WO2019134018A1 - Conjugués de vaccin et leurs utilisations - Google Patents

Conjugués de vaccin et leurs utilisations Download PDF

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Publication number
WO2019134018A1
WO2019134018A1 PCT/AU2018/051408 AU2018051408W WO2019134018A1 WO 2019134018 A1 WO2019134018 A1 WO 2019134018A1 AU 2018051408 W AU2018051408 W AU 2018051408W WO 2019134018 A1 WO2019134018 A1 WO 2019134018A1
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Prior art keywords
antigen
conjugate
endosomal
conjugate according
cell
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PCT/AU2018/051408
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English (en)
Inventor
Jason Waithman
Shane Stone
Paul Watt
Original Assignee
Telethon Kids Institute
Phylogica Limited
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Publication date
Priority claimed from AU2018900032A external-priority patent/AU2018900032A0/en
Application filed by Telethon Kids Institute, Phylogica Limited filed Critical Telethon Kids Institute
Publication of WO2019134018A1 publication Critical patent/WO2019134018A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001136—Cytokines
    • A61K39/001142—Chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • 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
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    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to conjugates, fusion polypeptides and compositions comprising those conjugates or fusion polypeptides for use in the generation of an immune response.
  • the immune response is generated to treat or prevent cancer or an infectious disease.
  • tumours Most cancer cells elicit an immune response that is evident by the presence of immune cell infiltrates and inflammation. This response, however, is not strong enough to overcome the cancer cell's defence strategies.
  • the lack of understanding of the complex interactions between tumours and the immune system has hindered the development of cancer immunotherapy. Approaches involving using purified tumour antigens and more complex mixtures of tumour antigens have often failed to stimulate adequate immune responses against tumours. The reasons for this are unknown, but may include the genetic instability of tumours and the ability of tumours to evade the immune system by presenting a "normal" appearance or releasing inhibitors.
  • Tumours can respond to an immune response by reducing the amount of targeted antigens, by masking antigens from the immune system or by expressing mutated versions of antigens that are no longer recognised. Such defensive strategies undermine the immune system, making it difficult to maintain an effective immune response at the level required to halt tumour growth and cause regression. Moreover, the responses may be inadequate since they fail to stimulate an adaptive immune response.
  • T cell-mediated anti-tumor immunity plays a role in regulating tumor growth, placing selective pressure on the antigenically-heterogeneous cancer cell population throughout disease progression.
  • Antigen specific immunotherapy aims to enhance or induce specific immune responses in patients and has been successfully used to control cancer diseases.
  • T cells play a central role in cell-mediated immunity in humans and animals.
  • the recognition and binding of a particular antigen is mediated by the T cell receptors (TCRs) expressed on the surface of T cells.
  • TCRs T cell receptors
  • the T cell receptor (TCR) of a T cell is able to interact with immunogenic peptides (epitopes) bound to major histocompatibility complex (MHC) molecules and presented on the surface of target cells.
  • MHC major histocompatibility complex
  • Humoral immunity Vaccines that stimulate antibody production (humoral immunity) have enjoyed success for more than two centuries.
  • Humoral immunity is of limited effectiveness against cancers because many tumor-associated antigens are intracellular and inaccessible to the antibody.
  • Effective cellular immune responses (cell- mediated immunity), especially cytotoxic T lymphocytes (CTLs), are the best weapons amongst the immune system's arsenal against these diseases.
  • CTLs cytotoxic T lymphocytes
  • the antigen In order for the antigen to be recognized by CD8 + T cells, it must be complexed with MHC class I molecules. Endogenous antigens almost always are degraded into peptides by proteasomes. The resultant small peptide fragments are picked up by TAP (transporter associated with antigen processing), and eventually complexed with MHC class I molecules, displayed on the surface of the cell. The antigen-MHC class I complex is recognized by CD8 + cytotoxic T cells.
  • Exogenous antigens are taken up by antigen-presenting cells (APCs), such as dendritic cells, by multiple mechanisms that include endocytosis or phagocytosis.
  • APCs antigen-presenting cells
  • the endosome or phagosome so formed predominantly mature to the point where they fuse with lysosomes, in which the antigen is degraded into peptide fragments which are then nestled within a class II histocompatibility molecule (MHC II) and displayed at the surface of the cell, and are recognized by CD4 + T cells.
  • MHC II class II histocompatibility molecule
  • the present invention provides a conjugate for inducing an immune response, the conjugate comprising:
  • an antigen wherein the antigen presenting cell targeting moiety is releasable from the moiety capable of endosomal escape and the antigen.
  • the antigen presenting cell is a cell capable of cross presentation. Even more preferably the antigen presenting cell is a professional antigen presenting cell such as a macrophage or dendritic cell.
  • the antigen presenting cell targeting moiety may localize to and bind a target molecule present on an antigen presenting cell.
  • the targeting moiety may be referred to as a dendritic cell targeting moiety.
  • the targeting moieties contemplated for use in any aspect of the invention include antibodies, polypeptides, peptides, aptamers, other ligands, or any combination thereof, that can bind a component of the target cell or molecule.
  • Exemplary dendritic cell targeting moieties are molecules that bind to any one of XCR1 , DEC-205 (CD205), Langerin (CD207), Clec9A receptors, DC-SIGN (CD209) and DNGR1.
  • a preferred dendritic cell targeting moiety binds to XCR1.
  • a moiety that binds to XCR1 comprises, consists essentially of or consists of an amino acid sequence of XCL1 as described herein.
  • the antigen presenting cell targeting moiety may be associated with the moiety capable of endosomal escape via a linker which is cleavable in an endosome compartment prior to the endosome maturing into a late endosome.
  • the linker is cleavable in an early endosome.
  • the linker which is cleavable in an early endosome comprises an amino acid sequence of a furin cleavage site.
  • the furin cleavage site may comprise, consist essentially of or consist of the amino acid sequence R-X-K/R-R (SEQ ID NO: 1 ). Even more preferably, the furin cleavage site may comprise, consist essentially of or consist of the amino acid sequence RKKR (SEQ ID NO: 2).
  • the linker between the antigen presenting cell targeting moiety and the moiety capable of endosomal escape may also be cleavable in the maturing or late endosome, or in the lysosome.
  • the linker which is also cleavable in a lysosome comprises an amino acid sequence of a cathepsin B cleavage site.
  • the cathepsin B cleavage site may comprise, consist essentially of or consist of the amino acid sequence V-citrulline (SEQ ID NO: 3; V denotes the amino acid valine) or FKFL (SEQ ID NO: 4).
  • the linker between the antigen presenting cell targeting moiety and the moiety capable of endosomal escape may comprise, consist essentially of or consist of a furin cleavage site as described herein and a cathepsin B cleavage site as described herein, preferably RKKR (SEQ ID NO: 2) and V-citrulline (SEQ ID NO: 3).
  • the dual furin/cathepsin B cleavage linker comprises, consists essentially of or consists of the amino acid sequence RKKRXV-citrulline, where X is any small amino acid, preferably a serine.
  • the dual furin/cathepsin B cleavage linker comprises, consists essentially of or consists of the amino acid sequence GSGTGGSTGRKKRSV-citrulline-GC (SEQ ID NO: 5).
  • the moiety capable of endosomal and/or lysosomal escape is capable of escaping from an endosomal and/or lysosomal compartment into the cytosol.
  • the moiety is capable of escaping from an endosomal and/or lysosomal compartment into the cytosol prior to the endosome maturing into a late endosome or lysosome. More preferably, the moiety is capable of escaping from an early endosomal compartment into the cytosol.
  • the moiety capable of endosomal and/or lysosomal escape mediates significantly greater endosomal escape than the TAT peptide.
  • the degree or level of endosomal escape may be determined by any method known in the art, including any method or assay described herein.
  • the level of endosomal escape may be determined using the beta-lactamase assay described herein.
  • a moiety capable of endosomal and/or lysosomal escape mediate significantly greater endosomal escape than the TAT peptide when measured using the beta-lactamase assay described herein.
  • a moiety capable of endosomal and/or lysosomal escape may result in greater than or equal to 5 %, 10 % or 15 % positive cells at 450 nM, at concentrations of 1 mM, 2 mM or 4 pM respectively, when measured using the beta-lactamase assay described herein.
  • endosomal escape may include early and/or late endosomal escape, preferably early endosomal escape.
  • Lysosomal escape may include early and/or late lysosomal escape, preferably early lysosomal escape.
  • the moiety capable of endosomal and/or lysosomal escape is a peptide.
  • the peptide comprises, consists essentially of, or consists of the amino acid sequence PLKPKKPKTQEKKKKQPPKPKKPKTQEKKKKQPPKPKR (SEQ ID NO: 6).
  • the peptide may comprise, consist essentially of or consist of any one or more of the amino acid sequences in WO2014205518 referred to therein as SEQ ID NO: 83-119 and which are herein incorporated in their entirety by reference.
  • the moiety capable of endosomal and/or lysosomal escape is associated with the antigen such that when the moiety escapes from an endosomal and/or lysosomal compartment into the cytosol, the antigen also enters the cytosol.
  • the moiety capable of endosomal and/or lysosomal escape and the antigen are chemically linked, for example, covalently linked.
  • the covalent linkage may be direct or via a linker.
  • the linker may comprise, consists essentially of or consist of a peptide, for example containing 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.
  • the amino acids are selected from alanine, glycine and serine.
  • the peptide comprises, consists essentially of, or consists of the amino acid sequence GAS (SEQ ID NO: 7).
  • the antigen presenting cell targeting moiety mediates internalization upon contact with an antigen presenting cell.
  • the moiety capable of endosomal and/or lysosomal escape mediates internalization upon contact with an antigen presenting cell.
  • the moiety capable of endosomal and/or lysosomal escape may be a cell penetrating peptide as described herein but also have the capacity to escape from endosomal or lysosomal compartments for delivery into the cytosol.
