GPI anchor refers to is a glycolipid that can be attached to the C- terminus of a protein during posttranslational modification. It is composed of a phosphatidylinositol group linked through a carbohydrate-containing linker (glucosamine and mannose glycosidically bound to the inositol residue) and via an ethanolamine phosphate (EtNP) bridge to the C-terminal amino acid of a mature protein. The two fatty acids within the hydrophobic phosphatidyl-inositol group anchor the protein to the cell membrane.
  • Glypiated (GPI-linked) proteins generally contain a signal peptide, thus directing them into the endoplasmic reticulum (ER).
  • the C-terminus is composed of hydrophobic amino acids that stay inserted in the ER membrane. The hydrophobic end is then cleaved off and replaced by the GPI-anchor.
  • the protein progresses through the secretory pathway, it is transferred via vesicles to the Golgi apparatus and finally to the extracellular space where it remains attached to the exterior leaflet of the cell membrane. Since the glypiation is the sole means of attachment of such proteins to the membrane, cleavage of the group by phospholipases will result in controlled release of the protein from the membrane. The latter mechanism is used in vitro; i.e., the membrane proteins released from the membranes in the enzymatic assay are glypiated protein.
  • PLC Phospholipase C
  • T-cell marker Thy-1 and acetylcholinesterase, as well as both intestinal and placental alkaline phosphatases, are known to be GPI-linked and are released by treatment with PLC.
  • GPI-linked proteins are thought to be preferentially located in lipid rafts, suggesting a high level of organization within plasma membrane microdomains.
  • Replication competent in the context of the present specification refers to a virus that possesses all the necessary machinery to replicate in cells in vitro and in vivo, i.e. without the assistance of a packaging cell line.
  • a viral vector, for example deleted in at least the EIA region, capable of replicating in a complementary packaging cell line is not a replication competent virus in the present context.
  • a viral vector is a replication deficient virus, which requires a packaging cell line (comprising a transgene) to replicate.
  • a replication capable virus as employed herein refers to a replication competent virus or a virus whose replication is dependent on a factor in the cancer cells, for example an upregulated factor, such as p53 or similar.
  • the adenovirus is a human adenovirus.
  • "Adenovirus”, “serotype” or adenoviral serotype” as employed herein refers to any adenovirus that can be assigned to any of the over 50 currently known adenoviral serotypes, which are classified into subgroups A-F, and further extends to any, as yet, unidentified or unclassified adenoviral serotypes. See, for example, Strauss, "Adenovirus infections in humans," in The Adenoviruses, Ginsberg, ea., Plenum Press, New York, NY, pp.
  • Adenoviruses are grouped based on their capsid.
  • the adenovirus is a subgroup B, for example independently selected from the group comprising or consisting of: Ad3, Ad7, Adll, Adl4, Adl6, Ad21, Ad34 and Ad51, such as Adll, in particular Adllp (the Slobitski strain).
  • the adenovirus of the invention has the capsid, such as the hexon and/or fibre of a subgroup B adenovirus, such as Adll, in particular Adllp.
  • the adenovirus is Adll or has the fibre and/or hexon and/or penton of Adll, such as Adllp.
  • the virus of the present disclosure is not a group A virus.
  • virus of the present disclsoure does not comprise an adeno death protein (ADP).
  • ADP adeno death protein
  • the virus of the present disclosure is not a group C virus.
  • the virus of the present disclosure does not comprise more and a fragment of part of an Ad5 virus.
  • Enadenotucirev is a chimeric oncolytic adenovirus, formerly known as ColoAdl (WO2005/118825), with fibre, penton and hexon from Adllp, hence it is a subgroup B virus. It has a chimeric E2B region, which comprises DNA from Adllp 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 additional genetic material whilst remaining viable. Furthermore, because EnAd is a subgroup B adenovirus, pre-existing immunity in humans is less common than, for example, Ad5. Other examples of chimeric oncolytic viruses with Adll fibre, penton and hexon include OvAdl and OvAd2 (see WO2006/060314).
  • EnAd seems to preferentially infect tumour cells, replicates rapidly in these cells and causes cell lysis. This, in turn, can generate inflammatory immune responses thereby stimulating the body to also fight the cancer. Part of the success of EnAd is hypothesised to be related to the fast replication of the virus in vivo.
  • EnAd can be administered systemically (e.g. by intravenous or intraperitoneal injection or infusion) and then subsequently selectively infect and express proteins within tumour cells.
  • This property of EnAd which may be shared by Adllp and other group B adenoviruses in particular those expressing the capsid proteins of Adllp (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 tumour, which is not feasible for many cancers.
  • EnAd selectively lyses tumour cells
  • transgenes such as a transgene which encodes a cell signalling protein or an antibody, or a transgene which encodes an entity which stimulates a cell signalling protein (s).
  • Advantageously arming a virus, with DNA encoding certain proteins that can be expressed inside the cancer cell may enable the body's own defenses to be employed to combat tumour cells more effectively, for example by making the cells more visible to the immune system or by delivering a therapeutic gene/protein preferentially to target tumour cells.
  • the oncolytic adenovirus of the present disclosure stimulates the patient's immune system to fight the tumor, for example by reducing the cancers ability to suppress immune responses.
  • the oncolytic virus has a fibre, hexon and penton proteins from the same serotype, for example Adll, in particular Adllp, for example found at positions 30812-31789, 18254-21100 and 13682-15367 of the genomic sequence of the latter wherein the nucleotide positions are relative to Genbank ID 217307399 (accession number: GC689208).
  • Adll hexon and penton proteins from the same serotype
  • Adllp for example found at positions 30812-31789, 18254-21100 and 13682-15367 of the genomic sequence of the latter wherein the nucleotide positions are relative to Genbank ID 217307399 (accession number: GC689208).
  • the adenovirus is enadenotucirev (also known as EnAd and formerly as ColoAdl).
  • Enadenotucirev refers the chimeric adenovirus of SEQ ID NO: 21. It is a replication competent oncolytic chimeric adenovirus which has enhanced therapeutic properties compared to wild type adenoviruses (see WO2005/118825).
  • EnAd has a chimeric E2B region, which features DNA from Adllp and Ad3, and deletions in E3/E4. The structural changes in enadenotucirev result in a genome that is approximately 3.5kb smaller than Adllp thereby providing additional "space" for the insertion of transgenes.
  • antibody refers to an immunoglobulin molecule capable of specific binding to a target antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, peptide etc., via at least one antigen recognition site (also referred to as a binding site herein), located in the variable region of the immunoglobulin molecule.
  • a target antigen such as a carbohydrate, polynucleotide, lipid, polypeptide, peptide etc.
  • at least one antigen recognition site also referred to as a binding site herein
  • antibody molecule includes antibodies and binding fragments thereof.
  • Antigen binding site refers to a portion of the molecule, which comprises a pair of variable regions, in particular a cognate pair that interact specifically with the target antigen.
  • Antibody binding fragment or antibody fragment refers to a fragment comprising a binding-domains which, such as a VH and/or VL which retains 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, bi, tri or tetra-valent antibodies, Bis- scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the same.
  • binding site that only recognises the antigen to which it is specific or a binding site that has significantly higher binding affinity to the antigen to which is specific compared to affinity to antigens to which it is non-specific, for example 5, 6, 7, 8, 9, 10 times higher binding affinity.
  • Antibody molecules as employed may comprise a complete antibody molecule having full length heavy and light chains, bispecific antibody format comprising full length antibodies or a fragment of any one of the same including, but are not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies, 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
  • Multi-valent antibodies may comprise multiple specificities e.g bispecific or may be monospecific (see for example WO 92/22853, WO05/113605, WO2009/040562 and WO2010/035012).
  • Humanised which include CDR-grafted antibodies
  • CDR-grafted antibodies refers to molecules having one or more complementarity determining regions (CDRs) from a non- human species and a framework region from a human immunoglobulin molecule (see, e.g. US 5,585,089; WO91/09967) . It will be appreciated that it may only be necessary to transfer the specificity determining residues of the CDRs rather than the entire CDR (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanised antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived.
  • the humanised antibody refers to an antibody molecule wherein the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g. a murine monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody).
  • a donor antibody e.g. a murine monoclonal antibody
  • acceptor antibody e.g. a human antibody.
  • a human antibody framework see for example, Kashmiri et al., 2005, Methods, 36, 25-34.
  • only the specificity determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework.
  • only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework.
  • any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions.
  • the humanised antibody according to the present disclosure has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs provided herein.
  • human frameworks which can be used in the present disclosure are KOL,
  • KOL and NEWM can be used for the heavy chain
  • REI can be used for the light chain and EU
  • LAY and POM can be used for both the heavy chain and the light chain.
