WO2022238363A1 - Constructions immunogènes et vaccins destinés à être utilisés dans le traitement prophylactique et thérapeutique de maladies infectieuses - Google Patents

Constructions immunogènes et vaccins destinés à être utilisés dans le traitement prophylactique et thérapeutique de maladies infectieuses Download PDF

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WO2022238363A1
WO2022238363A1 PCT/EP2022/062556 EP2022062556W WO2022238363A1 WO 2022238363 A1 WO2022238363 A1 WO 2022238363A1 EP 2022062556 W EP2022062556 W EP 2022062556W WO 2022238363 A1 WO2022238363 A1 WO 2022238363A1
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construct
unit
amino acids
seq
targeting
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PCT/EP2022/062556
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Agnete Brunsvik Fredriksen
Gunnstein NORHEIM
Elisabeth STUBSRUD
Audun Trygge Haugen BERSAAS
Stine GRANUM
Pierre DILLARD
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Nykode Therapeutics ASA
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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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/57—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
    • 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
    • A61K2039/575—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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • immunogenic constructs such as polynucleotides, polypeptides and multimeric proteins, such as dimeric proteins, and vaccines comprising such immunogenic constructs, which are useful for the prophylactic and therapeutic treatment of infectious diseases, as well as methods for producing and using the immunogenic constructs and vaccines.
  • Both B cell (humoral/antibody mediated) and immune system cell responses are important components of protective responses against infection caused by pathogens.
  • Specific antibodies against pathogen antigens can mediate a broad range of effector functions, such as e.g. a) direct neutralization of toxins or pathogens, b) neutralization of pathogen virulence factors, c) binding to and trapping of pathogens in mucins, d) activating complement to mediate anti-pathogen phagocytic clearance, degradation or lysis, e) activating neutrophil opsonophagocytosis, f) induce macrophage opsonophagocytosis g) activating natural killer (NK) cell degranulation to kill infected cells, h) enhancing antigen update, processing and presentation by dendritic cells to T and B cells, i) inducing degranulation of mast cells, basophils and eosinophils in the setting of parasitic infections (L. Lu et al, Nat Rev
  • T cell responses are critical for limiting viral replication and infection by killing the infected cells, inducing apoptosis, releasing antiviral substances, and/or inducing increased intracellular lysis in already infected cells and thus help to prevent, reduce severity of or cure the disease.
  • effective and long-lasting response in both arms of immunity usually require additional support from T-helper (Thl and Th2) lymphocytes.
  • Cytotoxic T lymphocytes also play a significant role (F. Sheperd et al, Int J Mol Sci 21, 2020, 6144) combating e.g. intracellular pathogens where MHC class I-restricted CD8+ T cells are critical for clearing bacterial infections and are known to provide protective immunity against a range of bacterial species.
  • MHC class II- restricted CD4+ T cells support memory CD8+ T cell responses and are important for protective immunity against bacterial infections.
  • Naive CD4+ T cells differentiate subsets of cells with effector capacity, such as T helper 1 (Thl) and Th2 cells.
  • Thl and Th2 cells After binding specific T cell epitopes on the surface of antigen-presenting cells (APCs), Thl and Th2 cells supply specific soluble cytokine signals that regulate the balance between antibody and CTL immunity.
  • effective immunity involves multiple antigen recognition events of specific pathogen immunogenic determinants (epitopes) by T-helper cells followed by molecular recognition by B cells, CTL, or both.
  • B cell epitopes can be categorized as linear or conformational epitopes, with linear epitopes often being parts of conformational B-cell epitopes in native proteins.
  • Conformational epitopes are exposed structural features on the surface of pathogens such as a viral envelope, bacterial outer membrane, or secreted bacterial toxins.
  • T cell epitopes are short peptides from any protein of a pathogen, which only have to conform to the host antigen-processing and MHC binding mechanisms, most notably class I or class II MHC haplotype restriction mechanisms.
  • Suitable T cell epitopes occur with an estimated frequency of about one per 200-500 amino acid sequence, depending on host population and pathogen. Therefore, it is likely that a naturally occurring protein antigen does not comprise or only comprises few suitable T cell epitopes, or has only suboptimal T cell epitopes.
  • T cell epitopes Combining in a vaccine one or more selected T cell epitopes and a B cell antigen is beneficial: while the presence of the antigen ensures the production of persistent and functional antibodies, the presence of T cell epitopes will elicit strong T cell response with a long-lasting memory population; in totality providing protection against subsequent infection and severe disease.
  • a vaccine may provide sufficient protection against infection and/or severe disease caused by a pathogen, even if the included B cell antigen is no longer optimal. If the included T cell epitopes are conserved T cell epitopes, e.g. between subgenus, species or strains, there is an even greater likelihood that the vaccine renders protection against future mutated pathogens and future similar pathogens. Combination of multiple antigen serotypes or T cell epitopes from divergent clades may be required to provide a broadly protective immune response across populations.
  • the vaccibody construct is a multimeric protein consisting of multiple polypeptides, such as a dimeric protein consisting of two polypeptides, each comprising a targeting unit, which targets or is capable of targeting antigen-presenting cells, a multimerization unit such as a dimerization unit and an antigenic unit and which, after administration to a subject, has shown to be efficient in generating an immune response against the antigens or epitopes comprised in the antigenic unit.
  • the vaccibody construct may be administered to a subject in the form of a polynucleotide (e.g. a DNA plasmid) comprising a nucleotide sequence encoding the polypeptide.
  • a polynucleotide e.g. a DNA plasmid
  • the polypeptide is expressed which, due to the multimerization unit, such as a dimerization unit, forms a multimeric protein, such as a dimeric protein.
  • the present disclosure provides a new immunogenic construct, which comprises an antigenic unit that comprises one or more T cell epitopes and one or more antigens which are arranged in a specific way.
  • the disclosure provides an immunogenic construct comprising:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or (ii) a polypeptide encoded by the nucleotide sequence defined in (i); or
  • a multimeric protein such as a dimeric protein, consisting of multiple polypeptides as defined in (ii), such as two polypeptides.