  • Those moieties having the capability of mediating intracellular delivery and escape from endosomal and/or lysosomal compartments for delivery into the cytosol may also be referred to as cytosolic delivery cell penetrating peptide (CPPs).
  • CPPs cytosolic delivery cell penetrating peptide
  • the antigen comprises a peptide capable of being presented by an HLA class I molecule.
  • the antigen also comprises a peptide capable of being presented by an HLA class II molecule.
  • the peptide capable of being presented by an HLA class I molecule comprises a CD8+ T cell epitope.
  • the peptide capable of being presented by an HLA class II molecule comprises a CD4+ T cell epitope.
  • Exemplary antigens include any tumour associated antigens, including those described herein.
  • the present invention also provides a chimeric or fusion polypeptide for inducing an immune response, the chimeric or fusion polypeptide comprising:
  • each of the antigen presenting cell targeting moiety, the moiety capable of endosomal and/or lysosomal escape and the antigen are peptides or polypeptides.
  • the means by which the antigen presenting cell targeting moiety is releasable from the moiety capable of endosomal escape and the antigen is also a peptide or polypeptide.
  • the chimeric or fusion polypeptide is a contiguous sequence of amino acids.
  • the chimeric or fusion polypeptide is expressed from a single expression construct.
  • a conjugate, or chimeric or fusion polypeptide of the invention may be produced synthetically or recombinantly.
  • a conjugate, or chimeric or fusion polypeptide of the invention may be substantially purified.
  • the present invention also provides a composition such as an antigenic composition, particularly a vaccine composition, comprising a conjugate, or chimeric or fusion polypeptide of the invention described herein, optionally in association with an adjuvant.
  • a composition such as an antigenic composition, particularly a vaccine composition, comprising a conjugate, or chimeric or fusion polypeptide of the invention described herein, optionally in association with an adjuvant.
  • the present invention also provides a composition such as an antigenic composition, particularly a vaccine composition, wherein the composition comprises a conjugate or a chimeric or fusion polypeptide as described herein, optionally in association with a virus-like particle, liposome or nanoparticle.
  • the invention provides a pharmaceutical composition for treating or preventing cancer in a subject comprising a conjugate, or chimeric or fusion polypeptide of the invention described herein and a pharmaceutically acceptable diluent, excipient or carrier.
  • the invention provides a pharmaceutical composition for treating or preventing cancer in a subject comprising as an active ingredient a conjugate, or chimeric or fusion polypeptide of the invention described herein, the composition further comprising a pharmaceutically acceptable diluent, excipient or carrier.
  • the invention provides a pharmaceutical composition for treating cancer in a subject comprising as a main ingredient a conjugate, or chimeric or fusion polypeptide of the invention described herein, the composition further comprising a pharmaceutically acceptable diluent, excipient or carrier.
  • the present invention also provides a conjugate, or chimeric or fusion polypeptide of the invention described herein for use in the treatment or prevention of cancer in a subject.
  • the invention also provides a nucleic acid molecule comprising, consisting essentially of or consisting of a nucleotide sequence encoding a conjugate, or chimeric or fusion polypeptide of the invention described herein.
  • such a nucleic acid is included in an expression construct in which the nucleic acid is operably linked to a promoter.
  • an expression construct can be in a vector, e.g., a plasmid.
  • the expression construct may comprise a promoter linked to a nucleic acid encoding that polypeptide chain a conjugate, or chimeric or fusion polypeptide of the invention described herein.
  • the invention provides a cell comprising a vector or nucleic acid described herein.
  • the cell is isolated, substantially purified or recombinant.
  • Examples of cells of the present invention include bacterial cells, yeast cells, insect cells or mammalian cells.
  • the present invention also provides a method for eliciting a cell-mediated immune response to an antigen in a subject, the method comprising providing a conjugate, chimeric or fusion polypeptide, or composition of the invention as described herein, and administering the conjugate, chimeric or fusion polypeptide, or composition to the subject, thereby eliciting a cell-mediated immune response to the antigen in a subject.
  • the present invention also provides a method for treating or preventing cancer or an infectious disease in a subject in need thereof, the method comprising administering a conjugate, chimeric or fusion polypeptide, or composition of the invention as described herein, thereby treating or preventing cancer or an infectious disease in the subject.
  • the present invention also provides a use of a conjugate, chimeric or fusion polypeptide, or composition of the invention as described herein in the manufacture of a medicament for the treatment or prevention of cancer or an infectious disease in a subject.
  • the cancer may be any one described herein.
  • the subject may be any one described herein.
  • the present invention also provides a method for inducing an immune response in a subject suitable for the treatment of cancer or infectious disease, the method including the steps of contacting antigen presenting cells with a conjugate, chimeric or fusion polypeptide, or composition of the invention as described herein ex Vo for a sufficient time and under conditions enabling the antigen to be presented on HLA-I of the antigen presenting cells; administering the antigen presenting cells to the subject, thereby forming an immune response in a subject suitable for the treatment of cancer or an infectious disease.
  • the present invention also provides a method for preparing an isolate or substantially purified composition of antigen presenting cells to treat or prevent cancer or an infectious disease in a subject, the method including the steps of providing a population of antigen presenting cells, and contacting the population of antigen presenting cells with a conjugate, chimeric or fusion polypeptide, or composition of the invention as described herein ex vivo for a sufficient time and under conditions enabling the antigen to be presented on HLA-I of the antigen presenting cells, thereby preparing an isolate or substantially purified composition of antigen presenting cells to treat or prevent cancer or an infectious disease in a subject.
  • the present invention also provides a method for forming an immune response in a subject suitable for the treatment or prevention of cancer or an infectious disease including the steps of obtaining antigen presenting cells from the subject, culturing the obtained antigen presenting cells to generate a population of antigen presenting cells, contacting the population of antigen presenting cells with a conjugate, chimeric or fusion polypeptide, or composition of the invention as described herein ex vivo for a sufficient time and under conditions enabling the antigen to be presented on HLA-I of the antigen presenting cells for forming an immune response, re-introducing the antigen presenting cells to the subject, thereby producing an immune response in a subject suitable for the treatment of cancer or an infectious disease.
  • the population of antigen presenting cells are contacted with a conjugate, chimeric or fusion polypeptide, or composition of the invention as described herein for a sufficient time and under conditions enabling the antigen to be presented on both HLA-I and HLA-II molecules on the cell surface.
  • the invention also provides a kit or article of manufacture comprising a conjugate, chimeric or fusion polypeptide, composition or nucleic acid of the invention described herein.
  • the kit further comprises written instructions for use of the conjugate, chimeric or fusion polypeptide, composition or nucleic acid of the invention described herein in a method or use of the invention as described herein.
  • the term "comprise” and variations of the term, such as “comprising”, “comprises” and “comprised” are not intended to exclude further additives, components, integers or steps.
  • Figure 1 provides a schematic of the proposed pathway for uptake of the conjugates, chimeric or fusion polypeptides of the invention (cytosolic delivery CPP referred to as FPP in this Figure).
  • Figure 2 provides a schematic of a moiety capable of endosomal escape linked to an antigen (cytosolic delivery CPP - 1746; antigen gD-GTG-gB).
  • Figure 3 provides a schematic of the assembly of the SpyTag and disulphide linker which may not be cleaved in a cross-presenting DCs endosome due to unique neutral pH in this intracellular compartment.
  • the disulphide bond would typically be cleaved in an acidic environment.
  • MCPA mercaptopropionic acid
  • MCPA mercaptopropionic acid
  • Figure 4 provides a schematic of the final V5-SpyT-Cys-mcpa-CPP-antigen conjugate.
  • Figure 5 shows an SDS-PAGE confirming SpyC/SpyT ligation.
  • Figure 6 provides a schematic of the experimental protocol of peptide vaccine using disulphide linkage technology, to measure specific T cell expansion.
  • Figure 7 shows the results that disulfide-linked vaccine constructs do not elicit a CD8+ gB-specific T cell expansion whereas disulfide-linked vaccine constructs elicit a CD4+ gD-specific T cell expansion.
  • Figure 8 provides a schematic of the SpyTag and cleavable linkers.
  • Figure 9 provides a schematic of the SpyTag-FCB-cytosolic delivery CPP- Antigen delivery module (cytosolic delivery CPP referred to as FPP in this Figure).
  • Figure 10 provides a schematic of the full peptide vaccine construct.
  • Figure 11 provides a schematic of the experimental protocol at two different amounts of peptide vaccine.
  • Figure 12 shows % gD-specific CD4+ T cells of total CD4+ T cells at 1 nmol and 2nmol doses.
  • Figure 13 shows % gB-specific CD8+ T cells of total CD8+ T cells at 1 nmol and 2nmol doses.
  • Figure 14 provides a schematic of the experimental prophylactic protocol in the B16 melanoma mouse model and results of percent tumour free survival.
  • Figure 15 shows B16-gB melanoma tumour growth kinetics 43 days post vaccination.
  • Figure 16 shows B16-gB melanoma percent survival 43 days post vaccination.
  • Figure 17 provides a schematic of the experimental therapeutic protocol in the B16 melanoma mouse model.
  • Figure 18 shows confocal microscopy images of T47D cells at 40X magnification after 1 hour incubation with CPP-SpyC/ SpyC-BLA conjugate. CCF2-AM incubated for 1 hour prior to protein incubation.
  • Figure 19 is a histogram showing the results of flow cytometry to quantify cytosolic CCF2-AM cleavage by internalized Beta-Lactamase. The ratio of blue to green fluorescence was assessed to determine the cell-penetrating and cytosolic delivery ability of the two CPP sequences 1746 and TAT. The flow cytometry data is further corroborated by confocal microscopy on live cells using the same assay conditions.
  • Figure 20 shows that CPP peptide vaccines provide efficient protection against HSV skin infection.
  • Cohorts of mice were primed (DO) subcutaneously with 2 nmol of XCL1 constructs linked to either gDgB or 1746-gDgB. All vaccinated groups and an additional unprimed group received 50 pg poly l:C as an adjuvant (DO). All groups were infected with HSV on the flank (D7) and viral titres in the skin were measured 6 days post infection (D13). Symbols represent individual mice with bars representing the mean.