  • human germline sequences may be used; these are available at: http://vbase.mrc-cpe.cam.ac.uk/
  • the acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.
  • the framework regions need not have exactly the same sequence as those of the acceptor antibody. For instance, unusual residues may be changed to more frequently- occurring residues for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions may be changed so that they correspond to the residue found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to recover the affinity of the donor antibody.
  • a protocol for selecting residues in the acceptor framework regions which may need to be changed is set forth in WO91/09967.
  • the antibody molecules of the present disclosure are fully human, in particular one or more of the variable domains are fully human.
  • Fully human molecules are those in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, not necessarily from the same antibody.
  • Examples of fully human antibodies may include antibodies produced, for example by the phage display methods described above and antibodies produced by mice in which the murine immunoglobulin variable and optionally the constant region genes have been replaced by their human counterparts eg. as described in general terms in EP0546073 Bl, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US5,770,429, EP 0438474 and EP0463151.
  • Suitable antibodies and antibody fragments may be agonistic or antagonistic and include those with anticancer activity and those which modify host cell responses to the cancer, for example: an agonist or antagonistic antibody or antibody fragment may decrease vascularization or normalise vascularization of the tumour.
  • agonistic antibodies or other encoded proteins may render the host cell more visible to the host's innate and adaptive immune responses, for example by expressing antigens, danger signals, cytokines or chemokines to attract and activate the same, or by binding to co-stimulatory or checkpoint pathway molecules to enhance adaptive immune responses.
  • Therapeutic antibody or antibody-binding fragment as employed herein refers to antibody or antibody-binding fragment which, when inserted in to the oncolytic virus, has a beneficial impact on a pathology in the patient, for example on the cancer being treated.
  • Beneficial impact as employed herein refers to a desirable and/or advantageous effect of the antibody being expressed in vivo.
  • Classes of therapeutic antibodies and antibody-binding fragments include: anti-EGF antibodies, anti-VEGF antibodies, anti-PDGF antibodies, anti-CTLA antibodies, anti-PDl antibodies, anti-PDLl antibodies and anti-FGF antibodies.
  • Registered therapeutic antibodies suitable for incorporation into viruses of the present disclosure include: abciximab, adalimumab, alemtzumab, basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, cetuximab, certolzumab, daclizumab, denosumab, eculzumab, efalixumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, muromonab-CD3, ofatumumab, palivizumab, panitumumab, ranibizumab, rituximab, tocilizumab, tositumomab and trastuzumab.
  • the antibody variable region sequences of an antibody or antibody fragment employed are between 95 and 100% similar or identical to the variable regions of bevacizumab (also known as Avastin®), such as 96, 97, 98 or 99% similar or identical.
  • Also suitable for incorporation into viruses of the present disclosure are the coding sequences for those antibodies and binding fragments thereof which are approved for a cancer indications, for example trastuzumab, tositumomab, rituximab, panitumumab, ofatumumab, ipilimumab, ibritumomab tiuxetan, gemtuzumab, denosumab, cetuximab, brentuximab vedotin, avastin and adalimumab.
  • the antibody variable region sequences of an antibody or antibody fragment employed are between 95 and 100% similar or identical to the variable regions of a known antibody or an antibody disclosed herein.
  • a transgene may encode a protein independently selected from the group comprising: IL-la, IL- ⁇ , IL-6, IL-9, IL-12, IL-13, IL-17, IL-18, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-33, IL-35, Interleukin-2 (IL-2), IL-4, IL-5, IL-7, IL-10, IL-15, IL-21, IL- 25, IL-1RA, IFNa, IFNp, IFNy, TNFa, TGFp, lymphotoxin a (LTA) and GM-CSF or an active fragment of any one of the same.
  • a chemokine encoded by a transgene is independently selected from the group comprising: IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, CCL21, CXCR2, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3, CXCR4, CXCR5 and CRTH2 or an active fragment of any one of the same.
  • a transgene according to the present disclosure encodes a protein independently selected from STAT3, STAT1, STAT4, STAT6, CTIIA, MyD88 and NFKB family or an active fragment of any one of the same.
  • a transgene may encode a cytokine independently selected from the TNF superfamily.
  • a transgene according to the present disclosure encodes ⁇ - ⁇ .
  • Linkers suitable for use in fusion proteins of the present disclosure include hinge linkers shown in SEQ ID NO: 31 to 39 and flexible linkers shown in SEQ ID NO: 40 to 80, wherein (S) is optional in sequences 42 to 46,
  • rigid linkers examples include the peptide sequences GAPAPAAPAPA (SEQ ID NO:
  • a bond refers to a covalent bond connecting the one DNA sequence to another DNA sequence, for example connecting one section of the virus genome to another.
  • a variable in formula (I) and (la) herein represents a bond the feature or element represented by the bond is absent i.e. deleted.
  • adenoviruses As the structure of adenoviruses is, in general, similar the elements below are discussed in terms of the structural elements and the commonly used nomenclature referring thereto, which are known to the skilled person.
  • an element When an element is referred to herein then we refer to the DNA sequence encoding the element or a DNA sequence encoding the same structural protein of the element in an adenovirus. The latter is relevant because of the redundancy of the DNA code. The viruses' preference for codon usage may need to be considered for optimised results.
  • Any structural element from an adenovirus employed in the viruses of the present disclosure may comprise or consist of the natural sequence or may have similarity over the given 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 person is aware that when making changes the reading frames of the virus must be not disrupted such that the expression of structural proteins is disrupted.
  • the given element is a full-length sequence i.e. the full-length gene.
  • Full length gene refers to at least the entirety of the coding sequence of a gene, but may include any associated non-coding regions, especially if they are relevant to the function of the gene.
  • the given element is less than a full-length and retains the same or corresponding function as the full-length sequence.
  • the DNA sequence may be less than a full-length and have no functionality, for example the E3 region may be totally or partly deleted. However, it may be useful to delete essentially all the E3 region as this optimises the space available for inserting transgenes.
  • the structural genes encoding structural or functional proteins of the adenovirus are generally linked by non-coding regions of DNA.
  • the genomic sequence of the structural element of interest especially non-coding regions thereof
  • the element will be considered a structural element of reference to the extent that it is fit for purpose and does not encode extraneous material.
  • the gene will be associated with suitable non-coding regions, for example as found in the natural structure of the virus.
  • an insert such as DNA encoding a restriction site and/or transgene, is inserted into a non-coding region of genomic virus DNA, such as an intron or intergenic sequence. Having said this some non-coding regions of adenovirus may have a function, for example in alternative splicing, transcription regulation or translation regulation, and this may need to be taken into consideration.
  • RNAi Ribonucleic acid
  • cytokines single chain or multimeric proteins
  • Gene as employed herein refers to coding and any non-coding sequences associated therewith, for example introns and associated exons.
  • a gene comprises or consists of only essential structural components, for example coding region.
  • ITR Inverted Terminal Repeat
  • the 5'ITR as employed herein 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 in an appropriate location.
  • the 5'ITR comprises or consists of the sequence from about lbp to 138bp of SEQ ID NO: 21 or a sequence 90, 95, 96, 97, 98 or 99% identical thereto along the whole length, in particular the sequence consisting of from about lbp to 138bp of SEQ ID NO: 17.
  • the 3'ITR as employed herein refers to part or all of an ITR from 3' end of an adenovirus which retains the function of the ITR when incorporated into an adenovirus in an appropriate location.
  • the 3'ITR comprises or consists of the sequence from about 32189bp to 32326bp of SEQ ID NO: 17 or a sequence 90, 95, 96, 97, 98 or 99% identical thereto along the whole length, in particular the sequence consisting of from about 32189bp to 32326bp of SEQ ID NO: 17.
  • Bl as employed herein refers to the DNA sequence encoding: part or all of an EIA from an adenovirus, part or all of the EIB region of an adenovirus, and independently part or all of EIA and EIB region of an adenovirus.
  • Bl is a bond then EIA and EIB sequences will be omitted from the virus.
  • Bl is a bond and thus the virus is a vector.
  • Bl further comprises a transgene. It is known in the art that the
  • El region can accommodate a transgene which may be inserted in a disruptive way into the El region (i.e. in the "middle" of the sequence) or part or all of the El region may be deleted to provide more room to accommodate genetic material.
  • EIA refers to the DNA sequence encoding part or all of an adenovirus EIA region. The latter here is referring to the polypeptide/protein EIA. It may be mutated such that the protein encoded by the EIA gene has conservative or non- conservative amino acid changes (e.g. 1, 2, 3, 4 or 5 amino acid changes, additions and/or deletions over the whole length) such that it has: the same function as wild-type (i.e. the corresponding non-mutated protein); increased function in comparison to wild-type protein; decreased function, such as no function in comparison to wild-type protein; or has a new function in comparison to wild-type protein or a combination of the same as appropriate.