  • the disclosure provides an immunogenic construct comprising:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides an immunogenic construct comprising:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • Such a construct will, once administered to a human in the form of a vaccine, elicit a rapid, strong and persistent T cell response and B cell response and is thus suitable as a prophylactic or therapeutic vaccine for an infectious disease.
  • This is of particular importance in a pandemic or epidemic, where time is of essence to stop a pathogen from spreading further or to mitigate health impact by reducing the extent of severe disease.
  • infected individuals may be asymptotic or only show mild and/or diffuse symptoms, there may not be time to test whether an individual is already infected or not.
  • tests may not be available, or may not be available at sufficient numbers, or may not be specific enough.
  • the time from exposure to infection, severe disease and/or ultimately death may vary according to pathogen species, and for some pathogen species this time window will allow a therapeutic use of the vaccine of same design as for preventive purpose (i.e. ranging from post-exposure prophylaxis to early stage treatment).
  • a therapeutic use of the vaccine of same design i.e. ranging from post-exposure prophylaxis to early stage treatment.
  • the immunogenic construct of the disclosure may be used in a vaccine, i.e. a composition comprising the construct of the disclosure and a pharmaceutically acceptable carrier, for use in the prophylactic or therapeutic treatment of an infectious disease, by administering the vaccine to a subject.
  • a vaccine i.e. a composition comprising the construct of the disclosure and a pharmaceutically acceptable carrier, for use in the prophylactic or therapeutic treatment of an infectious disease, by administering the vaccine to a subject.
  • Figure 1 shows an immunogenic construct of the disclosure which is a polypeptide and described as having an N-terminal start and a C-terminal end.
  • the elements of the polypeptide - targeting unit (TU), first joint region (1JR), first antigenic unit, second joint region (2 JR) and second antigenic unit - may be arranged in the polypeptide such that the second antigenic unit is located at the C-terminal end of the polypeptide (Fig. la) or at the N-terminal start of the polypeptide (Fig. lb). Further details are provided in the section with the title “Immunogenic construct”.
  • Figure 2 shows a schematic drawing of an embodiment of a dimeric protein, consisting of two polypeptides, each of which comprising a targeting unit, first joint region, first antigenic unit, second joint region and second antigenic unit, wherein the two polypeptides are linked via their respective first joint regions and via their respective second joint regions.
  • the joint regions comprise a flexible unit, which is closest to the targeting unit and second antigenic unit, respectively, and a binding unit, which is closest to the first antigenic unit.
  • Figure 3 shows the protein expression and secretion levels of the polypeptides encoded by DNA plasmids TECH004-IV007, TECH004-IV008, TECH004-IV011, TECH004-IV012, and TECH004-IV015 detected in the supernatant of Expi293F cells transfected with said DNA plasmids by the enzyme-linked immunosorbent assay (ELISA) using rabbit a-SARS-CoV-2/2019-nCoV RBD detection Ab (40592-T62), and goat a-human CCL3/MIP-1 alpha biotinylated antibody (AF-270-NA).
  • Figure 4 is a Western blot that shows full-length secretion of immunogenic proteins. Reduced supernatant samples from transfected Expi293F cells. Primary antibody: goat anti-human MIP-la (AF270). Secondary antibody: donkey anti-goat, Dylight 800 (SA5- 10092). Chemidoc channels Dylight 800 and 650 (for protein standard).
  • Figure 5 shows the immunogenicity of DNA plasmids TECH004-IV007, TECH004-IV008, TECH004-IV012, TECH004-IV013, and TECH004-IV015 against the encoded T cell epitopes in mice vaccinated with these plasmids by measuring the IFN-g secretion from T cells (total T cell response), compared to the negative control VB1026.
  • Figure 6 shows the immunogenicity of DNA plasmids TECH004-IV007, TECH004-IV008, TECH004-IV012, TECH004-IV013, and TECH004-IV015 against RBD antigenic unit in mice vaccinated with these plasmids by measuring the IFN-g secretion from T cells (total T cell response), compared to the negative control VB1026.
  • Figure 8 Shows the protein expression and secretion levels of the polypeptides encoded by DNA plasmids TECH004-IV028, TECH004-IV029, TECH004-IV030, TECH004-IV031, and TECH004-IV032 detected in the supernatant of Expi293F cells transfected with said DNA plasmids by the enzyme-linked immunosorbent assay (ELISA) using mouse anti-human IgG CH3 domain capture Ab (MCA878G), rabbit anti-influenza A H1N1 HA domain detection Ab (11684-R107), and goat anti-rabbit IgG secondary Ab (31460).
  • Figure 9 Shows the immunogenicity of DNA plasmids TECH004-IV028, TECH004-I V 029, TECH004-IV030, TECH004-IV031, and TECH004-IV032 against the encoded T cell epitopes in mice vaccinated with these plasmids by measuring the IFN-g secretion from T cells (total T cell response), compared to the negative control VB1026.
  • Figure 10 Shows the immunogenicity of DNA plasmids TECH004-IV028, TECH004-I V 029, TECH004-IV030, TECH004-IV031, and TECH004-IV032 against HA Antigenic unit in mice vaccinated with these plasmids by measuring the IFN-g secretion from T cells (total T cell response), compared to the negative control VB1026.
  • the disclosure provides an immunogenic construct comprising:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a multimeric protein such as a dimeric protein, consisting of multiple polypeptides as defined in (ii), such as two polypeptides.
  • the disclosure provides an immunogenic construct comprising:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides an immunogenic construct comprising:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • an “immunogenic construct” is one that elicits an immune response, particularly when administered to a subject in a form suitable for administration and in an amount effective to elicit the immune response (i.e. an immunologically effective amount).
  • a “subject” is an animal, e.g., a mouse, or a human.
  • a subject may be a patient, i.e. a human suffering from an infectious disease who is in need of a therapeutic treatment, or it may be a subject in need of prevention from being infected with an infectious disease, or it may be a subject suspected of suffering from an infectious disease.
  • the terms “subject” and “individual” are used interchangeably herein.