  • the present invention is based on the design and production of multicomponent conjugates that are useful in activating both CD8+ and CD4+ T cells for the treatment or prevention of cancer.
  • These conjugates target professional antigen presenting cells and result in presentation of peptides on HLA-I or HLA-II thus activating CD8+ and CD4+ T cells, respectively.
  • the HLA-I pathway is more stringent than the HLA-II pathway in that it generally requires cytosolic processing by the proteasome to generate the antigen to present to CD8+ T cells.
  • CD8+ T cell expansion can only occur if the antigen is presented by HLA-I cytosolic loading.
  • the conjugates and fusion polypeptides of the invention contain moieties capable of endosomal and/or lysosomal escape that traffic an associated antigen into the cytosol for processing and HLA-I cytosolic loading.
  • the conjugates and fusion polypeptides also allow the antigen to be located within the maturing late endosome, where it is loaded onto HLA-II via an endosomal processing pathway which is recognised by CD4+ T cells.
  • CD4+ T cells CD4+ T cells
  • the inventors have surprisingly found that for sufficient endosomal and/or lysosomal escape of the antigen to allow for efficient cross-presentation of antigenic peptides, the moiety capable of escape and the cargo antigen must be separated from the antigen cell targeting moiety.
  • linkers that release the targeting moiety from the endosomal and/or lysosomal escape moiety in the early endosome the endosomal and/or lysosomal escape moiety is then capable of trafficking an associated antigen into the cytosol for processing and HLA-I cytosolic loading.
  • the multicomponent conjugate surprisingly functions even each component is covalently linked in a single construct.
  • derived from shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
  • references herein to a range of, e.g., residues, will be understood to be inclusive.
  • reference to“a region comprising amino acids 56 to 65” will be understood in an inclusive manner, i.e. , the region comprises a sequence of amino acids as numbered 56, 57, 58, 59, 60, 61 , 62, 63, 64 and 65 in a specified sequence.
  • chimeric or fusion polypeptide is used to refer to a polypeptide that is composed of functional units, domains, sequences or regions of amino acids derived from different sources or that are derived from the same source and that have been assembled so as to have an organisation that is distinguished from that observed in a molecule from which the unit, domain, sequence or region is derived or related to.
  • substantially purified is meant the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.
  • recombinant shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody antigen binding domain, this term does not encompass an antibody naturally-occurring within a subject’s body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody antigen binding domain. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen binding domain. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.
  • polypeptide shall be taken to include a single polypeptide chain, i.e. , a series of contiguous amino acids, such as a polymer, linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex).
  • the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond.
  • non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
  • the polypeptide may include amino acid residues and variants and synthetic analogues of the same.
  • amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • synthetic non-naturally-occurring amino acid such as a chemical analogue of a corresponding naturally-occurring amino acid
  • Naturally-occurring amino acid polymers do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.
  • a polypeptide that contains at least one residue that is not naturally synthesised may be referred to as a “peptidomimetic”.
  • a peptidemimetic as used herein is a synthetic chemical compound that has substantially the same structure and/or functional characteristics of a conjugate, or chimeric or fusion polypeptide of the invention as described herein.
  • Non-natural components of peptidomimetic compounds may be according to one or more of: a) residue linkage groups other than the natural amide bond (‘peptide bond’) linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • Peptidomimetics can be synthesized using a variety of procedures and methodologies described in the scientific and patent literatures, e.g., Organic Syntheses Collective Volumes, Gilman et al.
  • a "fragment” is a portion of a conjugate, or chimeric or fusion polypeptide of the present invention that retains substantially similar functional activity or substantially the same biological function or activity as the polypeptide, as shown assays disclosed herein.
  • a “derivative” includes all modifications to a conjugate, or chimeric or fusion polypeptide of the present invention that substantially preserve the functions disclosed herein and include additional structure and attendant function.
  • a “variant” includes polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of a conjugate, or chimeric or fusion polypeptide of the present invention.
  • the term "sufficiently similar” means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity.
  • amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar.
  • variants will be sufficiently similar to the amino acid sequence of the preferred conjugates, or chimeric or fusion polypeptides of the present invention.
  • Variants include variants of a conjugate, or chimeric or fusion polypeptide of the present invention encoded by a polynucleotide that hybridizes to a polynucleotide of this invention, or a complement thereof, under stringent conditions. Such variants generally retain the functional activity of a conjugate, or chimeric or fusion polypeptide of the present invention.
  • Variants include polypeptides that differ in amino acid sequence due to mutagenesis.
  • Substantially similar functional activity and “substantially the same biological function or activity” each means that the degree of biological activity is within about 50% to 100% or more, within 80% to 100% or more, or within about 90% to 100% or more, of that biological activity demonstrated by the peptide to which it is being compared when the biological activity of each peptide is determined by the same procedure or assay.
  • Similarity between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide.
  • An amino acid of one peptide is similar to the corresponding amino acid of a second polypeptide if it is identical or a conservative amino acid substitution.
  • Conservative substitutions include those described in Dayhoff, M.O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, P. (1989) EMBO J. 8:779-785.
  • amino acids belonging to one of the following groups represent conservative changes or substitutions:
  • Val lie, Leu, Met, Ala, Phe;
  • Non-polar Ala, Val, Leu, lie, Pro, Met Phe, Trp Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gin
  • Aromatic Phe, Tyr, His Proton Donor: Asn, Gin, Lys, Arg, His, Trp
  • Proton Acceptor Glu, Asp, Thr, Ser, Tyr, Asn, Gin
  • any polypeptide of the invention as described herein that contains one or more conservative or non-conservative substitutions provided the conjugate, or chimeric or fusion polypeptide of the present invention retains the ability to activate or induce proliferation of CD8+ and CD4+ T cells. Determining whether a conjugate, or chimeric or fusion polypeptide of the present invention that contains one or more conservative or non-conservative substitutions retains the ability to activate or induce proliferation of CD8+ and CD4+ T cells can be readily determined empirically, for example by any method described herein.
  • the conjugate, or chimeric or fusion polypeptide may comprise the following components in order from N-terminus to C- terminus: an antigen presenting cell targeting moiety - X - a moiety capable of endosomal and/or lysosomal escape - an antigen.
  • the conjugate, or chimeric or fusion polypeptide may comprise the following components in order from N-terminus to C-terminus: a moiety capable of endosomal and/or lysosomal escape - an antigen - X - an antigen presenting cell targeting moiety.
  • the conjugate, or chimeric or fusion polypeptide may comprise the following components in order from N-terminus to C-terminus: an antigen - a moiety capable of endosomal and/or lysosomal escape - X - an antigen presenting cell targeting moiety.
  • X denotes that the antigen presenting cell targeting moiety is releasable from the moiety capable of endosomal escape and the antigen
  • the C-terminal residue of the moiety capable of endosomal and/or lysosomal escape may be covalently linked to the N-terminal residue of an antigen, or the N-terminal residue of the antigen may be covalently linked to the C-terminal residue of moiety capable of endosomal and/or lysosomal escape.
  • the moiety capable of endosomal and/or lysosomal escape and antigen are said to be“directly linked” or “adjacent”.
  • the conjugate or chimeric or fusion polypeptides include a linker for linking the moiety capable of endosomal and/or lysosomal escape to the antigen.
  • the linker may be any linker, including both amino acid and non-amino acid linkers.
  • the linker is non-immunogenic. Suitable linkers may be up to 15 amino acids in length, although less than five amino acids is preferred.
  • the linker may function to bring the moiety capable of endosomal and/or lysosomal escape to the antigen into a close spatial arrangement, alternatively, it may space the moiety capable of endosomal and/or lysosomal escape to the antigen apart.
  • the linker is not cleavable in an early endosome. Even more preferably, the linker is not cleavable in an endosomal compartment prior to maturation to a late endosome.
  • conjugate or chimeric or fusion polypeptides are displayed in the context of a virus-like particle, liposome or nanoparticle.
  • the antigen and cell-penetrating peptide are associated with a nanoparticle.
  • the conjugate or chimeric or fusion polypeptide includes a sequence enabling enzymatic ligation, including for example, to link the antigen presenting cell targeting moiety and the moiety capable of endosomal and/or lysosomal escape.
  • sequences include the peptide “SPYCATCHER”, which has the sequence:
  • immune response refers to an integrated bodily response to an antigen and in the present context preferably refers to an adaptive immune response including a cellular immune response.
  • the immune response may be protective/preventive/prophylactic and/or therapeutic.
  • inducing an immune response may mean that there was no immune response against a particular antigen before induction, but it may also mean that there was a certain level of immune response against a particular antigen before induction and after induction said immune response is enhanced.
  • inducing an immune response also includes “enhancing an immune response”.
  • said subject is protected from developing a disease such as a cancer disease or the disease condition is ameliorated by inducing an immune response.
  • an immune response against a tumour expressed antigen may be induced in a patient having cancer or in a subject being at risk of developing cancer. Inducing an immune response in this case may mean that the disease condition of the subject is ameliorated, that the subject does not develop metastases, or that the subject being at risk of developing a cancer disease does not develop a cancer disease.
  • conjugate, or chimeric or fusion polypeptide of the invention can be adapted to include any number of antigen presenting cell targeting moieties.
  • Preferred moieties target professional antigen presenting cells such as dendritic cells.
  • Antigen presenting cells are cells which present peptide fragments of protein antigens in association with HLA molecules on their cell surface. Some APCs may activate antigen specific T cells.
  • Antigen processing refers to the degradation of a polypeptide or antigen into procession products, which are fragments of said polypeptide or antigen (e.g., the degradation of a polypeptide into peptides) and the association of one or more of these fragments (e.g., via binding) with HLA molecules for presentation by cells, preferably antigen presenting cells, to specific T cells.