  • conservative or non- conservative amino acid changes e.g. 1, 2, 3, 4 or 5 amino acid changes, additions and/or deletions over the whole length
  • EIB refers to the DNA sequence encoding part or all of an adenovirus EIB region (i.e. polypeptide or protein), it may be mutated such that the protein encoded by the EIB 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 over the whole length) such that it has: the same function as wild-type (i.e. the corresponding non-mutated protein); increased function in comparison to wild-type protein; decreased function, such as no function in comparison to wild-type protein; or has a new function in comparison to wild-type protein or a combination of the same as appropriate.
  • conservative or non-conservative amino acid changes e.g. 1, 2, 3, 4 or 5 amino acid changes, additions and/or deletions over the whole length
  • Bl can be modified or unmodified relative to a wild-type El region, such as a wild-type E1A and/or E1B.
  • a wild-type E1A and/or E1B The skilled person can easily identify whether E1A and/or E1B are present or (part) deleted or mutated.
  • Wild-type as employed herein refers to a known adenovirus or a sequence from a known adenovirus.
  • a known adenovirus is one that has been identified and named, regardless of whether the sequence information is available.
  • Bl has the sequence from 139bp to 3932bp of SEQ ID NO: 17.
  • BA as employed herein refers to the DNA sequence encoding the E2B-L1-L2-L3-E2A-
  • L4 regions including any non-coding sequences including any non-coding sequences, as appropriate (in particular corresponding to the natural sequence from an adenovirus). Generally this sequence will not comprise a transgene. In one embodiment the sequence is substantially similar or identical to a contiguous sequence from a known adenovirus, for example a serotype shown in Table 1, in particular a group B virus, for example Ad3, Ad 7, Adl 1, Ad 14, Adl6, Ad21, Ad34, Ad35, Ad51 or a combination thereof, such as Ad3, Adll or a combination thereof.
  • a group B virus for example Ad3, Ad 7, Adl 1, Ad 14, Adl6, Ad21, Ad34, Ad35, Ad51 or a combination thereof, such as Ad3, Adll or a combination thereof.
  • E2B-L1-L2-L3-E2A-L4 refers to comprising these elements and other structural elements associated with the region, for example BA will generally include the sequence encoding the protein IV2a, for example as follows: IV2A IV2a-E2B-Ll-L2-L3-E2A-L4.
  • the E2B region is chimeric. That is, comprises DNA sequences from two or more different adenoviral serotypes, for example from Ad3 and Adll, such as Adllp.
  • Ad3 adenoviral serotypes
  • Adllp Adllp
  • the E2B region has the sequence from 5068bp to 10355bp of SEQ ID NO: 17 or a sequence 95%, 96%, 97%, 98% or 99% identical thereto over the whole length.
  • E2B in component B ⁇ comprises the sequences shown in
  • SEQ ID NO: 18 (which corresponds to SEQ ID NO: 3 disclosed in WO2005/118825).
  • B ⁇ has the sequence from 3933bp to 27184bp of SEQ ID NO: 18.
  • E3 refers to the DNA sequence encoding part or all of an adenovirus E3 region (i.e. protein/polypeptide), it may be mutated such 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 over the whole length), such that it has the same function as wild-type (the corresponding unmutated protein); increased function in comparison to wild-type protein; decreased function, such as no function in comparison to wild-type protein or has a new function in comparison to wild-type protein or a combination of the same, as appropriate.
  • conservative or non-conservative amino acid changes e.g. 1, 2, 3, 4 or 5 amino acid changes, additions and/or deletions over the whole length
  • wild-type the corresponding unmutated protein
  • increased function in comparison to wild-type protein decreased function, such as no function in comparison to wild-type protein or has a new function in comparison to wild-type protein or a combination of the same
  • the E3 region is form an adenovirus serotype given in Table 1 or a combination thereof, in particular a group B serotype, for example Ad3, Ad7, Adll (in particular Adl lp), Adl4, Adl6, Ad21, Ad34, Ad35, Ad51 or a combination thereof, such as Ad3, Adl l (in particular Adllp) or a combination thereof.
  • Ad3, Ad7, Adll in particular Adl lp
  • Adl4 Adl6, Ad21, Ad34, Ad35, Ad51 or a combination thereof, such as Ad3, Adl l (in particular Adllp) or a combination thereof.
  • the E3 region has a sequence shown in SEQ ID NO: 19.
  • the E3 region is partially deleted, for example is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% deleted.
  • B2 is a bond, wherein the DNA encoding the E3 region is absent.
  • the DNA encoding the E3 region can be replaced or interrupted by a transgene.
  • E3 region replaced by a transgene as employed herein includes part or all of the E3 region is replaced with a transgene.
  • the B2 region comprises the sequence from 27185bp to 28165bp of SEQ ID NO: 98.
  • B2 consists of the sequence from 27185bp to 28165bp of SEQ ID NO: 98.
  • ⁇ refers to the DNA sequence in the vicinity of the 5' end of the
  • L5 gene in BB In the vicinity of or proximal to the 5' end of the L5 gene as employed herein refers to: adjacent (contiguous) to the 5' end of the L5 gene or a non-coding region inherently associated herewith i.e. abutting or contiguous to the 5' prime end of the L5 gene or a non-coding region inherently associated therewith. Alternatively, in the vicinity of or proximal to may refer to being close the L5 gene, such that there are no coding sequences between the BX region and the 5' end of L5 gene.
  • ⁇ is joined directly to a base of L5 which represents, for example the start of a coding sequence of the L5 gene.
  • ⁇ is joined directly to a base of L5 which represents, for example the start of a non-coding sequence, or joined directly to a non-coding region naturally associated with L5.
  • a non-coding region naturally associated L5 as employed herein refers to part of all of a non-coding regions which is part of the L5 gene or contiguous therewith but not part of another gene.
  • ⁇ comprises the sequence of SEQ ID NO: 98.
  • This sequence is an artificial non-coding sequence wherein a DNA sequence, for example comprising a transgene (or transgene cassette), a restriction site or a combination thereof may be inserted therein.
  • This sequence is advantageous because it acts as a buffer in that allows some flexibility on the exact location of the transgene whilst minimising the disruptive effects on virus stability and viability.
  • the insert(s) can occur anywhere within SEQ ID NO: 98 from the 5' end, the 3' end or at any point between bp 1 to 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/
  • ⁇ comprises SEQ ID NO: 98 with a DNA sequence inserted between bp 27 and bp 28 or a place corresponding to between positions 28192bp and
  • ⁇ has the sequence from 28166bp to 28366bp of SEQ ID NO:
  • ⁇ is a bond
  • ⁇ refers to the DNA sequence encoding the L5 region.
  • L5 region refers to the DNA sequence containing the gene encoding the fibre polypeptide/protein, as appropriate in the context.
  • the fibre gene/region encodes the fibre protein which is a major capsid component of adenoviruses. The fibre functions in receptor recognition and contributes to the adenovirus' ability to selectively bind and infect cells.
  • the fibre can be from any adenovirus serotype and adenoviruses which are chimeric as result of changing the fibre for one of a different serotype are also envisaged with the present disclosure.
  • the fibre is from a group B virus, in particular Adll, such as Adllp.
  • ⁇ has the sequence from 28367bp to 29344bp of SEQ ID NO:
  • DNA sequence in relation to ⁇ refers to the DNA sequence in the vicinity of the 3' end of the L5 gene of ⁇ . In the vicinity of or proximal to the 3' end of the L5 gene as employed herein refers to: adjacent (contiguous) to the 3' end of the L5 gene or a non-coding region inherently associated therewith i.e. abutting or contiguous to the 3' prime end of the L5 gene or a non-coding region inherently associated therewith (i.e. all or part of an non-coding sequence endogenous to L5).
  • ⁇ is joined directly to a base of L5 which represents the
  • ⁇ is joined directly to a base of L5 which represents the
  • ⁇ comprises the sequence of SEQ ID NO: 99.
  • This sequence is a non-coding sequence wherein 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 a buffer in that allows some flexibility on the exact location of the transgene whilst minimising the disruptive effects on virus stability and viability.
  • the insert(s) can occur anywhere within SEQ ID NO: 18 from the 5' end, the 3' end or at any point between bp 1 to 35, 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.
  • ⁇ comprises SEQ ID NO: 99 with a DNA sequence inserted between positions bp 12 and 13 or a place corresponding to 29356bp and 29357bp in SEQ ID NO: 17.
  • the insert is a restriction site insert.
  • the restriction site insert comprises one or two restriction sites.
  • the restriction site is a 19bp restriction site insert comprising 2 restriction sites.
  • the restriction site insert is a 9bp restriction site insert comprising 1 restriction site.
  • 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.
  • restriction site is a 19bp restriction site insert comprising 2 restriction sites and at least one transgene, for example one or two transgenes.
  • restriction site insert is a 9bp restriction site insert comprising 1 restriction site and at least one transgene, for example one or two transgenes.
  • two restriction sites sandwich one or more, such as two transgenes (for example in a transgene cassette).
  • ⁇ comprises two restrictions sites the said restriction sites are different from each other.
  • said one or more restrictions sites in ⁇ are non-naturally occurring (such as unique) in the particular adenovirus genome into which they have been inserted.
  • said one or more restrictions sites in ⁇ are different to other restrictions sites located elsewhere in the adenovirus genome, for example different to naturally occurring restrictions sites or restriction sites introduced into other parts of the genome, such as ⁇ .
  • the restriction site or sites allow the DNA in the section to be cut specifically.