  • infectious disease is a disease caused by one or more pathogens, including viruses, bacteria, fungi and parasites.
  • a “treatment” is a prophylactic treatment or therapeutic treatment.
  • a “prophylactic treatment” is a treatment administered to a subject who does not or does not yet display signs or symptoms of, or displays only early signs or symptoms of, an infectious disease, such that treatment is administered for the purpose of preventing or decreasing the risk of developing the disease and/or symptoms associated with the disease.
  • a prophylactic treatment functions as a preventive treatment against an infectious disease, or as a treatment that inhibits or reduces further development or enhancement of the disease and/or its associated symptoms.
  • the terms “prophylactic treatment”, “prophylaxis” and “prevention” are used interchangeably herein.
  • a “therapeutic treatment” is a treatment administered to a subject who displays symptoms or signs of an infectious disease, in which treatment is administered to the subject for the purpose of diminishing or eliminating the disease and/or those signs or symptoms.
  • T cell epitope refers to a single T cell epitope or a part or region of an antigen containing multiple T cell epitopes, e.g. multiple minimal epitopes.
  • a “part” of an antigen is a fragment or portion of an antigen, i.e. part/fragment of the amino acid sequence of an antigen, or the nucleotide sequence encoding same, e.g. an epitope; preferably, the part or fragment of the antigen is immunogenic.
  • minimal epitope refers to a subsequence of an epitope predicted to bind to MHC I or MHC II.
  • the minimal epitope may be immunogenic, i.e. capable of eliciting an immune response.
  • the term minimal epitope thus may refer to short subsequences of an epitope, which are predicted to bind to MHC I or MHC II.
  • a 27-mer epitope may thus encompass several minimal epitopes, which may have a length shorter than 27 amino acids, but which each are immunogenic.
  • a minimal epitope could consist of the first 14 amino acids of the epitope, provided that it is predicted to bind to MHC I or MHC II, or it could consist of amino acids 9 to 18 of the epitope, or of amino acids 7 to 22, provided that these sequences are predicted to bind to MHC I or MHC II.
  • nucleotide sequence is a sequence consisting of nucleotides.
  • nucleotide sequence and “nucleic acid sequence” are used interchangeably herein.
  • the immunogenic construct of the disclosure can be a polypeptide and described as having an N-terminal start and a C-terminal end (illustrated in Fig. 1).
  • the elements of the polypeptide are: A targeting unit (TU), a first joint region (1JR), a first antigenic unit, a second joint region (2 JR) and a second antigenic unit.
  • the elements may be arranged in the polypeptide such that the second antigenic unit is located at the C-terminal end of the polypeptide (Fig. la) or at the N-terminal start of the polypeptide (Fig. lb).
  • the second antigenic unit is located at the C-terminal end of the polypeptide.
  • T2 T2 T cell epitopes
  • TL T cell epitope linker
  • L linker
  • the T cell epitopes are preferably separated by T cell epitope linkers.
  • a first antigenic unit that comprises n T cell epitopes preferably comprises n-1 T cell epitope linkers.
  • Fig. 2 illustrates an embodiment of the dimeric protein, wherein two polypeptides are linked via their respective first and second joint regions.
  • the various units of the construct will be discussed in detail. They are present in the polynucleotide as nucleotide sequences encoding the units while they are present in the polypeptide, multimeric or dimeric protein as amino acids sequences.
  • the units of the construct are mainly explained in relation to the polypeptide, /multimericdimeric protein, i.e. on the basis of their amino acid sequences.
  • the construct of the disclosure comprises a first joint region and a second joint region.
  • the first joint region and the second joint region may be any of the below described regions.
  • the multimeric protein of the disclosure is one where the multiple polypeptides are linked to each other via their joint regions.
  • the dimeric protein of the disclosure is one where the two polypeptides are linked to each other via their joint regions. In other embodiments, the multimeric protein of the disclosure is one where the multiple polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions.
  • the dimeric protein of the disclosure is one where the two polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions.
  • joint region refers to a sequence of amino acids between the first targeting unit and the first antigenic unit or between the first antigenic unit and the second antigenic unit. Any amino acid sequence that is capable of joining the multiple polypeptides, for example two polypeptides, but at the same time providing flexibility to the multimeric protein or dimeric protein, is a suitable joint region.
  • the joint regions provide flexibility to the multimeric protein or dimeric protein, such that the targeting units can interact with surface molecules on APCs, e.g. with surface molecules on the same APC, and bind to those surface molecules, even if they are located at variable distances and that the second antigenic units can interact with B cell receptors on B cells (BCRs) specific for the B cell epitopes in the second antigenic unit, either with both second antigenic units binding both arms of the same BCR or cross-binding neighboring BCRs.
  • BCRs B cell receptors on B cells
  • the joint regions join multiple of the polypeptides, such as two of the polypeptides, into a multimeric protein, such as a dimeric protein. Any amino acid sequence that fulfills one or more of these requirements is a suitable joint region.
  • the joint region comprise a flexible unit which provides flexibility and a binding unit which joins the multiple polypeptides, for example the two polypeptides, to form a multimer, for example a dimer.
  • the joint region comprise a flexible unit which provides flexibility and a binding unit which joins the multiple polypeptides to form a multimeric protein.
  • the joint region comprise a flexible unit which provides flexibility and a binding unit which joins the two polypeptides to form a dimeric protein.
  • the flexible unit in the first joint region is closest to the targeting unit and the binding unit is closest to the first antigenic unit.
  • the flexible unit in the second joint region is closest to the second antigenic unit and the binding unit is closest to the first antigenic unit.
  • the binding units of the first and second joint regions are different.
  • the binding unit comprised in the first joint region of one polypeptide molecule is able to bind to the binding unit comprised in the first joint region of another polypeptide molecule, whereby the two or multiple molecules are linked via their respective first joint regions.
  • the binding unit comprised in the second joint region of one polypeptide molecule is able to bind to the binding unit comprised in the second joint region of another polypeptide molecule, whereby the two or multiple molecules are linked via their respective second joint regions.