  • Professional antigen-presenting cells are very efficient at internalizing antigen, either by phagocytosis or by receptor-mediated endocytosis, and then displaying a fragment of the antigen, bound to a class II HLA molecule, on their membrane.
  • the T cell recognizes and interacts with the antigen-class II HLA molecule complex on the membrane of the antigen-presenting cell.
  • An additional co-stimulatory signal is then produced by the antigen-presenting cell, leading to activation of the T cell.
  • the expression of co-stimulatory molecules is a defining feature of professional antigen- presenting cells.
  • dendritic cells which have the broadest range of antigen presentation, and are probably the most important antigen-presenting cells, macrophages, B -cells, and certain activated epithelial cells.
  • DCs Dendritic cells
  • HLA class II and I antigen presentation pathways It is well known that dendritic cells are potent inducers of immune responses and the activation of these cells is a critical step for the induction of antitumoral immunity.
  • MHC major histocompatibility complex
  • MHC proteins or molecules are important for signalling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptides and present them for recognition by T cell receptors.
  • the proteins encoded by the MHC are expressed on the surface of cells, and display both self antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to a T cell.
  • MHC region is divided into three subgroups, class I, class II, and class III.
  • MHC class I proteins contain an a(alpha)-chain and b2 (beta2)-microglobulin (not part of the MHC encoded by chromosome 15). They present antigen fragments to cytotoxic T cells.
  • MHC class II proteins contain a- and b-chains and they present antigen fragments to T-helper cells.
  • MHC class III region encodes for other immune components, such as complement components and some that encode cytokines.
  • HLA genes in the MHC region that encode antigen-presenting proteins on the cell surface are referred to as human leukocyte antigen (HLA) genes.
  • MHC is often used to refer to HLA gene products.
  • HLA genes include the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1 , HLA-DPB1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, and HLA-DRB 1.
  • an MHC molecule is an HLA molecule.
  • an“antigen presenting cell targeting moiety” refers to a molecule(s) that has the ability, or capability, to localize to and bind a target molecule present on an antigen presenting cell.
  • a“dendritic cell targeting moiety” refers to a molecule(s) that has the ability to localize to and bind a target molecule present on a dendritic cell.
  • conjugates, fusion proteins or compositions of the invention comprising such an antigen presenting cell targeting moiety, for example a dendritic cell targeting moiety, can bind to a targeted cell or molecule (directly or indirectly).
  • the targeting moieties of the invention contemplated for use with the biologically active agents include antibodies, polypeptides, peptides, aptamers, other ligands, or any combination thereof, that can bind a component of the target cell or molecule.
  • Exemplary dendritic cell targeting moieties are molecules that bind to any one of DEC-205 (CD205), Langerin (CD207), Clec9A receptors, DC-SIGN (CD209) and DNGR1.
  • the antigen presenting cell is a professional antigen presenting cell. Even more preferably the professional antigen presenting cell is a dendritic cell or a macrophage.
  • the antigen presenting cell targeting moiety may localize to and bind a target molecule present on an antigen presenting cell.
  • the targeting moiety may be referred to as a dendritic cell targeting moiety.
  • the targeting moieties contemplated for use in any aspect of the invention include antibodies, polypeptides, peptides, aptamers, other ligands, or any combination thereof, that can bind a component of the target cell or molecule.
  • the dendritic cell (DC)-targeting moiety is a ligand for a receptor presented on the surface of a DC.
  • Exemplary dendritic cell targeting moieties are molecules that bind to any one of XCR1 , DEC-205 (CD205), Langerin (CD207), Clec9A receptors, DC-SIGN (CD209), DNGR1 and Clec12A.
  • a preferred dendritic cell targeting moiety binds to XCR1.
  • a moiety that binds to XCR1 comprises, consists essentially of or consists of an amino acid sequence of XCL1 as described herein.
  • the antigen presenting cell targeting moiety may be associated with the moiety capable of endosomal escape via a linker which is cleavable in an endosome compartment prior to the endosome maturing into a late endosome.
  • the linker is cleavable in an early endosome.
  • the linker which is cleavable in an early endosome comprises, consists essentially of or consists of a cleavage site for an intracellular protease located in a lysosomal/endosomal compartment.
  • proteases The three main classes of intracellular proteases residing in the lysosomal/endosomal compartments and participating in antigen degradation are cysteine (cathepsin B, F, H, L, S, Z, and AEP, for asparaginylendopeptidase), aspartate (cathepsin D, E), and serine (cathepsin A, G) proteases.
  • cysteine cathepsin B, F, H, L, S, Z, and AEP, for asparaginylendopeptidase
  • aspartate cathepsin D, E
  • serine cathepsin A, G proteases.
  • the protease nomenclature designates the amino acid of the protease active site that catalyzes hydrolysis of the substrate peptide bond.
  • the linker is cleavable in an endosome, even more preferably an early endosome.
  • the linker which is cleavable in an early endosome comprises an amino acid sequence of a furin (also known as dibasic processing enzyme, Tachypleus kexin, PACE, PCSK3 peptidase, prohormone convertase, SPC1 , prohormone convertase 3, proprotein convertase subtilisin/kexin type 3) cleavage site.
  • the furin cleavage site may comprise, consist essentially of or consist of the amino acid sequence R-X-K/R-R (SEQ ID NO: 1 ). Even more preferably, the furin cleavage site may comprise, consist essentially of or consist of the amino acid sequence RKKR (SEQ ID NO: 2).
  • the linker between the antigen presenting cell targeting moiety and the moiety capable of endosomal escape may also be cleavable in late stage or maturing endosome, or a lysosome.
  • the linker which is also cleavable in late stage or maturing endosome, or a lysosome comprises an amino acid sequence of a cathepsin B cleavage site.
  • the cathepsin B cleavage site may comprise, consist essentially of or consist of the amino acid sequence V-citrulline (SEQ ID NO: 3; V denotes the amino acid valine) or FKFL (SEQ ID NO: 4).
  • the linker between the antigen presenting cell targeting moiety and the moiety capable of endosomal escape may comprise, consist essentially of or consist of a furin cleavage site as described herein and a cathepsin B cleavage site as described herein, preferably RKKR (SEQ ID NO: 2) and V-citrulline (SEQ ID NO: 3).
  • the dual furin/cathepsin B cleavage linker comprises, consists essentially of or consists of the amino acid sequence RKKRXV-citrulline, where X is any small amino acid, preferably a serine.
  • the dual furin/cathepsin B cleavage linker comprises, consists essentially of or consists of the amino acid sequence GSGTGGSTGRKKRSV-citrulline-GC (SEQ ID NO: 5).
  • the antigen presenting cell targeting moiety mediates internalization upon contact with an antigen presenting cell.
  • CPP cell penetrating peptide
  • a CPP refers to a peptide that is capable of crossing a cellular membrane.
  • a CPP is capable of translocating across a mammalian cell membrane and entering into a cell.
  • a CPP may direct a conjugate to a desired subcellular compartment.
  • a CPP may direct or facilitate penetration of a molecule of interest across a phospholipid, mitochondrial, endosomal, lysosomal, vesicular, or nuclear membrane.
  • CPPs that are able to“escape” the endosomal and lysosomal compartments for cytosolic delivery can be referred to as “cytosolic delivery cell penetrating peptides” as described herein.
  • a CPP may be translocated across the membrane with its amino acid sequence complete and intact, or alternatively partially degraded.
  • a CPP may direct a molecule of interest from outside a cell through the plasma membrane, and into the cytoplasm or a desired subcellular compartment.
  • a CPP may direct a molecule of interest across the blood-brain, trans- mucosal, hematoretinal, skin, gastrointestinal and/or pulmonary barriers.
  • the capability to translocate across membranes of a CPP may be energy dependent or independent, and/or receptor dependent or independent.
  • the CPP is a peptide which is demonstrated to translocate across a plasma membrane as determined by the methods described herein.
  • CPPs encompass peptides that not only become internalized by cells, but also are efficiently able to escape endosomal and/or lysosomal compartments once internalized by cells. Some CPPs, such as TAT, exhibit low to moderate levels of endosomal escape, while others can exhibit superior levels of cytoplasmic delivery. The latter are also referred to as “cytosolic delivery cell penetrating peptides” (cytosolic delivery CPPs) as described herein.
  • cytosolic delivery CPPs refers to a subset of CPPs that in addition being able to mediate intracellular delivery, is also able to escape from endosomal and/or lysosomal compartments for delivery into cytosol.
  • Endosomes comprise three different compartments: early endosomes, late endosomes, and recycling endosomes. They are distinguished by the time it takes for endocytosed material to reach them, and by markers such as Rabs. They also have different morphology. Once endocytic vesicles have uncoated, they fuse with early endosomes. Early endosomes then mature into late endosomes before fusing with lysosomes.
  • the moiety capable of endosomal and/or lysosomal escape is capable of escaping from an endosomal and/or lysosomal compartment into the cytosol.
  • the moiety is capable of escaping from an endosomal and/or lysosomal compartment into the cytosol prior to the endosome maturing into a late endosome. More preferably, the moiety is capable of escaping from an early endosomal compartment into the cytosol.
  • the moiety capable of endosomal and/or lysosomal escape is a peptide.
  • the peptide is the TAT peptide, derived from the HIV virus: GRKKRRQRRRPQ (SEQ ID NO: 8).
  • the peptide comprises, consists essentially of, or consists of the amino acid sequence PLKPKKPKTQEKKKKQPPKPKKPKTQEKKKKQPPKPKR (SEQ ID NO: 6).
  • the peptide may comprise, consist essentially of or consist of any one or more of the amino acid sequences in WO2014205518 referred to therein as SEQ ID NO: 83-119 which are herein incorporated by reference.
  • the moiety capable of endosomal and/or lysosomal escape is associated with the antigen such that when the moiety escapes from an endosomal and/or lysosomal compartment into the cytosol, the antigen also enters the cytosol.