  • ⁇ has the sequence from 29345bp to 29379bp of SEQ ID NO: 17. In one embodiment ⁇ is a bond. In one embodiment the insert is after bp 12 in SEQ ID NO: 99.
  • the insert is at about position 29356bp of SEQ ID NO: 17.
  • the insert is a transgene cassette comprising one or more transgenes, for example 1, 2 or 3, such as 1 or 2.
  • E4 refers to the DNA sequence encoding part or all of an adenovirus E4 region (i.e. polypeptide/protein region), which may be mutated such that the protein encoded by the E4 gene has conservative or non-conservative amino acid changes (e.g. 1, 2, 3, 4 or 5 amino acid changes, additions and/or deletions), and has the same function as wild-type (the corresponding non-mutated protein); increased function in comparison to wild-type protein; decreased function, such as no function in comparison to wild-type protein or has a new function in comparison to wild-type protein or a combination of the same as appropriate.
  • conservative or non-conservative amino acid changes e.g. 1, 2, 3, 4 or 5 amino acid changes, additions and/or deletions
  • the E4 region is partially deleted, for example is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% deleted.
  • the E4 region has the sequence from 32188bp to 29380bp of SEQ ID NO: 17.
  • E4 is present except for the E4orf4 region which is deleted.
  • B3 is a bond, i.e. wherein E4 is absent.
  • B3 has the sequence consisting of from 32188bp to 29380bp of SEQ ID NO: 17.
  • formulas herein such as formula (I), (la) are contiguous and may embody non-coding DNA sequences as well as the genes and coding DNA sequences (structural features) mentioned herein.
  • the formulas of the present disclosure are attempting to describe a naturally occurring sequence in the adenovirus genome.
  • the formula is referring to the major elements characterising the relevant section of genome and is not intended to be an exhaustive description of the genomic stretch of DNA.
  • E1A, E1B, E3 and E4 as employed herein each independently refer to the wild-type and equivalents thereof, mutated or partially deleted forms of each region as described herein, in particular a wild-type sequence from a known adenovirus.
  • Insert refers to a DNA sequence that is incorporated either at the 5' end, the 3' end or within a given DNA sequence reference segment such that it interrupts the reference sequence.
  • inserts generally occur within either SEQ ID NO: 98 or SEQ ID NO: 99.
  • An insert can be either a restriction site insert, a transgene cassette or both. When the sequence is interrupted the virus will still comprise the original sequence, but generally it will be as two fragments sandwiching the insert.
  • the transgene or transgene cassette does not comprise a non- biased inserting transposon, such as a TN7 transposon or part thereof.
  • Tn7 transposon as employed herein refers to a non-biased insertion transposon as described in WO2008/080003.
  • the transgene or transgene cassette further comprises a regulatory element or sequence.
  • Restriction sites in the locations disclosed here are useful in viruses and constructs of the present disclosure, such as plasmids, because they allow the transgene to be changed rapidly and, for example selectively when the restriction sites around the transgene(s) are unique.
  • the transgene or transgene cassette comprises a restriction site at each terminus, thereby allowing the cassette to be replaced.
  • restriction site is a location in a DNA sequence that can be cut by a restriction enzyme, usually an enzyme specific to the sequence.
  • the restriction site comprises 3 to 22 base pairs, for example 4 to 22, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 base pairs.
  • restriction sites cut by restriction enzymes include but are not limited to:
  • restriction enzymes that cut the same recognition sites may also be suitable.
  • one or more restrictions sites in ⁇ and ⁇ are independently selected from a restriction site specific to an enzyme described herein, for example Notl,
  • Fsel, AsiSI, Sgfl and Sbfl, in particular the restriction sites inserted are all different, such as sites specific for Notl and sites specific for Fsel located in ⁇ and Sgfl and Sbfl located in ⁇ .
  • the region ⁇ and/or ⁇ do not comprise a restriction site.
  • the viruses and constructs of the present disclosure can be prepared without restriction sites, for example using synthetic techniques. These techniques allow a great flexibility in the creation of the viruses and constructs. Furthermore, the present inventors have established that the properties of the viruses and constructs are not diminished when they are prepared by synthetic techniques.
  • Promoter as employed herein means a region of DNA that initiates transcription of a particular gene or genes. Promoters are generally located proximal to the genes they transcribe, on the same strand and upstream (i.e. 5') on the DNA. Proximal as employed in this context means sufficiently close to function as a promoter. In one embodiment the promoter is within 100 bp of the transcription start site. Thus endogenous promoter as employed herein refers to a promoter that naturally occurs in (i.e. is native to) the adenovirus (or construct) into which the transgene, is being inserted.
  • the endogenous promoter employed is the naturally occurring promoter in the virus in its original location in the virus genome, in particular this is the primary or only promoter employed in the expression of the transgene or transgenes.
  • the endogenous promoter used to promote the translation and optionally the transcription of the transgene is one resident, i.e. is one integrated in the genome of the adenovirus and not previously introduced by recombinant techniques.
  • transgene/transgene cassette is inserted in the appropriate orientation to be under the control of said endogenous promoter. That is, where the promoter is generally on the antisense strand, the cassette is inserted, for example in the antisense orientation.
  • genes can be expressed in one of two orientations. However, generally one orientation provides increased levels of expression over the other orientation, for a given (particular) transgene.
  • the cassette is in the sense orientation. That is, is transcribed in a 5' to 3' direction. In one embodiment the cassette is in the antisense orientation. That is, transcribed in the 3' to 5' orientation.
  • the endogenous promoters in the virus can, for example, be utilised by employing a gene encoding a transgene and a splice acceptor sequence.
  • the cassette will comprise a splice acceptor sequence when under the control of an endogenous promoter.
  • the coding sequence for example the sequence encoding the antibody or antibody binding fragment further comprises a splice acceptor sequence.
  • transgene, transgenes, or transgene cassette are under the control of an E4 promoter or a major late promoter, such as the major late promoter (ML promoter).
  • E4 promoter or a major late promoter, such as the major late promoter (ML promoter).
  • ML promoter major late promoter
  • the transgene is activated, i.e. transcribed, when a particular promoter dictates.
  • the Major Late Promoter refers to the adenovirus promoter that controls expression of the "late expressed" genes, such as the L5 gene.
  • the MLP is a "sense strand" promoter. That is, the promoter influences genes that are downstream of the promoter in the 5'-3' direction.
  • the major late promoter refers the original major late promoter located in the virus genome.
  • E4 promoter refers to the adenovirus promoter of the E4 region.
  • the E4 region is an antisense region; therefore the promoter is an antisense promoter. That is, the promoter is upstream of the E4 region in the 3'-5' direction. Therefore any transgene cassette under control of the E4 promoter may need to be oriented appropriately.
  • the cassette under the control of the E4 promoter is in the antisense orientation.
  • the cassette is under the control of the E4 promoter in the sense orientation.
  • the E4 promoter as employed herein refers to the original E4 promoter located in the virus genome.
  • a replication competent oncolytic adenovirus serotype 11 (such as Adllp) or virus-derivative thereof wherein the fibre, hexon and capsid are serotype 11 (such as Adllp)
  • the virus genome comprises a DNA sequence encoding a therapeutic antibody or antibody-binding fragment, wherein said DNA sequence under the control of a promoter endogenous to the adenovirus selected from consisting of E4 and the major late promoter (i.e. the E4 promoter or the major late promoter), such that the transgene does not interfere with virus replication, for example is associated with the L5 region (i.e. before or after said region), such as located after L5 in the virus genome.
  • an endogenous promoter is introduced into the viral genome at a desired location by recombinant techniques, for example is introduced in the transgene cassette.
  • this arrangement will generally be referred to as an exogenous promoter.
  • the transgene cassette comprises an exogenous promoter.
  • Exogenous promoter refers to a promoter that is not naturally occurring in the adenovirus into which the transgene is being inserted. Typically exogenous promoters are from other viruses or are mammalian promoters.
  • Exogenous promoter as employed herein means a DNA element, usually located upstream of the gene of interest, that regulates the transcription of the gene.
  • the regulator of gene expression is an exogenous promoter, for example CMV (cytomegalovirus promoter), CBA (chicken beta actin promoter) or PGK (phosphoglycerate kinase 1 promoter), such as CMV promoter.
  • CMV cytomegalovirus promoter
  • CBA chicken beta actin promoter
  • PGK phosphoglycerate kinase 1 promoter
  • a replication competent oncolytic adenovirus serotype 11 such as Adllp
  • virus-derivative thereof wherein the fibre, hexon and capsid are serotype 11 (such as Adllp)
  • the virus genome comprises a DNA sequence encoding a therapeutic antibody or antibody-binding fragment located in a part of the virus genome which is expressed late in the virus replication cycle and such that the transgene does not interfere with virus replication, wherein said DNA sequence under the control of a promoter exogenous to the adenovirus (for example the CMV promoter).