  • the multiple polypeptide molecules are linked to each other via their respective first joint regions and via their respective second joint regions, forming a multimeric protein.
  • the two polypeptide molecules are linked to each other via their respective first joint regions and via their respective second joint regions, which results in formation of a dimeric protein.
  • the first- and/or second joint region comprises at least one naturally occurring sequence. In some embodiments, the first- and/or second joint region comprises at least one artificial sequence.
  • the binding unit is a covalent binding unit, in other embodiments, the binding unit is a non-covalent binding unit.
  • sequence of one or both joint regions is a non- immunogenic sequence, preferably, the sequences of both joint regions are non- immunogenic sequences.
  • the targeting unit, first antigenic unit, second antigenic unit, T cell epitopes, antigens and parts thereof are as described herein elsewhere.
  • Flexible unit
  • each joint region comprises a flexible unit and a binding unit.
  • the flexible unit is between the targeting unit and the binding unit.
  • the flexible unit is a non-immunogenic sequence.
  • the amino acid sequence of the flexible unit is a naturally occurring peptide sequence.
  • the flexible unit is derived from an immunoglobulin.
  • the flexible unit is a hinge region of an immunoglobulin, which hinge region does not comprise cysteine residues.
  • the flexible unit is an artificial sequence.
  • the flexible unit comprises small, non-polar (e.g. glycine, alanine or leucine) or polar (e.g. serine or threonine) amino acids.
  • the small size of these amino acids provides flexibility and allows for mobility of the connected amino acid sequences.
  • the incorporation of serine or threonine can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the linker and antigens.
  • the flexible unit is an artificial sequence, e.g. a serine (S) and/or glycine (G) rich linker, i.e.
  • GGGGSGGGSS SEQ ID NO: 8
  • GGGSG SEQ ID NO: 9
  • SGSSGS SEQ ID NO: 10
  • GGSGG SEQ ID NO: 7
  • GGGGSGGGGS SEQ ID NO: 11
  • GGGGS GGGS
  • GGGSS GGSS
  • SEQ ID NO: 13 GGSG
  • SEQ ID NO: 14 GGSG
  • SEQ ID NO: 15 SGSSGS
  • the serine and/or glycine rich linker further comprises at least one leucine (L) residue, such as at least 1 or at least 2 or at least 3 leucine residues, e .g. 1, 2, 3 or 4 leucine residues.
  • L leucine
  • the flexible unit comprises or consists of LGGGS (SEQ ID NO: 16), GLGGS (SEQ ID NO: 17), GGLGS (SEQ ID NO: 18), GGGLS (SEQ ID NO: 19) or GGGGL (SEQ ID NO: 20).
  • the flexible unit comprises or consists of LGGSG (SEQ ID NO: 21), GLGSG (SEQ ID NO: 22), GGLSG (SEQ ID NO: 23), GGGLG (SEQ ID NO: 24) or GGGSL (SEQ ID NO: 25).
  • the flexible unit comprises or consists of LGGSS (SEQ ID NO: 26), GLGSS (SEQ ID NO: 27) or GGLSS (SEQ ID NO: 29).
  • the flexible unit comprises or consists of LGLGS (SEQ ID NO: 29), GLGLS (SEQ ID NO: 30), GLLGS (SEQ ID NO: 31), LGGLS (SEQ ID NO: 32), GLGGL (SEQ ID NO: 33) or (GLGGL)m (SEQ ID NO: 108).
  • the flexible unit comprises or consists of LGLSG (SEQ ID NO: 34), GLLSG (SEQ ID NO: 35), GGLSL (SEQ ID NO: 36), GGLLG (SEQ ID NO: 37) or GLGSL (SEQ ID NO: 38).
  • the flexible unit comprises or consists of LGLSS (SEQ ID NO: 39) or GGLLS (SEQ ID NO: 40).
  • the flexible unit is serine-glycine linker that has a length of 10 amino acids and comprises 1 or 2 leucine residues.
  • the flexible unit comprises or consists of LGGGSGGGGS (SEQ ID NO: 41), GLGGSGGGGS (SEQ ID NO: 42), GGLGSGGGGS (SEQ ID NO: 43), GGGLSGGGGS (SEQ ID NO: 44) or GGGGLGGGGS (SEQ ID NO: 45).
  • the flexible unit comprises or consists of LGGSGGGGSG (SEQ ID NO: 46), GLGSGGGGSG (SEQ ID NO: 47), GGLSGGGGSG (SEQ ID NO: 48), GGGLGGGGSG (SEQ ID NO: 49) or GGGSLGGGSG (SEQ ID NO: 50).
  • the flexible unit comprises or consists of LGGSSGGGSS (SEQ ID NO: 51), GLGSSGGGSS (SEQ ID NO: 52), GGLSSGGGSS (SEQ ID NO: 53), GGGLSGGGSS (SEQ ID NO: 54) or GGGSLGGGSS (SEQ ID NO: 55). In further embodiments, the flexible unit comprises or consists of
  • the flexible unit comprises or consists of LGGSGLGGSG (SEQ ID NO: 61), GLGSGGLGSG (SEQ ID NO: 62), GGLSGGGLSG (SEQ ID NO: 63), GGGLGGGGLG (SEQ ID NO: 64) or
  • the flexible unit comprises or consists of LGGSSLGGSS (SEQ ID NO: 66), GLGSSGLGSS (SEQ ID NO: 67) or GGLSSGGLSS (SEQ ID NO: 68). In yet other embodiments, the flexible unit comprises or consists of GSGGGA (SEQ ID NO: 69), GSGGGAGSGGGA (SEQ ID NO: 70),
  • the flexible unit comprises or consists of SGGGSSGGGS (SEQ ID NO: 74), GGGGSGGGGS (SEQ ID NO: 75), SSGGGSSGGG (SEQ ID NO: 76), GGSGGGGSGG (SEQ ID NO: 77), GSGSGSGSGS (SEQID NO: 78), GGGSSGGGSG (SEQ ID NO: 79, corresponding to amino acids 121-130 of SEQ ID NO: 1), GGGSSS (SEQ ID NO: 80), GGGSSGGGSSGGGSS (SEQ ID NO: 81) or GLGGLAAA (SEQ ID NO: 82).