  • the moiety capable of endosomal and/or lysosomal escape and the antigen are chemically linked, for example, covalently linked.
  • the covalent linkage may be direct or via a linker.
  • the linker may comprise, consists essentially of or consist of a peptide, for example containing 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.
  • the amino acids are selected from alanine, glycine and serine.
  • the peptide comprises, consists essentially of, or consists of the amino acid sequence GAS (SEQ ID NO: 7).
  • the moiety capable of endosomal and/or lysosomal escape mediates internalization upon contact with an antigen presenting cell.
  • the moiety capable of endosomal and/or lysosomal escape may be a cell penetrating peptide as described herein but also have the capacity to escape from endosomal or lysosomal compartments for delivery into the cytosol.
  • Those moieties having the capability of mediating intracellular delivery and escape from endosomal and/or lysosomal compartments for delivery into the cytosol may also be referred to as cytosolic delivery cell penetrating peptide (cytosolic delivery CPPs).
  • An exemplary assay to measure peptide cell-penetration and cytosolic delivery, or endosomal or lysosomal escape may be by a fluorescent Beta-Lactamase enzymatic assay as described below in Example 5.
  • a moiety capable of endosomal and/or lysosomal escape may mediate significantly greater endosomal escape than the TAT peptide.
  • the degree or level of endosomal escape may be determined by any method known in the art, including any method or assay described herein, such as the the beta-lactamase described below in Example 5.
  • the level of endosomal escape may be determined using the beta-lactamase assay described herein.
  • a moiety capable of endosomal and/or lysosomal escape mediate significantly greater endosomal escape than the TAT peptide when measured using the beta-lactamase assay described herein.
  • a moiety capable of endosomal and/or lysosomal escape may result in greater than or equal to 5%, 10% or 15% positive cells at 450nM, at concentrations of 1 mM, 2mM or 4mM respectively, when measured using the beta-lactamase assay described herein.
  • an “antigen” covers any substance that will elicit an immune response.
  • an “antigen” relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T cells).
  • the term "antigen” comprises any molecule which comprises at least one epitope.
  • an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune reaction, which is preferably specific for the antigen (including cells expressing the antigen).
  • any suitable antigen may be used, which is a candidate for an immune reaction, wherein the immune reaction is preferably a cellular immune reaction.
  • the antigen is preferably presented by a cell, preferably by an antigen presenting cell which includes a diseased cell, in particular a cancer cell, in the context of MHC/HLA molecules, which results in an immune reaction against the antigen.
  • An antigen is preferably a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens include tumour antigens.
  • the antigen peptides according to the invention are MHC class I and/or class II presented peptides or can be processed to produce MHC class I and/or class II presented peptides.
  • the antigen peptides comprise an amino acid sequence substantially corresponding to the amino acid sequence of a fragment of an antigen.
  • the antigen comprises a peptide capable of being presented by an HLA class I molecule.
  • the antigen also comprises a peptide capable of being presented by an HLA class II molecule.
  • the peptide capable of being presented by an HLA class I molecule comprises a CD8+ T cell epitope.
  • the peptide capable of being presented by an HLA class II molecule comprises a CD4+ T cell epitope.
  • the antigen typically comprises a HLA-l-restricted T cell epitope and a HLA-ll-restricted T cell epitope.
  • the antigen comprises a CD4+ T cell epitope and a CD8+ T cell epitope that result in activation and/or proliferation of a CD4+ T cell and a CD8+ T cell respectively.
  • exemplary antigens are any tumour associated antigens, including those described herein.
  • the antigen results in an immune response being raised against cells characterized by expression of the antigen and preferably by presentation of the antigen such as diseased cells, in particular cancer cells or infected cells (i.e. viral or bacterial infected).
  • the antigen may be a pathogen derived antigen.
  • an antigen from a virus, bacterium, parasite or fungi may be a pathogen derived antigen.
  • the antigen is a tumour antigen, i.e., a part of a tumour cell such as a protein or peptide expressed in a tumour cell which may be derived from the cytoplasm, the cell surface or the cell nucleus, in particular those which primarily occur intracellularly or as surface antigens of tumour cells.
  • a tumour antigen preferably comprises any antigen which is expressed in and optionally characteristic with respect to type and/or expression level for tumours or cancers as well as for tumour or cancer cells.
  • the term "tumour antigen” or “tumour-associated antigen” relates to proteins that are under normal conditions specifically expressed in a limited number of tissues and/or organs or in specific developmental stages, for example, the tumour antigen may be under normal conditions specifically expressed in stomach tissue, preferably in the gastric mucosa, in reproductive organs, e.g., in testis, in trophoblastic tissue, e.g., in placenta, or in germ line cells, and are expressed or aberrantly expressed in one or more tumour or cancer tissues.
  • "a limited number” preferably means not more than 3, more preferably not more than 2.
  • tumour antigens in the context of the present invention include, for example, differentiation antigens, preferably cell type specific differentiation antigens, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain differentiation stage, cancer/testis antigens, i.e., proteins that are under normal conditions specifically expressed in testis and sometimes in placenta, and germ line specific antigens.
  • differentiation antigens preferably cell type specific differentiation antigens, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain differentiation stage
  • cancer/testis antigens i.e., proteins that are under normal conditions specifically expressed in testis and sometimes in placenta
  • germ line specific antigens i.e., the tumour antigen or the aberrant expression of the tumour antigen identifies cancer cells.
  • the tumour antigen that is expressed by a cancer cell in a subject is preferably a self-protein in said subject.
  • the tumour antigen in the context of the present invention is expressed under normal conditions specifically in a tissue or organ that is non-essential, i.e. , tissues or organs which when damaged by the immune system do not lead to death of the subject, or in organs or structures of the body which are not or only hardly accessible by the immune system.
  • tumor antigen “tumor antigen”
  • tumor expressed antigen “cancer antigen”
  • cancer expressed antigen can include multiple tumor epitopes or antigens encoded within the same contiguous sequence.
  • Exemplary antigens include those: (a) comprising the minimal HLA-class-l restricted T cell epitopes described in Sachin et al. Nature (2017) 547:222-226, preferably in the Extended Data Table 3; (b) comprising the class I epitopes described in Ott et al. Nature (2017) 547: 217-221 ; (c) comprising the peptide neoantigens described in Creaney et al. (2015) Oncoimmunology. 4(7):e1011492; (d) comprising the neoantigens described in McGranaha et al. Science (2016) 351 (6280): 1463-9; comprising the neoantigens described in Rizvi et al. (2015) 351 (6280): 1463-9.
  • a peptide If a peptide is to be presented directly, i.e., without processing, in particular without cleavage, it has a length which is suitable for binding to an HLA molecule, in particular a class I HLA molecule, and preferably is 7-20 amino acids in length, more preferably 7-12 amino acids in length, more preferably 8-11 amino acids in length, in particular 9 or 10 amino acids in length.
  • the peptide produced by processing has a length which is suitable for binding to an HLA molecule, in particular a class I HLA molecule, and preferably is 7-20 amino acids in length, more preferably 7-12 amino acids in length, more preferably 8-11 amino acids in length, in particular 9 or 10 amino acids in length.
  • the sequence of the peptide which is to be presented following processing is derived from the amino acid sequence of an antigen, i.e. , its sequence substantially corresponds and is preferably completely identical to a fragment of an antigen.
  • Peptides having amino acid sequences substantially corresponding to a sequence of a peptide which is presented by the class I HLA may differ at one or more residues that are not essential for T cell receptor (TCR) recognition of the peptide as presented by the class I HLA, or for peptide binding to HLA.
  • TCR T cell receptor
  • Such substantially corresponding peptides are also capable of stimulating an antigen-responsive CTL and may be considered immunologically equivalent.
  • an antigen peptide when presented by HLA should be recognizable by a T cell receptor.
  • the antigen peptide if recognized by a T cell receptor is able to induce in the presence of appropriate co-stimulatory signals, clonal expansion of the T cell carrying the T cell receptor specifically recognizing the antigen peptide.
  • antigen peptides in particular if presented in the context of HLA molecules, are capable of stimulating an immune response, preferably a cellular response against the antigen from which they are derived or cells characterized by expression of the antigen and preferably characterized by presentation of the antigen.
  • an antigen peptide is capable of stimulating a cellular response against a cell characterized by presentation of the antigen with class I HLA and preferably is capable of stimulating an antigen- responsive CTL.
  • Activation and/or expansion of CD4+ or CD8+ T cells may be determined by any method known in the art, including any method described herein.
  • epitope refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by a T cell, in particular when presented in the context of HLA molecules.
  • An epitope of a protein such as a tumour antigen preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, most preferably between 10 and 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 amino acids in length. It is particularly preferred that the epitope in the context of the present invention is a T cell epitope.
  • the chimeric or fusion proteins of the invention may be produced by recombinant expression systems (such as recombinant DNA technology) or by chemical synthesis (such as solid phase peptide synthesis), or by a combination thereof and joined via chemical or enzymatic ligation chemistry, These techniques are well known in the art.
  • the present invention provides a nucleic acid molecule including a nucleotide sequence encoding a conjugate, or chimeric or fusion polypeptide of the invention as described herein, optionally operatively linked to at least one regulatory element.
  • the nucleic acid is provided in isolated or substantially purified form.
  • the nucleic acid molecule may, for example, be inserted into a suitable expression vector for production of the chimeric or fusion polypeptide as a recombinant protein by insertion of the expression vector into a prokaryotic or eukaryotic host cell.
  • Successful expression of the recombinant protein requires that the expression vector contains the necessary regulatory elements for transcription and translation which are compatible with, and recognised by the particular host cell system used for expression.
  • a variety of host cell systems may be utilized to express the recombinant protein, which include, but are not limited to bacteria transformed with a bacteriophage vector, plasmid vector, or cosmid DNA; yeast containing yeast vectors; fungi containing fungal vectors; insect cell lines infected with virus (e.g.