  • the DNA sequence encoding an antibody or fragment is associated with the L5 region as described elsewhere herein.
  • Regulator of gene expression refers to a genetic element, such as a promoter, enhancer or a splice acceptor sequence that plays a role in gene expression, typically by initiating or enhancing transcription or translation.
  • Splice acceptor sequence refers to a regulatory sequence determining when an mRNA molecule will be recognised by small nuclear ribonucleoproteins of the spliceosome complex. Once assembled the spliceosome catalyses splicing between the splice acceptor site of the mRNA molecule to an upstream splice donor site producing a mature mRNA molecule that can be translated to produce a single polypeptide or protein.
  • splice acceptor sequences may be employed in the present invention and these can be described as short splice acceptor (small), splice acceptor (medium) and branched splice acceptor (large).
  • SSA refers to a short splice acceptor, typically comprising just the splice site, for example 4 bp.
  • SA refers to a splice acceptor, typically comprising the short splice acceptor and the polypyrimidine tract, for example 16 bp.
  • bSA refers to a branched splice acceptor, typically comprising the short splice acceptor, polypyrimidine tract and the branch point, for example 26 bp.
  • the splice acceptor employed in the constructs of the disclosure are CAGG or SEQ ID NO: 10 or 11.
  • the SSA has the nucleotide sequence of CAGG.
  • the SA has the nucleotide sequence of SEQ ID NO: 10.
  • the bSA has the nucleotide sequence of cagg.
  • the splice acceptor sequence is independently selected from the group comprising: SEQ ID NO: 10, SEQ ID NO: 11, and cagg.
  • the splice site is immediately proceeded (i.e. followed in a 5' to 3' direction) by a consensus Kozak sequence comprising CCACC.
  • the splice site and the Kozak sequence are interspersed (separated) by up to 100 or less bp.
  • the Kozak sequence has the nucleotide sequence of CCACC.
  • the coding sequence when under the control of an endogenous or exogenous 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 initiation codon (AUG), for example is in the context of the sequence (gcc)gccRccAUGg [SEQ ID NO: 20] the start of the start of the coding sequences is indicated by the bases in bold.
  • a lowercase letter denotes common bases at this position (which can nevertheless vary) and upper case letters indicate highly-conserved bases, i.e.
  • the 'AUGG' sequence is constant or rarely, if ever, changes; 'R' indicates that a purine (adenine or guanine) is usually observed at this position and the sequence in brackets (gcc) is of uncertain significance.
  • the initiation codon AUG is incorporated into a Kozak sequence.
  • Internal Ribosome Entry DNA Sequence refers to a DNA sequence encoding an Internal Ribosome Entry Sequence (IRES).
  • IRES as employed herein means a nucleotide sequence that allows for initiation of translation a messenger RNA (mRNA) sequence, including initiation starting within an mRNA sequence. This is particularly useful when the cassette encodes polycistronic mRNA. Using an IRES results in a polycistronic mRNA that is translated into multiple individual proteins or peptides.
  • the Internal Ribosome Entry DNA sequence has the nucleotide sequence of SEQ ID NO: 6.
  • a particular IRES is only used once in the genome. This may have benefits with respect to stability of the genome.
  • “High self-cleavage efficiency 2A peptide” or “2A peptide” as employed herein refers to a peptide which is efficiently cleaved following translation. Suitable 2A peptides include P2A, F2A, E2A and T2A. The present inventors have noted that once a specific DNA sequence encoding a given 2A peptide is used once, the same specific DNA sequence may not be used a second time. However, redundancy in the DNA code may be utilised to generate a DNA sequence that is translated into the same 2A peptide. Using 2A peptides is particularly useful when the cassette encodes polycistronic mRNA. Using 2A peptides results in a single polypeptide chain being translated which is modified post-translation to generate multiple individual proteins or peptides.
  • the encoded P2A peptide employed has the amino acid sequence of SEQ ID NO: 4.
  • the encoded F2A peptide employed has the amino acid sequence of SEQ ID NO: 103.
  • the encoded E2A peptide employed has the amino acid sequence of SEQ ID NO: 21.
  • the encoded T2A peptide employed has the amino acid sequence of SEQ ID NO: 5.
  • an mRNA or each mRNA encoded by transgene is/are comprise a polyadenylation signal sequence, such as typically at the end of an mRNA sequence, for example as shown in SEQ ID NO: 6.
  • the transgene or the transgene cassette comprises at least one sequence encoding a polyadenylation signal sequence.
  • PolyA Polyadenylation signal
  • polyadenylation sequence means a DNA sequence, usually containing an AATAAA site, that once transcribed can be recognised by a multiprotein complex that cleaves and polyadenylates the nascent mRNA molecule.
  • polyadenylation sequence has the nucleotide sequence of SEQ ID NO: 6.
  • the construct does not include a polyadenylation sequence.
  • the regulator of gene expression is a splice acceptor sequence.
  • the sequence encoding a protein/polypeptide/peptide, such as an antibody or antibody binding fragment further comprises a polyadenylation signal.
  • virus or construct with a sequence disclosed herein, for example wherein the virus is NG-500 (SEQ ID NO: 7).
  • the present disclosure relates also extends to a pharmaceutical formulation of a virus as described herein.
  • liquid parenteral formulation for example for infusion or injection, of a replication capable oncolytic according to the present disclosure wherein the formulation provides a dose in the range of lxlO 10 to lxlO 14 viral particles per volume of dose.
  • Parenteral formulation means a formulation designed not to be delivered through 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.
  • parenteral formulation is in the form of an injection.
  • Injection includes intravenous, subcutaneous, intra-tumoral or intramuscular injection.
  • Injection as employed herein means the insertion of liquid into the body via a syringe.
  • the method of the present disclosure does not involve intra-tumoral injection.
  • parenteral formulation is in the form of an infusion.
  • Infusion as employed herein means the administration of fluids at a slower rate by drip, infusion pump, syringe driver or equivalent device.
  • the infusion is administered over a period in the range of 1.5 minutes to 120 minutes, such as about 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.
  • one dose of the formulation less than lOOmls, for example 30mls, such as administered by a syringe driver.
  • the injection is administered as a slow injection, for example over a period of 1.5 to 30 minutes.
  • the formulation is for intravenous (i.v.) administration.
  • This route is particularly effective for delivery of oncolytic virus because it allows rapid access to the majority of the organs and tissue and is particular useful for the treatment of metastases, for example established metastases especially those located in highly vascularised regions such as the liver and lungs.
  • Therapeutic formulations typically will be sterile and stable under the conditions of manufacture and storage.
  • the composition can 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, particular as a single dose.
  • the formulation will generally comprise a pharmaceutically acceptable diluent or carrier, for example a non-toxic, isotonic carrier that is compatible with the virus, and in which the virus is stable for the requisite period of time.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can 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.
  • a dispersant or surfactant such as lecithin or a non-ionic surfactant such as polysorbate 80 or 40.
  • isotonic agents include sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • parenteral formulations employed may comprise one or more of the following a buffer, for example 4-(2-hydroxyethyl)-l-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 thought to prevent one or more pathways of possible degradation.
  • the formulation will comprise purified oncolytic virus according to the present disclosure, for example lxlO 10 to lxlO 14 viral particles per dose, such as 1 xlO 10 to lxlO 12 viral particles per dose.
  • concentration of virus in the formulation is in the range 2xl0 8 to 2xl0 14 vp/ml, such as 2 x 10 12 vp/ml.
  • the parenteral formulation comprises glycerol.
  • the formulation comprises oncolytic adenovirus as described herein, HEPES (N-2-hydroxyethylpiperazine-N ' -2-ethanesulfonic acid), glycerol and buffer.
  • the parenteral formulation consists of virus of the disclosure, HEPES for example 5mM, glycerol for example 5-20% (v/v), hydrochloric acid, for example to adjust the pH into the range 7-8 and water for injection.
  • HEPES for example 5mM
  • glycerol for example 5-20% (v/v)
  • hydrochloric acid for example to adjust the pH into the range 7-8 and water for injection.
  • 0.7 mL of virus of the disclosure at a concentration of 2 x 10 12 vp/mL is formulated in 5 mM HEPES, 20% glycerol with a final pH of 7.8.
  • the formulation is provided as a formulation for topical administrations including inhalation.
  • Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free from propellant gases.
  • Inhalable powders according to the disclosure will generally contain a virus as described herein with a physiologically acceptable excipient.
  • These inhalable powders may include monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo- and polysaccharides (e.g. dextranes), polyalcohols (e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these with one another.
  • monosaccharides e.g. glucose or arabinose
  • disaccharides e.g. lactose, saccharose, maltose
  • oligo- and polysaccharides e.g. dextranes
  • polyalcohols e.g. sorbitol, mannitol, xylitol
  • salts e.g. sodium chloride, calcium carbonate
  • Particles for deposition in the lung require a particle size less than 10 microns, such as 1-9 microns for example from 0.1 to 5 ⁇ , in particular from 1 to 5 ⁇ .
  • the size of the particle carrying the virus is of primary importance and thus in one embodiment the virus according to the present disclosure may be adsorbed or absorbed onto a particle, such as a lactose particle of the given size.