  • the flexible unit comprises or consists of the sequence TQKSLSLSPGKGLGGL (SEQ ID NO: 83). In other embodiments, the flexible unit comprises or consists of the sequence SLSLSPGKGLGGL (SEQ ID NO: 84). In other embodiments, the T cell epitope linker comprises or consists of AAY or GPGPG (SEQ ID NO: 109).
  • the flexible unit in the second joint region is a GSAT linker, i.e. a linker comprising one or more glycine, serine, alanine and threonine residues, e.g. a linker comprising or consisting of the sequence GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 87) or a SEG linker, i.e. a linker comprising one or more serine, glutamic acid and glycine residues, e.g.
  • a linker comprising or consisting of the sequence GGS GGGSEGGGSEGGGSEGGGSEGGGSEGGGS GGGS (SEQ ID NO: 88) or ELKTPLGDTTHT (SEQ ID NO: 85; corresponding to amino acids 94-105 of SEQ ID NO: 1).
  • the flexible unit is not a target of proteases.
  • the flexible unit consists of up to 20 amino acids, such as up to 15 amino acids, such as 14 amino acids, such as 13 amino acids, such as up to 12 amino acids, such as 11 amino acids or such as up to 10 amino acids.
  • the flexible unit comprised in the second joint region, i.e. closest to the second antigenic unit consists of from 5 to 60 amino acids, e.g. from 7 to 55 amino acids or 8 to 50 amino acids or 9 to 45 amino acids or 10 to 40 amino acids or 11 to 35 amino acids or 12 to 30 amino acids or 13 to 20 amino acids.
  • the flexible unit comprises or consists of an amino acid sequence having at least 50% sequence identity to the amino acid sequence 94-105 of SEQ ID NO: 1, such as 60% or such as 70% or such as 80% or such as 90% sequence identity.
  • the flexible unit comprises or consists of hinge exon hi of IgG3.
  • the flexible unit comprises or consists of the amino acid sequence 94-105 of SEQ ID NO: 1.
  • the flexible unit comprises or consists of an amino acid sequence having at least 50% sequence identity to the amino acid sequence 16-23 of SEQ ID NO: 2 or SEQ ID NO: 114, such as 60% or such as 70% or such as 80% or such as 90% sequence identity.
  • the flexible unit comprises or consists of the amino acid sequence 16-23 of SEQ ID NO: 2 or SEQ ID NO: 114, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted , with the proviso that no more than 5 amino acids have been so altered, such as 4 amino acids, no more than 3 amino acids, such as 2 amino acids or no more than 1 amino acid.
  • the flexible unit comprises or consists of the lower hinge region of IgGl.
  • the flexible unit comprises or consists of the amino acid sequence 16-23 of SEQ ID NO: 2.
  • the joint region comprises a covalent binding unit.
  • the covalent binding unit comprises one or more cysteine residues, and the polypeptides described herein are linked via one or more disulfide bonds formed between the cysteine residue(s) comprised in the covalent binding units.
  • the covalent binding unit consists of or comprises a cysteine rich sequence.
  • the covalent binding unit comprises at least 2 cysteine residues, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 cysteine residues.
  • the covalent binding unit of the first joint region comprises a different number of cysteine residues than the covalent binding unit of the second joint region.
  • the cysteine residues of the covalent binding unit of first joint region are positioned differently than the cysteine residues of the covalent binding unit of the second joint region.
  • the number of amino acid residues between the cysteine residues of the covalent binding unit of the first joint region is different than that of the second joint region.
  • the number of cysteine residues is based on the length of the antigenic unit: the more amino acid residues comprised in the antigenic unit, the higher the number of cysteine residues in the covalent binding unit.
  • the covalent binding unit comprises the sequence EPKSCDTPPPCPRCP (SEQ ID NO: 86; corresponding to amino acids 106-120 of SEQ ID NO: 1).
  • the covalent binding unit is a non-immunogenic sequence. In some embodiments, the covalent binding unit is an artificial sequence.
  • the amino acid sequence of the covalent binding unit is a naturally occurring sequence.
  • the covalent binding unit consists of from 2 to 100 amino acids, such as 3 to 70 amino acids, such as 4 to 50 amino acids or 5 to 30 amino acids. In further embodiments, the covalent binding unit consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In preferred embodiments, the covalent binding unit consists of 15 amino acids. In other preferred embodiments, the covalent binding unit consists of 15 amino acids, whereof 3 are cysteine residues.
  • the covalent binding unit is derived from an immunoglobulin.
  • the covalent binding unit is a hinge region derived from an immunoglobulin, such as exon h4 of IgG3 or the middle hinge region of IgGl.
  • the hinge region may be Ig derived, such as derived from IgG, e.g. IgGl, IgG2 or IgG3.
  • the hinge region is derived from IgM, e.g. comprising or consisting of the nucleotide sequence with SEQ ID NO: 89 or an amino acid sequence encoded by said nucleotide sequence.
  • the covalent binding unit comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence 106-120 of SEQ ID NO: 1, such as at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity.
  • the covalent binding unit comprises or consists of the amino acid sequence 106-120 of SEQ ID NO: 1, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 6 amino acids have been so altered, such as 5 amino acids, such as 4 amino acids, no more than 3 amino acids, such as 2 amino acids or no more than 1 amino acid.
  • the covalent binding unit is hinge exon h4 of IgG3.
  • the covalent binding region consists of amino acid sequence 106-120 of SEQ ID NO: 1
  • the covalent binding unit comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence 5-15 of SEQ ID NO: 2 or SEQ ID NO: 114, provided that the cysteine residues are retained in their number and position, such as at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity.
  • the covalent binding unit comprises or consists of the amino acid sequence 5-15 of SEQ ID NO: 2 or SEQ ID NO: 114, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 5 amino acids have been so altered, such as 4 amino acids, no more than 3 amino acids, such as 2 amino acids or no more than 1 amino acid.