  • baculovirus and mammalian cell lines transfected with plasmid or viral expression vectors, or infected with recombinant virus (e.g. vaccinia virus, adenovirus, adeno-associated virus, retrovirus, etc).
  • recombinant virus e.g. vaccinia virus, adenovirus, adeno-associated virus, retrovirus, etc.
  • various promoters and enhancers can be incorporated into the expression vector, to increase the expression of the recombinant protein, provided that the increased expression of the amino acid sequences is compatible with (for example, non-toxic to) the particular host cell system used.
  • promoter will depend on the expression system used. Promoters vary in strength, i.e. ability to facilitate transcription. Generally, it is desirable to use a strong promoter in order to obtain a high level of transcription of the coding nucleotide sequence and expression into recombinant protein. For example, bacterial, phage, or plasmid promoters known in the art from which a high level of transcription have been observed in a host cell system including E.
  • coli include the lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters, lacUV5, ompF, bla, Ipp, and the like, may be used to provide transcription of the inserted nucleotide sequence encoding amino acid sequences.
  • Enhancer sequences are DNA elements that appear to increase transcriptional efficiency in a manner relatively independent of their position and orientation with respect to a nearby coding nucleotide sequence. Thus, depending on the host cell expression vector system used, an enhancer may be placed either upstream or downstream from the inserted coding sequences to increase transcriptional efficiency.
  • Other regulatory sites such as transcription or translation initiation signals, can be used to regulate the expression of the coding sequence.
  • nucleic acid encoding same can be cloned into expression constructs or vectors, which are then transfected into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce the protein.
  • host cells such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce the protein.
  • exemplary cells used for expressing a protein are CHO cells, myeloma cells or HEK cells.
  • Molecular cloning techniques to achieve these ends are known in the art and described, for example in Ausubel et ai, (editors), Current Protocols in Molecular Biology, Greene Pub.
  • promoter is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner.
  • promoter is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked.
  • Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid.
  • operably linked to means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a protein (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence.
  • a signal sequence e.g., a sequence encoding a protein (e.g., derived from the information provided herein)
  • an enhancer element e.g., derived from the information provided herein
  • a promoter e.g., derived from the information provided herein
  • a transcription termination sequence e.g., a transcription termination sequence.
  • Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).
  • prokaryotic secretion signals e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II
  • yeast secretion signals e.g., invertase leader, a factor leader, or acid phosphatase leader
  • mammalian secretion signals e.g., herpes simplex gD signal.
  • Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1 -a promoter (EF1 ), small nuclear RNA promoters (U1 a and U 1 b), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, b-actin promoter; hybrid regulatory element comprising a CMV enhancer/ b- actin promoter or an immunoglobulin promoter or active fragment thereof.
  • CMV-IE cytomegalovirus immediate early promoter
  • EF1 human elongation factor 1 -a promoter
  • U1 a and U 1 b small nuclear RNA promoters
  • a-myosin heavy chain promoter Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, b-actin promote
  • Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).
  • COS-7 monkey kidney CV1 line transformed by SV40
  • human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture
  • baby hamster kidney cells BHK, ATCC CCL 10
  • Chinese hamster ovary cells CHO
  • Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL 1 promoter, the GAL4 promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.
  • Means for introducing the isolated nucleic acid or expression construct comprising same into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., Wl, USA) amongst others.
  • the host cells used to produce the protein may be cultured in a variety of media, depending on the cell type used.
  • Commercially available media such as Ham's FI0 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells.
  • Media for culturing other cell types discussed herein are known in the art.
  • supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • supernatants can be filtered and/or separated from cells expressing the protein, e.g., using continuous centrifugation.
  • the conjugate, or chimeric or fusion polypeptide prepared from the cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing.
  • affinity chromatography e.g., protein A affinity chromatography or protein G chromatography
  • a protein can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine tag, or a influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag.
  • a poly-histidine tag e.g., a hexa-histidine tag, or a influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag.
  • HA hemagglutinin
  • V5 Simian Virus 5
  • FLAG tag e.g., a FLAG tag
  • GST glutathione S-transferase
  • a protein comprising a hexa-his tag is purified by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein.
  • Ni-NTA nickel-nitrilotriacetic acid
  • a ligand or antibody that binds to a tag is used in an affinity purification method.
  • the conjugate, chimeric or fusion polypeptide, composition or nucleic acid of the present invention as described herein are useful in the treatment or prevention of any hyperproliferative condition.
  • An example of a condition is cancer.
  • Exemplary cancers include cystic and solid tumors, bone and soft tissue tumors, including tumors in anal tissue, bile duct, bladder, blood cells, bowel, brain, breast, carcinoid, cervix, eye, esophagus, head and neck, kidney, larynx, leukemia, liver, lung, lymph nodes, lymphoma, melanoma, mesothelioma, myeloma, ovary, pancreas, penis, prostate, skin (e.g.
  • Soft tissue tumors include Benign schwannoma Monosomy, Desmoid tumor, lipo-blastoma, lipoma, uterine leiomyoma, clear cell sarcoma, dermatofibrosarcoma, Ewing sarcoma, extraskeletal myxoid chondrosarcoma, liposarcooma myxoid, Alveolar rhabdomyosarcoma and synovial sarcoma.
  • Specific bone tumors include nonossifying fibroma, unicameral bone cyst, enchon-droma, aneurismal bone cyst, osteoblastoma, chondroblastoma, chondromyxofibroma, ossifying fibroma and adamantinoma, Giant cell tumor, fibrous dysplasia, Ewing’s sarcoma eosinophilic granuloma, osteosarcoma, chondroma, chondrosarcoma, malignant fibrous histiocytoma and metastatic carcinoma.
  • Leukemias include acute lymphoblastic, acute myeloblastic, chronic lymphocytic and chronic myeloid.
  • breast tumors colorectal tumors, adenocarcinomas, mesothelioma, bladder tumors, prostate tumors, germ cell tumor, hepatoma/cholongio, carcinoma, neuroendocrine tumors, pituitary neoplasm, small round cell tumor, squamous cell cancer, melanoma, atypical fibroxanthoma, seminomas, nonseminomas, stromal leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors and Wilm's tumor.
  • an infectious disease is a condition that is associated with or caused by an infectious agent.
  • an infectious agent is a virus, bacterium, parasite or fungi.
  • Exemplary virus is influenza and Herpes Simplex Virus (HSV).
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment may not necessarily result in the complete clearance of cancer or infected cells but may reduce or minimise complications and side effects of infection, or the presence or progression of cancer. The success or otherwise of treatment may be monitored by physical examination of the subject, cytopathological, serological DNA, or mRNA detection techniques.
  • Preventing refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of a condition, disease, disorder, or phenotype, including an abnormality or symptom.
  • a subject in need of prevention may be prone to develop the condition.
  • the present invention also includes methods of preventing the development of cancer in a subject.
  • the subject for whom prevention of cancer is required may be considered to be at risk of developing cancer, but does not yet have detectable cancer.
  • a subject at risk of the development of cancer may be a subject with a family history of cancer, and/or a subject for whom genetic testing or other testing indicates a high risk or high likelihood of the development of cancer.
  • the subject may have cancer stem cells but does not yet have any detectable tumours. It will be understood that methods of preventing the development of cancer include methods of delaying the onset of cancer in a subject.
  • ameliorate or “amelioration” refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.
  • a subject in need of treatment may already have the condition, or may be prone to have the condition or may be one in whom the condition is to be prevented.
  • a method of the present invention comprises administering a prophylactically or therapeutically effective amount of a conjugate, chimeric or fusion polypeptide, composition or nucleic acid of the invention as described herein.
  • the phrase‘therapeutically effective amount’ generally refers to an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
  • dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
  • prophylactically effective amount shall be taken to mean a sufficient quantity of a protein to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition.
  • an amount will vary depending on, for example, the specific antigen binding site(s) administered and/or the particular subject and/or the type or severity or level of condition and/or predisposition (genetic or otherwise) to the condition. Accordingly, this term is not to be construed to limit the present invention to a specific quantity, e.g., weight or amount of antigen binding site(s), rather the present invention encompasses any amount of the antigen binding site(s) sufficient to achieve the stated result in a subject.
  • Suitable dosages of a conjugate, chimeric or fusion polypeptide, composition or nucleic acid of the invention as described herein will vary depending on the specific the condition to be treated and/or the subject being treated. It is within the ability of a skilled physician to determine a suitable dosage, e.g., by commencing with a sub-optimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment/prophylaxis, data from the cell culture assays or animal studies are used, wherein a suitable dose is within a range of circulating concentrations that include the ED 50 of the active compound with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • a therapeutically/prophylactically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e. , the concentration or amount of the compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e. , the concentration or amount of the compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma maybe measured, for example, by high performance liquid chromatography.
  • the invention is also useful for veterinary purposes.
  • the invention is useful for domestic animals such as cattle, sheep, horses and poultry; for companion animals such as cats and dogs; and for zoo animals. Therefore, the general term "subject” or“subject to be / being treated” is understood to include all animals (such as humans, apes, dogs, cats, horses, and cows) that require an enhanced immune response, for example subjects having cancer.
  • administered means administration of a therapeutically effective dose of the aforementioned conjugate, chimeric or fusion polypeptide, composition or nucleic acid of the present invention as described herein to the subject.
  • Subjects requiring treatment include those already having a benign, pre- cancerous, or non-metastatic tumour as well as those in which the occurrence or recurrence of cancer is to be prevented.
  • Subjects may have metastatic cells, including metastatic cells present in the ascites fluid and/or lymph node.
  • a subject in need of treatment may be one diagnosed with, or at risk of developing, any one of the cancers described herein.
  • one or more of the following effects may be observed: reduction in the reoccurrence of malignant tumours, reduction in metastasis of malignant tumours, reduction in number or size of tumours, differentiation of tumour cells, expression of b-catenin and E-cadherin in malignant tumours to facilitate cell-to- cell adhesion and reduction in metastasis, reduction in tumour cells ability to prevent immunorecognition.