  • propellant gases which can be used to prepare the inhalable aerosols are known in the art.
  • Suitable propellant gases are selected from among 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.
  • hydrocarbons such as n- propane, n-butane or isobutane
  • halohydrocarbons such as chlorinated and/or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane.
  • the above-mentioned propellant gases may be used on their own or in mixtures thereof.
  • Particularly suitable propellant gases are halogenated alkane derivatives selected from among TG 11, TG 12, TG 134a and TG227.
  • halogenated alkane derivatives selected from among TG 11, TG 12, TG 134a and TG227.
  • TG134a 1,1,1,2-tetrafluoroethane
  • TG227 1,1,2,3,3,3- heptafluoropropane
  • mixtures thereof are particularly suitable.
  • the propellant gas-containing inhalable aerosols may also contain other ingredients, such as co-solvents, stabilisers, surface-active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All these ingredients are known in the art.
  • the propellant gas-containing inhalable aerosols according to the invention may contain up to 5 % by weight of active substance. 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.
  • topical administrations to the lung may also be by administration of a liquid solution or suspension formulation, for example employing a device such as a nebulizer, for example, a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus(R) nebulizer connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).
  • a nebulizer for example, a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus(R) nebulizer connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).
  • the virus of the invention can be delivered dispersed in a solvent, e.g. in the form of a solution or a suspension, for example as already described above for parenteral formulations. It can be suspended in an appropriate physiological solution, e.g., saline or other pharmacologically acceptable solvent or a buffered solution.
  • a physiological solution e.g., saline or other pharmacologically acceptable solvent or a buffered solution.
  • Buffered solutions known in the art may contain 0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mg to 0.30 mg anhydrous citric acid, and 0.45 mg to 0.55 mg sodium citrate per 1 ml of water so as to achieve a pH of about 4.0 to 5.0.
  • the therapeutic suspensions or solution formulations can 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 can be encapsulated in liposomes or biodegradable microspheres.
  • the formulation will generally be provided in a substantially sterile form employing sterile manufacture processes.
  • This may include production and sterilization by filtration of the buffered solvent/solution used for the formulation, aseptic suspension of the antibody in the sterile buffered solvent solution and dispensing of the formulation into sterile receptacles by methods familiar to those of ordinary skill in the art.
  • Nebulisable formulation according to the present disclosure may be provided, for example, as single dose units (e.g., sealed plastic containers or vials) packed in foil envelopes. Each vial contains a unit dose in a volume, e.g., 2 mL, of solvent/solution buffer.
  • the present disclosure also extends to liquid solutions or suspensions delivered intra-nasally, for example employing a device as disclosed in WO2009/068877 and US2004/0153033 both incorporated herein by reference. Treatment
  • 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.
  • the method of treatment is for use in the treatment of a tumour.
  • Tumour as employed herein is intended to refer to an abnormal mass of tissue that results from excessive cell division that is uncontrolled and progressive, also called a neoplasm. Tumours may be either benign (not cancerous) or malignant. Tumour encompasses all forms of cancer and metastases. In one embodiment the tumour is cancerous.
  • the tumour is a solid tumour.
  • the solid tumour may be localised or metastasised.
  • tumour is of epithelial origin.
  • the tumour is a malignancy, such as colorectal cancer, hepatoma, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, renal cancer, bladder cancer, head and neck cancer or lung cancer.
  • a malignancy such as colorectal cancer, hepatoma, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, renal cancer, bladder cancer, head and neck cancer or lung cancer.
  • the tumour is a colorectal malignancy.
  • Malignancy as employed herein refers to cancerous cells.
  • the oncolytic adenovirus is employed in the treatment or prevention of metastasis.
  • the method or formulation herein is employed in the treatment of drug resistant cancers.
  • the virus is administered in combination with the administration of a further cancer treatment or therapy.
  • a virus or formulation according to the present disclosure for use in the manufacture of a medicament for the treatment of cancer, for example a cancer described above.
  • a method of treating cancer 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.
  • the oncolytic virus or formulation herein is administered in combination with another therapy.
  • “In combination” as employed herein is intended to encompass where the oncolytic virus is administered before, concurrently and/or post cancer treatment or therapy. However, generally the treatment regimens for the combination thera
  • Cancer therapy includes surgery, radiation therapy, targeted therapy and/or chemotherapy.
  • Cancer treatment refers to treatment with a therapeutic compound or biological agent, for example an antibody intended to treat the cancer and/or maintenance therapy thereof.
  • the cancer treatment is selected from any other anti-cancer therapy including a chemotherapeutic agent; a targeted anticancer agent, such as an antibody drug conjugate; radiotherapy, radio-isotope therapy or any combination thereof.
  • a chemotherapeutic agent such as an antibody drug conjugate
  • a targeted anticancer agent such as an antibody drug conjugate
  • radiotherapy radio-isotope therapy or any combination thereof.
  • the virus of the present disclosure such as an oncolytic adenovirus may be used as a pre-treatment to the therapy, such as a surgery (neoadjuvant therapy), to shrink the tumour, to treat metastasis and/or prevent metastasis or further metastasis.
  • the oncolytic adenovirus may be used after the therapy, such as a surgery (adjuvant therapy), to treat metastasis and/or prevent metastasis or further metastasis.
  • a virus or formulation of the present disclosure is employed in maintenance therapy.
  • the administration of the 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.
  • the virus is administered in combination with the administration of a chemotherapeutic agent.
  • Chemotherapeutic agent as employed herein is intended to refer to specific antineoplastic chemical agents or drugs that are selectively destructive to malignant cells and tissues.
  • alkylating agents for example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumour agents.
  • specific chemotherapeutic agents include doxorubicin, 5-fluorouracil (5-FU), paclitaxel, capecitabine, irinotecan, and platins such as cisplatin and oxaliplatin.
  • the dose may be chosen by the practitioner based on the nature of the cancer being treated.
  • the therapeutic agent is ganciclovir, which may assist in controlling immune responses and/or tumour vascularisation.
  • one or more therapies employed in the method herein are metronomic, that is a continuous or frequent treatment with low doses of anticancer drugs, often given concomitant with other methods of therapy.
  • Subgroup B oncolytic adenoviruses in particular Adll and those derived therefrom such as EnAd may be particularly synergistic with chemotherapeutics because they seem to have a mechanism of action that is largely independent of apoptosis, killing cancer cells by a predominantly necrolytic mechanism. Moreover, the immunosuppression that occurs during chemotherapy may allow the oncolytic virus to function with greater efficiency.
  • Therapeutic dose as employed herein refers to the amount of virus, such as oncolytic adenovirus that is suitable for achieving the intended therapeutic effect when employed in a suitable treatment regimen, for example ameliorates symptoms or conditions of a disease, in particular without eliciting dose limiting 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: tumour or metastatic growth is slowed or stopped, or the tumour or metastasis is found to shrink in size, and/or the life span of the patient is extended.
  • Suitable therapeutic doses are generally a balance between therapeutic effect and tolerable toxicity, for example where the side-effect and toxicity are tolerable given the benefit achieved by the therapy.
  • one or more doses (for example each dose) of virus or composition comprising the same is administered such that the rate of viral particle delivery is in the range of 2xl0 10 particles per minute to 2xl0 12 particles per minute.
  • a virus or therapeutic construct according to the present disclosure is administered weekly, for example one week 1 the dose is administered on day 1, 3, 5, followed by one dose each subsequent week.
  • a virus or therapeutic construct according to the present disclosure is administered bi-weekly or triweekly, for example is administered in week 1 one on days 1, 3 and 5, and on week 2 or 3 is also administered on days 1, 3 and 5 thereof.
  • This dosing regimen may be repeated as many times as appropriate.
  • a virus or therapeutic construct according to the present disclosure is administered monthly, for example in a treatment cycle or as maintenance therapy.
  • viruses and constructs of the present disclosure are prepared by recombinant techniques.
  • the armed adenovirus genome can be manufactured by other technical means, including entirely synthesising the genome or a plasmid comprising part of all of the genome.
  • the region of insertion may not comprise the restriction site nucleotides as the latter are artefacts following insertion of genes using cloning methods.
  • the armed adenovirus genome is entirely synthetically manufactured, for example as per SEQ ID NO: 7, which was employed with transgene cassettes in SEQ ID NO: 8.
  • the disclosure herein further extends to an adenovirus of formula (I) or a sub- formula thereof, obtained or obtainable from inserting a transgene or transgene cassette.