  • the covalent binding unit is the middle hinge region of IgGl.
  • the covalent binding unit consists of or comprises the amino acid sequence 5-15 of SEQ ID NO: 2 or SEQ ID NO: 114.
  • the joint region as described herein comprises a non- covalent binding unit.
  • the non-covalent binding unit contributes to multimerization, for example to dimerization, through non-covalent interactions, e.g. hydrophobic interactions.
  • the non-covalent binding unit has the ability to form multimeric proteins via non-covalent interactions.
  • the non-covalent binding unit has the ability to form dimers via non-covalent interactions.
  • the amino acid sequence of the non-covalent binding unit is a non-immunogenic sequence.
  • the amino acid sequence of the non-covalent binding unit is a naturally occurring sequence.
  • the non-covalent binding unit is an artificial sequence.
  • the non-covalent binding unit consists of or comprises an immunoglobulin domain, such as an immunoglobulin constant domain (C domain), such as a carboxyterminal constant domain (i.e. a CH3 domain), a CHI domain or a CH2 domain, or a sequence that is substantially identical to the C domain or a variant thereof.
  • C domain immunoglobulin constant domain
  • CH3 domain carboxyterminal constant domain
  • CHI domain CHI domain
  • CH2 domain a sequence that is substantially identical to the C domain or a variant thereof.
  • the non-covalent binding unit consists of or comprises SEQ ID NO: 113.
  • the non-covalent binding unit is a CH3 domain derived from IgG, such as derived from IgG3 or IgGl, preferably derived from IgGl.
  • the non-covalent binding unit in one joint region comprises a CH3 domain, it does not comprise a CH2 domain in addition and vice versa.
  • the non-covalent binding unit comprises or consists of a CH3 domain derived from IgG3 with an amino acid sequence having at least 80% sequence identity to the amino acid sequence 131-237 of SEQ ID NO: 1 or SEQ ID NO: 113.
  • the non-covalent binding unit comprises or consists of a carboxyterminal C domain derived from IgG3 with the amino acid sequence 131-237 of SEQ ID NO: 1 or SEQ ID NO: 113, wherein any one of the amino acids of the non- covalent binding unit has been substituted, deleted or inserted, with the proviso that no more than 22 amino acids have been so altered, such as no more than 21 amino acids , such as no more than 20 amino acids, such as no more than 19 amino acids, such as no more than 18 amino acids, such as no more than 17 amino acids, such as no more than 16 amino acids, such as no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no more than 5 amino acids, such as
  • the non-covalent binding unit comprises or consists of a CH3 domain derived from IgG3 with an amino acid sequence having at least 85% sequence identity to the amino acid sequence 131-237 of SEQ ID NO: 1 or SEQ ID NO: 113, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity.
  • the non-covalent binding unit comprises or consists of a CH3 domain derived from IgG3 with the amino acid sequence 131-237 of SEQ ID NO: 1 or SEQ ID NO: 113.
  • the non-covalent binding unit comprises or consists of a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence with SEQ ID NO: 90.
  • the non-covalent binding unit comprises or consists of a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence with SEQ ID NO: 90, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%,
  • the non-covalent binding unit comprises or consists of the nucleotide sequence of SEQ ID NO: 90. In some embodiments, the non-covalent binding unit comprises or consists of a CH3 domain derived from IgGl with an amino acid sequence having at least 80 % sequence identity to the amino acid sequence according to SEQ ID NO: 3.
  • the non-covalent binding unit comprises or consists of a a CH3 domain derived from IgGl with the amino acid sequence of SEQ ID NO: 3, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 22 amino acids have been so altered, such as no more than 21 amino acids, such as no more than 20 amino acids, such as no more than 19 amino acids, such as no more than 18 amino acids, such as no more than 17 amino acids, such as no more than 16 amino acids, such as no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no more than 5 amino acids, such as no more than 4 amino acids, such as no more than 3 amino acids, such as
  • the non-covalent binding unit comprises or consists of a CH3 domain from IgGl with an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 3, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity.
  • the non-covalent binding unit is or comprises CH3 of IgGl.
  • the non-covalent binding unit is or comprises a leucine zipper motif.
  • a leucine zipper is a common three-dimensional structural motif in proteins where leucine side chains from one alpha helix interdigitate with those from another alpha helix, facilitating dimerization.
  • Leucine zippers are a dimerization motif of the bZIP (basic-region leucine zipper) class of eukaryotic transcription factors.
  • the bZIP domain is 60 to 80 amino acids in length with a highly conserved DNA binding basic region and a more diversified leucine zipper dimerization region.
  • the non-covalent binding unit is or comprises a leucine zipper motif derived from the bZIP class of eukaryotic transcription factors.
  • the non-covalent binding unit is or comprises a leucine zipper motif.
  • the non-covalent binding unit is or comprises a Jun/Fos- based leucine zipper. In some embodiments, the non-covalent binding unit is or comprises a ATF6-based leucine zipper. In some embodiments, the non-covalent binding unit is or comprises a PAR-based leucine zipper. In some embodiments, the non-covalent binding unit is or comprises a C/EBPa-based leucine zipper. In some embodiments, the non-covalent binding unit is or comprises an OASIS-based leucine zipper.
  • the non-covalent binding unit is or comprises a leucine zipper motif (amino acids 308-336) from the CREB transcription factor (SEQ ID NO: 4).
  • the non-covalent binding unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4, such as at least 81% or at least 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • the non-covalent binding unit comprises or consists of the amino acid sequence of SEQ ID NO: 4, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 12 amino acids have been so altered, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no more than 5 amino acids, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid.
  • amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 12 amino acids have been so altered, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no more
  • the non- covalent binding unit facilitates multimerization of/joins multiple polypeptides, such as two, three, four or more polypeptides, into a multimeric protein, such as a dimeric protein, a trimeric protein, or a tetrameric protein.
  • the non-covalent binding unit joins multiple polypeptides, such as two, three, four or more polypeptides, into a multimeric protein, such as a dimeric protein, a trimeric protein, or a tetrameric protein.