  • the objective or outcome of treatment may be to reduce the number of cancer cells; reduce the primary tumour size; inhibit (i.e. , slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
  • Efficacy of treatment can be measured by assessing the duration of survival, time to disease progression, the response rates (RR), duration of response, and/or quality of life.
  • the method is particularly useful for extending time to disease progression.
  • the method is particularly useful for extending survival of the human, including overall survival as well as progression free survival.
  • the method is particularly useful for providing a complete response to therapy whereby all signs of cancer in response to treatment have disappeared. This does not always mean the cancer has been cured.
  • the method is particularly useful for providing a partial response to therapy whereby there has been a decrease in the size of one or more tumours or lesions, or in the extent of cancer in the body, in response to treatment.
  • the objective or outcome of treatment may be any one or more of the following: to reduce the number of cancer cells; reduce the primary tumour size; inhibit (i.e. , slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; relieve to some extent one or more of the symptoms associated with the disorder.
  • subjects requiring treatment include those having a benign, pre-cancerous, non-metastatic tumour.
  • the cancer is pre-cancerous or pre -neoplastic.
  • the cancer is a secondary cancer or metastases.
  • the secondary cancer may be located in any organ or tissue, and particularly those organs or tissues having relatively higher hemodynamic pressures, such as lung, liver, kidney, pancreas, bowel and brain.
  • the secondary cancer may be detected in the ascites fluid and/or lymph nodes.
  • the cancer may be substantially undetectable.
  • Pre-cancerous or“pre-neoplasia” generally refers to a condition or a growth that typically precedes or develops into a cancer.
  • a "pre -cancerous" growth may have cells that are characterized by abnormal cell cycle regulation, proliferation, or differentiation, which can be determined by markers of cell cycle.
  • the cancer is pre-cancerous or pre -neoplastic.
  • the cancer is a secondary cancer or metastases.
  • the secondary cancer may be located in any organ or tissue, and particularly those organs or tissues having relatively higher hemodynamic pressures, such as lung, liver, kidney, pancreas, bowel and brain.
  • the objective or outcome of treatment may be a reduction in viral, bacterial or fungal titre or symptom associated with the disease.
  • phrases ‘pharmaceutically acceptable’ indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the subject being treated therewith.
  • adjuvants are known for use in conjunction with vaccine compositions.
  • the adjuvants aid by modulating the immune response and in attaining a more durable and higher level of immunity using smaller amounts of vaccine antigen or fewer doses than if the vaccine antigen were administered alone.
  • adjuvants examples include incomplete Freund's adjuvant (IFA), Adjuvant 65 (containing peanut oil, mannide monooleate and aluminium monostearate), oil emulsions, Ribi adjuvant, the pluronic polyols, polyamines, Avridine, Quil A, saponin, MPL, QS-21 , mineral gels such as aluminium salts and calcium salts, nanoparticles such as hydroxyapatite, calcium phosphate, aluminium salts, sugar oligomers and polymers such as mannan, chitosan.
  • IFA incomplete Freund's adjuvant
  • Adjuvant 65 containing peanut oil, mannide monooleate and aluminium monostearate
  • oil emulsions examples include oil emulsions, Ribi adjuvant, the pluronic polyols, polyamines, Avridine, Quil A, saponin, MPL, QS-21 , mineral gels such as aluminium salts and calcium salts, nanop
  • the vaccine composition may include conventional pharmaceutically acceptable carriers, excipients, fillers, buffers or diluents as appropriate.
  • the skilled person will also be familiar with compounds or compositions that are known to bolster anti-tumor T cell immunity such as including, but not limiting to co- administration with checkpoint blockade drugs. Consequently, also contemplated are co-administration of an inhibitor of immune checkpoint regulators such as PD-1 , PDL-1 etc. As such, the present invention also includes the administration of a vaccine composition as herein described, in conjunction with such an inhibitor.
  • a vaccine composition to a subject, including but not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, sub-lingual, buccal and oral administration. These routes of administration are particularly useful for vaccination. Other routes include delivery into tumors, subcutaneous delivery, and intranodal,
  • a vector comprising a nucleic acid molecule of the invention as described herein may be a viral or bacterial vaccine vector, and used to provide a recombinant viral vaccine, a recombinant bacterial vaccine, a recombinant attenuated bacterial vaccine, or an inactivated recombinant viral vaccine.
  • Vaccinia virus is the best known example, in the art, of an infectious virus that is engineered to express vaccine antigens derived from other organisms.
  • the recombinant live vaccinia virus which is attenuated or otherwise treated so that it does not cause disease by itself, is used to immunize the host. Subsequent replication of the recombinant virus within the host provides a continual stimulation of the immune system with the vaccine antigens thereby providing long lasting immunity.
  • virus-like particles, liposomes, nanoparticles can be substituted for live viruses, particularly in vaccines with applications such as described herein.
  • the invention also provides for the use of a nucleic acid molecule including a nucleotide sequence encoding a conjugate, or chimeric or fusion polypeptide of the invention directly as the vaccine formulation.
  • Nucleotide sequences encoding the chimeric or fusion polypeptides, operatively linked to one or more regulatory elements, can be introduced directly to vaccinate a subject ("direct gene transfer").
  • Direct gene transfer into a vaccinated subject resulting in expression of the genetic material by the vaccinated subject's cells such as vascular endothelial cells as well as the tissue of the major organs, has been demonstrated by techniques in the art such as by injecting intravenously an expression plasmid:cationic liposome complex [Zhu et ai, 1993, Science 261 :209-211 ]
  • Other effective methods for delivering vector DNA into a target cell are known in the art.
  • purified recombinant plasmid DNA containing viral genes has been used to inoculate (whether parenterally, mucosally, or via gene- gun immunization) vaccines to induce a protective immune response [Fynan et at.
  • cells removed from a subject can be transfected or electroporated by standard procedures known in the art, resulting in the introduction of the recombinant vector DNA into the target cell.
  • Cells containing the recombinant vector DNA may then be selected for using methods known in the art, such as by use of a selection marker expressed in the vector, and the selected cells may then be re-introduced into the subject to express the recombinant protein.
  • compositions of this invention are particularly useful for parenteral administration, such as intravenous administration.
  • the compositions for administration will commonly comprise a solution of a conjugate, chimeric or fusion polypeptide, composition or nucleic acid of the invention dissolved in a pharmaceutically acceptable carrier, for example an aqueous carrier.
  • a pharmaceutically acceptable carrier for example an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline and the like.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of a conjugate, chimeric or fusion polypeptide, composition or nucleic acid of the invention in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Nonaqueous vehicles such as mixed oils and ethyl oleate may also be used.
  • Liposomes may also be used as carriers.
  • the vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
  • the present invention additionally comprises a kit comprising one or more of the following a conjugate, chimeric or fusion polypeptide, composition or nucleic acid of the invention as described herein.
  • the kit can additionally comprise a pharmaceutically acceptable carrier.
  • kit of the invention is packaged with instructions for use in a method or use of the invention as described herein.
  • Example 1 Methods for construction of peptide vaccines
  • the muXCL1 -SpyC DNA sequence was synthesized and cloned (ATUM) into Ncol and Xhol sites of pET28a+ vector (Merck Millipore). Recombinant proteins were expressed as 6xHis-N-terminally tagged fusion proteins in E. coli strain BL21 (DE3) Gold (Agilent Technologies). The protein was purified using IMAC as previously described (Milech et al 2015, Sci Rep 5, 18329) with an additional Ion Exchange Chromatography (IEX).
  • IMAC Ion Exchange Chromatography
  • the purification step was performed after IMAC.
  • the protein was desalted into 20 mM Tris, pH 8.0 binding buffer and purified through a HiTrap Q HP 5ml_ column (GE Healthcare) and were eluted using a 0-1 M NaCI gradient.
  • the final protein was desalted into PBS pH 7.4, and purity was confirmed by analysis on 4-16 % SDS-PAGE stained with Gel Code Blue Reagent.
  • Peptide 3 mcpa-PYC35-gDgB ( ⁇ 5 mg at 90%) (46 Amino Acids) (Mercapto propionic acid) -
  • Peptide 4 mcpa-TAT-gDgB ( ⁇ 5 mg at 90%) (40 Amino Acids)
  • Cross-presenting DCs are an important cell uniquely capable of presenting external antigens on both MFIC-I and -II, thus activating both CD8+ and CD4+ T cells to the dangers of a new infection.
  • These cross-presenting DCs exclusively express a cell- surface marker called XCR1 , which allows us to specifically target them using its endogenous ligand XCL1.
  • Coupling XCL1 in a peptide vaccine construct eg: XCL1- cytosolic delivery CPP-Antigen targets the vaccine to the cross-presenting XCR1 + DCs.
  • the vaccine Upon binding of the peptide vaccine XCL1 - cytosolic delivery CPP-Ag, to the XCR1 cell surface marker, the vaccine is internalised by endocytosis inside the cell within an organelle called the endosome. Once inside the endosome, the cytosolic delivery CPP-Ag component is decoupled from the targeting XCL1 component. Two outcomes can now occur: a proportion of the released cytosolic delivery CPP-Ag component can escape into the cytoplasm of the DC where it is loaded onto MHC-I, which is recognized by CD8+ T cells. A proportion of the cytosolic delivery CPP- Antigen is located within the maturing to late stage endosome, where it is loaded onto MHC-II via an endosomal processing pathway which is recognised by CD4+ T cells.
  • Figure 1 provides a schematic of the proposed pathway for uptake of the conjugates, chimeric or fusion polypeptides of the invention.
  • Example 2 Disulfide-linked CPP antigen evokes a qDT-specific CD4+ T cell expansion but no qBT-specific CD8+ expansion
  • Peptide vaccines were constructed as shown in Figures 2 to 4. SpyC/SpyT ligation was confirmed by SDS-PAGE (see Figure 5). A disulphide bond may not be cleaved in a cross-presenting DCs endosome due to unique neutral pH in this intracellular compartment. The disulphide bond would typically be cleaved in an acidic environment. As a result, the detectable label will remain bound to the endosomal escape peptide (1746: SEQ ID NO: 6) such that the endosomal escape peptide and associated antigen cannot escape the endosome and therefore cannot enter the cytosol.