  • Example 1 Production of an enadenotucirev (EnAd) virus expressing the T cell activating antigens CD80 and a membrane-anchored single chain Fv fragment antibody to the ⁇ chain of the human CD3 complex (CD3E) together with the chemokine, ⁇ as a third transgene
  • the plasmid pEnAd2.4 was used to generate the plasmid pNG-500 by direct insertion of a cassette encoding the human T cell activating antigen CD80 (SEQ ID NO: 1), a membrane-anchored chimeric form of the single chain Fv anti-human CD3s (SEQ ID NO: 2) and the human macrophage inflammatory protein la (MlPla, isoform LD78p, SEQ ID NO: 3) .
  • the pNG-500 cassette contains; a 5' short splice acceptor sequence (CAGG); membrane- anchored anti-human CD3e ScFv cDNA; a high efficiency self-cleavable P2A peptide sequence (SEQ ID NO: 4); human CD80 cDNA sequence; a high efficiency self-cleavable T2A peptide sequence (SEQ ID NO: 5) and a 3' polyadenylation sequence (SEQ ID NO: 6) .
  • a Schematic of the inserted transgene cassette is shown in Figure 1A. Construction of the plasmid was confirmed by DNA sequencing.
  • the plasmid pNG-500 was linearised by restriction digest with the enzyme Ascl to produce the virus genome NG-500 (SEQ ID NO: 7).
  • the virus NG-500 was amplified and purified according to the methods given below.
  • Digested DNA was purified by phenol/chloroform extraction and precipitated for 16hrs, -20°C in 300 ⁇ 1 >95% molecular biology grade ethanol and ⁇ 3M Sodium Acetate.
  • the precipitated DNA was pelleted by centrifuging at 14000rpm, 5 mins and was washed in 500 ⁇ 70% ethanol, before centrifuging again, 14000rpm, 5mins.
  • the clean DNA pellet was air dried, resuspended in 500 ⁇ 1 OptiMEM containing 15 ⁇ 1 lipofectamine transfection reagent and incubated for 30 mins, RT. The transfection mixture was then added drop wise to a T-25 flask containing 293 cells grown to 70% confluency.
  • the transfected 293 cells were monitored every 24hrs and were supplemented with additional media every 48-72hrs.
  • the production of virus was monitored by observation of a significant cytopathic effect (CPE) in the cell monolayer.
  • CPE cytopathic effect
  • the NG-500 virus was harvested from 293 cells by three freeze-thaw cycles.
  • the harvested viruses were used to re-infect 293 cells in order to amplify the virus stocks. Viable virus production during amplification was confirmed by observation of significant CPE in the cell monolayer.
  • the amplified stock was used for further amplification before the virus was purified by double caesium chloride banding to produce a NG-500 virus stock (lot number NG-500-R01 was used in the experiments described in Examples 2-6).
  • Example 2 Oncolytic activity and replication of the NG-500 virus in colon carcinoma cells
  • HT-29 colon carcinoma cells were seeded in 96 well plates at a cell density of 2.5e4 cells/well. Plates were incubated for 4 hrs, 37°C, 5% CO2, before cells were either infected with EnAd or NG-500 virus particles at an infection density range of 100-0.39 particles per cell (ppc).
  • HT-29 cell viability was assessed using Cell Titre 96 MTS Reagent (Promega: G3581) 72 hrs post infection. Quantification of the % cell survival at each infection density demonstrated that NG-500 oncolytic potency was comparable to the non-transgene expressing parental virus, EnAd ( Figure IB).
  • A549 lung carcinoma cells infected for 24 or 48 hrs with lOOppc EnAd or NG-500 or left uninfected (UIC) were used for quantification of viral DNA by qPCR.
  • Cell supernatants were collected and clarified by centrifuging for 5 mins, 1200rpm.
  • DNA was extracted from 10 ⁇ of supernatant using the Sigma Genelute DNA extraction Kit, according to the manufacturer's protocol.
  • a standard curve using EnAd virus particles 2.5el0-2.5e5vp was also prepared and extracted using the Sigma Genelute Kit. Each extracted sample or standard was analysed by qPCR using an EnAd E3 gene specific primer-probe set.
  • Example 3 Cell surface expression of the T cell activating antigen, CD80, NG-500 infected A549 cell line
  • CD80 transgene expression was compared in NG-500 and EnAd treated A549 lung carcinoma cells.
  • A549 cells were seeded in 12 well plates at cell densities of 7.5e5 cells/well. Plates were incubated for 18 hrs, 37°C, 5% C0 2 , before cells were either infected with EnAd or NG-500 virus at 100 particles per cell (ppc) or were left uninfected.
  • CD80 protein expression was compared on the surface of A549 cells at 24 and 46 hrs post-infection. At each time point cells were harvested and stained according to methods detailed below.
  • CD80 expression could only be detected on A549 cells infected with NG- 500 virus. Analysis of CD80 expression at 46 hrs post infection showed CD80 could be detected on the surface of >95% of NG-500 treated cells. By 48 hrs approximately 70% of the A549 cells treated with either EnAd or NG-500 were dead (leaky membranes) but still intact, as defined by the live/dead staining ( Figure 2A).
  • Example 4 Production of secreted ⁇ - ⁇ in NG-500 infected carcinoma cell lines
  • a Jurkat T-cell reporter cell line (Jurkat DualTM cells, InVivogen), which permits study of NF-KB pathway activation by analysis of the activity of a secreted luciferase reporter protein driven by an NF- ⁇ transcriptional response element, was used to study the functional activity of the CD80 and anti-CD3 ScFv transgenes in the NG-500 virus. The activity was compared to another virus, NG-348, which expresses CD80 and anti-CD3 ScFv but not ⁇ . A549 cells were seeded into 96 well plates at a density of 8e4 cells/well.
  • Example 6 Human T-cell activation and degranulation mediated by NG-500 infected carcinoma cell lines
  • A549 lung carcinoma cells either infected with NG-500, NG-348 or EnAd virus particles or left uninfected, were co-cultured with CD3+ T-cells isolated from human PBMC donors. Both NG-348 and NG-500 viruses express the membrane-anchored chimeric form of the single chain Fv anti-human CD3s and CD80 but, unlike NG-500, NG-348 does not also encode ⁇ .
  • T-cell activation was assessed by analysing cell surface activation marker CD25 (by Flow cytometry), CD107a cell surface expression as a marker for degranulation (by Flow cytometry) and secretion of stimulatory cytokine, IFNy (by ELISA).
  • A549 cells were seeded into 12 well plates at a density of 5e5 cells/well. Plates were incubated for 18 hrs, 37°C, 5% C0 2 , before cells were either infected with EnAd, NG-500 or NG-348 at 10 ppc or were left uninfected.
  • CD3 + T-cells isolated by negative selection from PBMCs (MACs), were added to the A549 cell monolayers at a ratio of 8 T-cells: 1 tumour cell. The co-culture was carried out for 16 hrs, after which point cellular supernatants were collected for ELISA analysis and T-cells harvested for Flow cytometry analysis.
  • Culture media containing non-adherent cells was removed from co-culture wells and centrifuged (300xg). The supernatant was carefully removed, diluted 1 in 2 with PBS 5% BSA and stored for ELISA analysis. The adherent cell monolayers were washed once with PBS and then detached using trypsin. The trypsin was inactivated using complete media and the cells were added to the cell pellets that had been collected from the culture supernatants. The cells were centrifuged (300xg), the supernatant discarded and the cell pellet washed in 200 ⁇ , of PBS.
  • the cells were centrifuged again and then re-suspended in 50 ⁇ , of FACs buffer (5% BSA PBS) containing Live/Dead Aqua (Life tech) for 15 mins at room temperature.
  • the cells were washed once in FACs buffer before staining with panels of directly conjugated antibodies: anti-CD3 conjugated to BV605; anti-CD25 conjugated to BV421; anti-CD107a conjugated to FITC.
  • a sample of cells from each co-culture condition was also stained with relevant isotype control antibodies. All staining was carried out in FACs buffer in a total volume of 5C ⁇ L/well for 15 mins, 4°C. Cells were then washed with FACs buffer (200 ⁇ ) before resuspension in 200 ⁇ , of FACs buffer and analysis by Flow cytometry (Attune).
  • T-cell activation marker CD25 was assessed by expression of the T-cell activation marker CD25 and the marker for T-cell degranulation, CD107a on live, CD3 + , single cells.
  • CD4+ CD3+CD8-
  • CD3+CD8+ T- cells expressing CD25 and CD107a was significantly higher for T cells cultured with NG-500 infected A549 cells than EnAd or uninfected controls and NG-500 and NG-348 demonstrated equivalent levels of T cell activation ( Figure 4A and 4B).
  • BSA/PBS assay buffer in a range of 1:100 to 1:1000
  • ELISA was carried out using the Human IFN gamma Ready Set Go kit (Affymetrix) according to the manufacturer's protocol.
  • the concentration of secreted IFNy was determined by interpolating from the standard curve. Expression of IFNy could only be detected in the supernatants of co-cultures using NG-348 or NG-500 infected A549 cells and was not detectable in either the EnAd, or untreated controls (Figure 5).
  • the above examples demonstrate that the oncolytic adenovirus of the present disclosure, NG-500 i) retains the oncolytic properties and replication competency of the original parental adenovirus EnAd; ii) successfully expressed CD80 on the cell surface of the cancer cells it infected; iii) successfully produced secreted ⁇ - ⁇ in the cancer cells it infected; and iv) were able to upregulate T cell activation to a significantly greater extent than EnAd.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un adénovirus oncolytique déficient en réplication ou apte à la réplication, comprenant : (i) un premier transgène comprenant une séquence d'ADN codant pour une forme à ancrage membranaire d'une protéine immunomodulatrice ou d'un fragment actif de celle-ci, et (ii) au moins un deuxième transgène codant pour une deuxième protéine ou un fragment actif de celle-ci, les transgènes étant situés entre le gène de fibre virale L5 et le gène E4 du virus et lesdits transgènes étant sous le contrôle d'un promoteur endogène au virus, tels que le promoteur tardif majeur. L'invention concerne également des compositions les comprenant, ainsi que l'utilisation de ces virus et compositions dans le traitement du cancer, en particulier.
PCT/EP2017/078218 2016-11-03 2017-11-03 Adénovirus oncolytique codant pour des transgènes Ceased WO2018083259A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1618572.0 2016-11-03
GB201618572 2016-11-03