  • the non-covalent binding unit is or comprises a trimerization unit, such as a collagen-derived trimerization unit, such as a human collagen-derived trimerization domain, such as human collagen derived XVIII trimerization domain (see for instance A. Alvarez-Cienfuegos et al, Sci Rep 6, 28643 (2016)) or human collagen XV trimerization domain.
  • the non-covalent binding unit is a trimerization unit that comprises or consists of the nucleotide sequence with SEQ ID NO: 91, or an amino acid sequence encoded by said nucleotide sequence.
  • the trimerization unit is the C-terminal domain of T4 fibritin.
  • the non-covalent binding unit is a trimerization unit that comprises or consists of the amino acid sequence with SEQ ID NO: 92, or a nucleotide sequence encoding said amino acid sequence.
  • the non-covalent binding unit is or comprises a tetramerization unit, such as a domain derived from p53, optionally further comprising a flexible unit or a covalent binding unit as described above.
  • the non-covalent binding unit is a tetramerization unit that comprises or consists of the nucleotide sequence with SEQ ID NO: 93, or an amino acid sequence encoded by said nucleotide sequence, optionally further comprising a flexible unit or a covalent binding unit as described above.
  • the joint region comprises a flexible unit and a binding unit which is either a covalent or non-covalent binding unit.
  • the joint region comprises a binding unit which comprises both, a covalent binding unit and a non-covalent binding unit.
  • the joint region comprises a flexible unit, a covalent binding unit and a non-covalent binding unit.
  • the non-covalent binding unit is located between the antigenic unit and the covalent binding unit.
  • the covalent binding unit is located between the antigenic unit and the non-covalent binding unit.
  • the joint region comprises a flexible unit and a non- covalent binding unit. In other embodiments, the joint region comprises a flexible unit and a covalent binding unit. In preferred embodiments, the flexible unit is located closest to the targeting unit, i.e. between the targeting unit and covalent- and/or non- covalent binding unit.
  • the joint region further comprises a linker.
  • the linker is located between the covalent binding unit, and the non- covalent binding unit.
  • the joint region comprises hinge exon hi and hinge exon h4 of IgG3.
  • the joint region comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence 94-120 of SEQ ID NO: 1, provided that the cysteine residues in the sequence are retained in their number and position, such as at least 50% sequence identity, at least 60%, at least 70%, at least 80% or at least 90% sequence identity.
  • the joint region comprises or consists of the amino acid sequence 94-120 of SEQ ID NO: 1, provided that the cysteine residues in the sequence are retained in their number and position, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted , with the proviso that no more than 16 amino acids have been so altered, such as no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no more than 5 amino acids, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid.
  • no more than 16 amino acids have been so altered, such as no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13
  • the joint region is hinge exon hi and hinge exon h4 of IgG3. In other embodiments, the joint region consists of or comprises the amino acid sequence 94-120 of SEQ ID NO: 1.
  • the joint region comprises or consists of a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence with SEQ ID NO: 94.
  • the joint region comprises or consists of a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence with SEQ ID NO: 94, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • the joint region comprises or consists of the nucleotide sequence of SEQ ID NO: 94.
  • said joint region comprises the hinge exons in the order h4 to hi, i.e. the above-described sequence is “flipped”, such that the flexible unit, hi, is closest to the second antigenic unit.
  • the joint region comprises the middle and lower hinge regions of IgGl.
  • the joint region comprises or consist of an amino acid sequence having at least 40% sequence identity to the amino acid sequence 5-23 of SEQ ID NO: 2 or SEQ ID NO: 114, provided that the cysteine residues in the sequence are retained in their number and position, such as at least 50% sequence identity, at least 60%, at least 70%, at least 80% or at least 90% sequence identity.
  • the joint region comprises or consists of the amino acid sequence 5-23 of SEQ ID NO: 2 or SEQ ID NO: 114, provided that the cysteine residues in the sequence are retained in their number and position, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 11 amino acids have been so altered, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no more than 5 amino acids, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid.
  • the joint region is the middle and lower hinge region of IgGl.
  • the joint region consists of or comprises the amino acid sequence 5-23 of SEQ ID NO: 2 or SEQ ID NO: 114.
  • said joint region comprises the hinge regions in the order lower hinge region to middle hinge region, i.e. the above-described sequence is “flipped”, such that the flexible unit, the lower hinge region, is closest to the targeting unit.
  • the joint region comprising hinge exon hi and hinge exon h4 of IgG3 and/or the joint region comprising the middle and lower hinge region of IgGl may further comprise a non-covalent binding unit, e.g. the afore-described non-covalent binding units, preferably an immunoglobulin constant domain.
  • a non-covalent binding unit e.g. the afore-described non-covalent binding units, preferably an immunoglobulin constant domain.
  • the antigenic unit comprises epitopes having a length suitable for specific presentation on MHC class I or MHC class II.
  • the epitope has a length of from 7 to 11 amino acids for MHC class I presentation. In other embodiments, the epitope has a length of 15 amino acids for MHC class II presentation.
  • First antigenic unit comprising one or more T cell epitopes
  • the construct of the disclosure comprises a first antigenic unit comprising one or more T cell epitopes.
  • the one or more T cell epitopes are disease-relevant T cell epitopes, i.e. they are comprised (or naturally found) in proteins of the pathogen which causes the disease or is involved in causing it, e.g. eggs of a parasite which do not cause the disease but develop into larvae which cause it, and which pathogen is the target of a vaccine comprising the immunogenic construct of the disclosure including such T cell epitopes.
  • the first antigenic unit comprises one T cell epitope, In other embodiments, the first antigenic unit comprises multiple T cell epitopes, which can be identical or different, preferably the several T cell epitopes are multiple different T cell epitopes.
  • the first antigenic unit comprises one or more T cell epitopes of a pathogen, i.e. one T cell epitope of a pathogen or more than one T cell epitope of a pathogen, i.e. multiple T cell epitopes of a pathogen.
  • the multiple T cell epitopes are of the same pathogen, i.e. (naturally) comprised in the same or different proteins of the pathogen.