  • gBT.I The CD8 + T cells in this TCR transgenic animal express a FI-2K b -restricted T cell receptor specific for herpes simplex virus (FISV) glycoprotein B (gB).
  • FISV herpes simplex virus
  • gDT.II The CD4 + T cells in this TCR transgenic animal express a l-A b -restricted T cell receptor specific for FISV glycoprotein D (gD).
  • Peptide vaccine administration schedule
  • mice On day 0 (priming), 3 x B6 mice were injected with: XCL1 -SpyC/V5-SpyT-PYC35-gDgB;
  • Peptide concentration was 10 mM.
  • the peptide amount per injection was 0.5 nmol.
  • the Poly l:C concentration was 1 pg/mI.
  • the Injection volume was 50 pi.
  • Poly l:C and peptides were delivered subcutaneously.
  • Disulfide-linked vaccine constructs do not elicit gB-specific T cell expansion. Disulfide-linked vaccine constructs elicit gD-specific T cell expansion. ( Figure 7). This highlights the importance of a cleavable linker in this conjugate / fusion polypeptide design to allow endosomal escape.
  • Example 3 Assembly and efficacy of a peptide vaccine comprising a cleavable linker and a moietv capable of endosomal escape
  • Figures 8 to 10 show the approach to design and construction of a peptide vaccine comprising a cleavable linker moiety linking the dendritic cell targeting moiety and endosomal escape peptide-antigen moiety.
  • the final peptide vaccine construct comprised a dendritic cell targeting moiety (in the form of XCL-1 ), a cleavable linker (in the form of a dual furin-cathepsin B (FCB) cleavable linker comprised of a Furin recognition sequence and V-citrulline, linked to cysteine), and a moiety capable of endosomal escape (1746) and associated antigen (gD and gB).
  • a dendritic cell targeting moiety in the form of XCL-1
  • FCB furin-cathepsin B
  • the peptide vaccine was injected into mice using a similar protocol to that described in Example 2. Briefly: On day -2, 1 x 10 4 gDT. II transgenic T cells were adoptively transferred via intravenous injection.
  • mice On day 0 (priming), 3 x B6 mice were injected with:
  • Peptide concentration was 10 mM.
  • the peptide amount per injection was 0.5 nmol.
  • the Poly l:C concentration was 1 pg/mI.
  • the Injection volume was 50 pi.
  • Poly l:C and peptides were delivered subcutaneously.
  • cytosolic delivery cell penetrating peptide in the form of “1746”
  • CD8 + T cell proliferation compared with the use of a conventional CPP such as TAT which cannot escape the early endosome.
  • the efficacy of the peptide vaccine was further assessed using a subcutaneous B16 melanoma model engineered to express glycoprotein B (gB) from Flerpes Simplex Virus.
  • the peptide vaccine tested includes a well characterized CD8+ T cell gB peptide epitope with and without CPP.
  • results demonstrate that the CPP peptide vaccines result in prolonged tumour-free survival than non-CPP control (gD gB - without CPP capable of endosomal escape).
  • the results in Figure 15 also show that the peptide vaccine shown as 1746-gDgB with XCL1 targeting, a cleavable furin/cathepsin B linker and the CPP 1746, results in a delay in detectable tumour and a reduction in tumour growth compared to control and a peptide construct without CPP (shown as gDgB).
  • the 1746-gDgB delayed onset of a detectable tumour equivalent to HSV.
  • Figure 16 shows a significantly greater percent survival of 1746-gDgB compared to peptide control (gD gB without 1746). Notably, the survival is the same as FISV immunization.
  • Example 5 Exemplary assay to measure cell penetration and cytosolic delivery
  • Beta-Lactamase is a bacterial enzyme that catalyses the opening of Beta-lactam rings. It does not occur naturally in eukaryotic cells. Beta-Lactamase is also not intrinsically cell penetrating and requires the addition of a cell-internalising agent to access the eukaryotic cytoplasm.
  • CCF2-AM (Thermofisher Scientific, Australia) is a Fluorescence Resonance Energy Transfer (FRET) substrate that is enzymatically cleaved by Beta-Lactamase.
  • FRET Fluorescence Resonance Energy Transfer
  • CCF2-AM is an esterified form of 7-hydroxycoumarin linked to fluorescein by a cephalosporin core. Esterification facilitates cell entry of the molecule. Once inside, the molecule is transformed into its anionic form by endogenous cytoplasmic esterases which trap the molecule inside the cell’s cytoplasm. When excited at 409 nm, uncleaved CCF2-AM emits a FRET signal at 520 nm (green).
  • Beta-Lactamase activity is quantified by measuring the ratio of blue fluorescence to green fluorescence using flow cytometry.
  • the method described here outlines the measurement of a peptide’s cell- penetrating and cytosolic delivery ability by coupling it to Beta-Lactamase and measuring cytoplasmic Beta-Lactamase enzyme activity by cleavage of cytosolic CCF2, where increased enzyme activity is indicative of increased cell penetration and cytoplasmic delivery of Beta-Lactamase.
  • Beta-Lactamase was expressed as C-terminal SpyC fusion, making SpyC-BLA (SEQ ID No: 12).
  • the cell-penetrating peptides, 1746 and TAT were synthesized as N- terminal fusions to SpyTag to make CPP-SpyT fusion peptides (SEQ ID Nos: 13 and 14).
  • SpyC-BLA was independently reacted with each CPP-SpyT peptide to be tested to form a CPP conjugate as schematically illustrated in Figure 18 and 19.
  • the cell-penetrating and cytosolic delivery ability of 1746 and TAT as CPP- SpyT/SpyC-BLA conjugates was assessed by flow cytometry to determine cytosolic CCF2-AM cleavage by internalized Beta-Lactamase.
  • the ratio of blue to green fluorescence was assessed to determine the cell-penetrating and cytosolic delivery ability of the two CPP sequences.
  • the flow cytometry data is further corroborated by confocal microscopy on live cells using the same assay conditions
  • Example 6 Peptide vaccine and viral challenge
  • mice were treated on DO & D13 with the following:
  • HSV Herpes Simplex Virus

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Abstract

La présente invention concerne des conjugués, des polypeptides de fusion et des compositions comprenant ces conjugués ou polypeptides de fusion, destinés à être utilisés dans la génération d'une réponse immunitaire. En particulier, la réponse immunitaire est générée pour traiter ou prévenir le cancer ou une maladie infectieuse. La présente invention concerne un conjugué pour induire une réponse immunitaire, le conjugué comprenant : (a) une fraction de ciblage de cellule présentatrice d'antigène ; (b) une fraction permettant un échappement endosomal et/ou lysosomal ; et (c) un antigène ; la fraction de ciblage de cellule présentatrice d'antigène pouvant être libérée à partir de la fraction permettant un échappement endosomal et à partir de l'antigène.
PCT/AU2018/051408 2018-01-05 2018-12-24 Conjugués de vaccin et leurs utilisations WO2019134018A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021113574A1 (fr) * 2019-12-05 2021-06-10 Yale University Immunothérapie à base de lymphocytes t pour des infections virales du système nerveux central et des tumeurs
WO2021203608A1 (fr) * 2020-04-09 2021-10-14 诺未科技（北京）有限公司 Vaccin contre le cancer métastatique ciblant vegfr2
CN117797253A (zh) * 2024-02-29 2024-04-02 诺未科技（北京）有限公司 一种能扩展t细胞抗原表位的肿瘤疫苗及其制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007015704A2 (fr) * 2004-10-29 2007-02-08 University Of Rochester Vecteurs de bacteriophage modifie et utilisation de ceux-ci
WO2016146260A1 (fr) * 2015-03-16 2016-09-22 Amal Therapeutics Sa Nouveau complexe comprenant un peptide de pénétration cellulaire, un cargo et un agoniste des peptides tlr
WO2017025057A1 (fr) * 2015-08-11 2017-02-16 Coherent Biopharma Conjugués de médicament multi-ligands et leurs utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007015704A2 (fr) * 2004-10-29 2007-02-08 University Of Rochester Vecteurs de bacteriophage modifie et utilisation de ceux-ci
WO2016146260A1 (fr) * 2015-03-16 2016-09-22 Amal Therapeutics Sa Nouveau complexe comprenant un peptide de pénétration cellulaire, un cargo et un agoniste des peptides tlr
WO2017025057A1 (fr) * 2015-08-11 2017-02-16 Coherent Biopharma Conjugués de médicament multi-ligands et leurs utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN, X. ET AL.: "Fusion protein linkers: Property, design and functionality", ADVANCED DRUG DELIVERY REVIEWS, vol. 65, 2013, pages 1357 - 1369, XP028737352, DOI: doi:10.1016/j.addr.2012.09.039 *
REDDINGTON, S. ET AL.: "Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher", CURRENT OPINION IN CHEMICAL BIOLOGY, vol. 29, 2015, pages 94 - 99, XP029342251, DOI: doi:10.1016/j.cbpa.2015.10.002 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021113574A1 (fr) * 2019-12-05 2021-06-10 Yale University Immunothérapie à base de lymphocytes t pour des infections virales du système nerveux central et des tumeurs
CN115038462A (zh) * 2019-12-05 2022-09-09 耶鲁大学 用于中枢神经系统病毒感染和肿瘤的基于t细胞的免疫疗法
WO2021203608A1 (fr) * 2020-04-09 2021-10-14 诺未科技（北京）有限公司 Vaccin contre le cancer métastatique ciblant vegfr2
CN117797253A (zh) * 2024-02-29 2024-04-02 诺未科技（北京）有限公司 一种能扩展t细胞抗原表位的肿瘤疫苗及其制备方法

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