Publications (1)

Publication Number Publication Date
WO2018083259A1 true WO2018083259A1 (fr) 2018-05-11

Family

ID=60450595

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/078218 Ceased WO2018083259A1 (fr) 2016-11-03 2017-11-03 Adénovirus oncolytique codant pour des transgènes

Country Status (1)

Country Link
WO (1) WO2018083259A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018191545A1 (fr) * 2017-04-12 2018-10-18 Epicentrx, Inc. Adénovirus recombinant à transgènes multiples
WO2018218151A1 (fr) * 2017-05-25 2018-11-29 University Of Central Florida Research Foundation, Inc. Nouveaux virus oncolytiques pour sensibiliser des cellules tumorales à la destruction par des cellules tueuses naturelles
US10849945B2 (en) 2015-04-30 2020-12-01 Psioxus Therapeutics Limited Oncolytic adenovirus encoding a B7 protein or active fragment
US11077156B2 (en) 2013-03-14 2021-08-03 Salk Institute For Biological Studies Oncolytic adenovirus compositions
US11130968B2 (en) 2016-02-23 2021-09-28 Salk Institute For Biological Studies High throughput assay for measuring adenovirus replication kinetics
US11155622B2 (en) 2015-12-17 2021-10-26 Psioxus Therapeutics Limited Virus encoding an anti-TCR-complex antibody or fragment
US11401529B2 (en) 2016-02-23 2022-08-02 Salk Institute For Biological Studies Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics
US11439678B2 (en) 2013-10-25 2022-09-13 Psioxus Therapeutics Limited Oncolytic adenoviruses armed with heterologous genes
US11813337B2 (en) 2016-12-12 2023-11-14 Salk Institute For Biological Studies Tumor-targeting synthetic adenoviruses and uses thereof
US11840702B2 (en) 2017-08-28 2023-12-12 Akamis Bio Limited Adenovirus armed with bispecific T cell activator
US12049513B2 (en) 2016-08-29 2024-07-30 Akamis Bio Limited Oncolytic group B adenovirus expressing a stroma-targeted bispecific t-cell engager
US12365878B2 (en) 2018-04-09 2025-07-22 Salk Institute For Biological Studies Oncolytic adenovirus with enhanced replication properties comprising modifications in E1A, E3, and E4

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015059303A1 (fr) * 2013-10-25 2015-04-30 Psioxus Therapeutics Limited Adénovirus oncolytiques pourvus de gènes hétérologues
WO2016174200A1 (fr) * 2015-04-30 2016-11-03 Psioxus Therapeutics Limited Adénovirus oncolytique codant une protéine b7
WO2017103290A1 (fr) * 2015-12-17 2017-06-22 Psioxus Therapeutics Limited Adénovirus du groupe b codant pour un anticorps ou fragment contre le complexe tcr

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015059303A1 (fr) * 2013-10-25 2015-04-30 Psioxus Therapeutics Limited Adénovirus oncolytiques pourvus de gènes hétérologues
WO2016174200A1 (fr) * 2015-04-30 2016-11-03 Psioxus Therapeutics Limited Adénovirus oncolytique codant une protéine b7
WO2017103290A1 (fr) * 2015-12-17 2017-06-22 Psioxus Therapeutics Limited Adénovirus du groupe b codant pour un anticorps ou fragment contre le complexe tcr

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BRIAN CHAMPION ET AL: "Developing tumor-localized, combination immunotherapies", July 2016 (2016-07-01), XP055439476, Retrieved from the Internet <URL:http://psioxus.com/wp-content/uploads/2016/12/AACR-Poster-Apr-2016.pdf> [retrieved on 20180110] *
CHOI K-J ET AL: "Concurrent delivery of GM-CSF and B7-1 using an oncolytic adenovirus elicits potent antitumor effect", GENE THERAPY, NATURE PUBLISHING GROUP, GB, vol. 13, no. 13, July 2006 (2006-07-01), pages 1010 - 1020, XP002571567, ISSN: 0969-7128, [retrieved on 20060309], DOI: 10.1038/SJ.GT.3302759 *
STEPHANE PAUL ET AL: "The combination of a chemokine, cytokine and TCR-based T cell stimulus for effective gene therapy of cancer", CANCER IMMUNOLOGY, IMMUNOTHERAPY, vol. 51, no. 11-12, December 2002 (2002-12-01), Berlin/Heidelberg, pages 645 - 654, XP055355169, ISSN: 0340-7004, DOI: 10.1007/s00262-002-0314-2 *
Y.-S. LEE ET AL: "Enhanced Antitumor Effect of Oncolytic Adenovirus Expressing Interleukin-12 and B7-1 in an Immunocompetent Murine Model", CLINICAL CANCER RESEARCH, vol. 12, no. 19, October 2006 (2006-10-01), pages 5859 - 5868, XP055057666, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-06-0935 *
YANG ZHANG-MIN ET AL: "Anti-CD3 scFv-B7.1 fusion protein expressed on the surface of HeLa cells provokes potent T-lymphocyte activation and cytotoxicity", BIOCHEMISTRY AND CELL BIOLOGY. BIOCHIMIE ET BIOLOGIE CELLULAIRE, NRC RESEARCH PRESS, CA, vol. 85, no. 2, 31 March 2007 (2007-03-31), pages 196 - 202, XP009501722, ISSN: 0829-8211 *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11077156B2 (en) 2013-03-14 2021-08-03 Salk Institute For Biological Studies Oncolytic adenovirus compositions
US11938159B2 (en) 2013-10-25 2024-03-26 Akamis Bio Limited Oncolytic adenoviruses armed with heterologous genes
US11439678B2 (en) 2013-10-25 2022-09-13 Psioxus Therapeutics Limited Oncolytic adenoviruses armed with heterologous genes
US10849945B2 (en) 2015-04-30 2020-12-01 Psioxus Therapeutics Limited Oncolytic adenovirus encoding a B7 protein or active fragment
US11000559B2 (en) 2015-04-30 2021-05-11 Psioxus Therapeutics Limited Oncolytic adenovirus encoding a B7 protein
US11155622B2 (en) 2015-12-17 2021-10-26 Psioxus Therapeutics Limited Virus encoding an anti-TCR-complex antibody or fragment
US11970536B2 (en) 2015-12-17 2024-04-30 Akamis Bio Limited Group B adenovirus encoding an anti-TCR-complex antibody or fragment
US12281324B2 (en) 2016-02-23 2025-04-22 Salk Institute For Biological Studies Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics
US11130968B2 (en) 2016-02-23 2021-09-28 Salk Institute For Biological Studies High throughput assay for measuring adenovirus replication kinetics
US11401529B2 (en) 2016-02-23 2022-08-02 Salk Institute For Biological Studies Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics
US12049513B2 (en) 2016-08-29 2024-07-30 Akamis Bio Limited Oncolytic group B adenovirus expressing a stroma-targeted bispecific t-cell engager
US12258417B2 (en) 2016-08-29 2025-03-25 Akamis Bio Limited Adenovirus armed with bispecific T cell engager
US11813337B2 (en) 2016-12-12 2023-11-14 Salk Institute For Biological Studies Tumor-targeting synthetic adenoviruses and uses thereof
US12036247B2 (en) 2017-04-12 2024-07-16 Epicentrx, Inc. Multiple transgene recombinant adenovirus
WO2018191545A1 (fr) * 2017-04-12 2018-10-18 Epicentrx, Inc. Adénovirus recombinant à transgènes multiples
US11684637B2 (en) 2017-05-25 2023-06-27 University Of Central Florida Research Foundation, Inc. Oncolytic viruses for sensitizing tumor cells to killing by natural killer cells
WO2018218151A1 (fr) * 2017-05-25 2018-11-29 University Of Central Florida Research Foundation, Inc. Nouveaux virus oncolytiques pour sensibiliser des cellules tumorales à la destruction par des cellules tueuses naturelles
US12312393B2 (en) 2017-05-25 2025-05-27 University Of Central Florida Research Foundation, Inc. Oncolytic viruses for sensitizing tumor cells to killing by natural killer cells
US11840702B2 (en) 2017-08-28 2023-12-12 Akamis Bio Limited Adenovirus armed with bispecific T cell activator
US12365878B2 (en) 2018-04-09 2025-07-22 Salk Institute For Biological Studies Oncolytic adenovirus with enhanced replication properties comprising modifications in E1A, E3, and E4

Similar Documents

Publication Publication Date Title
US20240247061A1 (en) Group b adenovirus encoding an anti-tcr-complex antibody or fragment
US20250026851A1 (en) Adenovirus armed with bispecific t cell activator
US10849945B2 (en) Oncolytic adenovirus encoding a B7 protein or active fragment
WO2018083259A1 (fr) Adénovirus oncolytique codant pour des transgènes
WO2018083258A1 (fr) Adénovirus oncolytique codant pour au moins trois transgènes
WO2018083257A1 (fr) Adénovirus oncolytiques codant pour des transgènes
US11142580B2 (en) Oncolytic virus and method
HK1263149A1 (en) Oncolytic adenovirus encoding a b7 protein
HK1249009B (en) Oncolytic adenovirus encoding a b7 protein

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17803814

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17803814

Country of ref document: EP

Kind code of ref document: A1