  • the multiple T cell epitopes are of multiple different pathogens, i.e. (naturally) comprised in proteins of different pathogens.
  • a “different pathogen” may, for example be a different virus or bacterium or a different strain of the same virus or bacterium or it may be the same strain, but comprising one or more mutations.
  • the T cell epitopes comprised in the first antigenic unit of the construct of the disclosure have a length of from 7 to about 200 amino acids, with the longer T cell epitopes possibly including hotspots of minimal epitopes.
  • a hotspot of minimal epitopes is a region that contains several minimal epitopes (e.g. having a length of from 8-15 amino acids) that are predicted to be presented by different HLA alleles to cover a broad range of world population.
  • the first antigenic unit comprises T cell epitopes with a length of from 7 to 150 amino acids, preferably of from 7 to 100 amino acids, e.g. from about 10 to about 100 amino acids or from about 15 to about 100 amino acids or from about 20 to about 75 amino acids or from about 25 to about 50 amino acids.
  • T cell epitopes having a length of about 60 to 200 amino acids may be split into shorter sequences and included into the first antigenic unit separated by the linkers which are described herein.
  • a T cell epitope having a length of 150 amino acids may be split into 3 sequences of 50 amino acids each, and included into the first antigenic unit, preferably with a T cell epitope linker separating the 3 sequences from each other.
  • the T cell epitope has a length suitable for presentation by MHC (major histocompatibility complex).
  • MHC major histocompatibility complex
  • MHC class I and MHC II are interchangeably used herein with HLA class I and HLA class II.
  • HLA human leukocyte antigen
  • the first antigenic unit comprises T cell epitopes having a length suitable for specific presentation on MHC class I or MHC class II.
  • the T cell epitope has a length of from 7 to 11 amino acids for MHC class I presentation.
  • the T cell epitope has a length of from 9 to 60 amino acids, such as from 9 to 30 amino acids, such as 15 to 60 amino acids, such as 15 to 30 amino acids, such as 11 to 15 amino acids, such as 12 to 20 amino acids for MHC class II presentation. In other embodiments, the T cell epitope has a length of 15 amino acids for MHC class II presentation.
  • the number of T cell epitopes in the first antigenic unit may vary, and depends on the length and number of other elements included in the first antigenic unit, e.g. T cell epitope linkers as described in this application, and on the length and number of the antigens and other elements included in the second antigenic unit.
  • the first antigenic unit comprises up to 3500 amino acids, such as from 60 to 3500 amino acids, e.g. from about 80 or about 100 or about 150 amino acids to about a 3000 amino acids, such as from about 200 to about 2500 amino acids, such as from about 300 to about 2000 amino acids or from about 400 to about 1500 amino acids or from about 500 to about 1000 amino acids.
  • the first antigenic unit comprises 1 to 10 T cell epitopes such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 or 10 T cell epitopes or 11 to 20 T cell epitopes, such as 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 T cell epitopes or 21 to 30 T cell epitopes, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 T cell epitopes or 31 to 40 T cell epitopes, such as 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 T cell epitopes or 41 to 50 T cell epitopes, such as 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 T cell epitopes.
  • the T cell epitope may be comprised in any of the pathogen’s proteins, i.e. structural proteins, including surface proteins, and non- structural proteins; in other words, the T cell epitope may be found in the proteins naturally present in said pathogen.
  • the T cell epitope is from a conserved region of the pathogen, i.e. conserved between several subgenera, species or strains of respective pathogens.
  • the T cell epitope may be encoded by a nucleotide sequence which is found in a conserved region of the genome of the pathogen, i.e. conserved between several subgenus, species or strains of respective pathogens.
  • the T cell epitope may thus be conserved between several subgenus, species or strains of respective pathogens, i.e. the amino acid sequence of the T cell epitope is conserved between these.
  • the T cell epitope may be from a conserved region of a betacoronavirus, e.g. a region which is conserved between viruses from the same subgenus, such as the subgenus Sarbecovirus, e.g. conserved between SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19) and SARS-CoV, which causes severe acute respiratory syndrome (SARS).
  • a vaccine comprising the construct will, or is at least expected to, also provide protection against multiple variants of a betacoronavirus, e.g.
  • variants of SARS-CoV or variants of SARS-CoV-2 which is important for the efficacy of such a vaccine against future variants.
  • Viruses are known to mutate, e.g. undergo viral antigen drift or antigen shift.
  • conserved regions across the genomes of the betacoronavirus genus indicate that these conserved regions are needed to maintain essential structures or functions.
  • future mutations will take place in the less-conserved regions.
  • the vaccinated individual By raising an immune response against the conserved regions, the vaccinated individual will be protected also against future variants, or at least is expected to have a higher likelihood of being protected also against future variants and/or protected against severe disease caused by future variants.
  • the T cell epitope may be from a region of a human papilloma virus (HPV), e.g. from HPV16 or HPV18.
  • HPV antigens may be any antigens selected from the list consisting of El, E2, E6, E7, LI and L2, e.g. E6 and/or E7 of HPV16 and/or HPV18.
  • a vaccine comprising the construct will provide protection against HPV.
  • HPV infections are involved in certain cancers, such as squamous cell carcinoma of the head and neck, cervical cancer and vulvar squamous cell carcinoma. Indeed, HP VI 6 viral antigens are expressed in about 50% of all patients with said cancers.
  • the T cell epitope may be from a region of a human Influenza virus, such as human Influenza virus A, human Influenza virus B, human Influenza virus C and human Influenza virus D.
  • the human Influenza virus may be a specific hemagglutinin (HA) subtype, such as HI, H2, and H3, and/or a specific neuraminidase (NA) subtype, such as N1 or N5.
  • the human Influenza virus may be a H1N1 subtype.
  • the T cell epitope may be selected from the group consisting of T cell epitopes comprising or consisting of any of SEQ ID NO: 116-135. In some embodiments, the T cell epitope may be selected from the group consisting of T cell epitopes comprising or consisting of any of SEQ ID NO: 147-156. In some embodiments, the T cell epitopes are randomly arranged in the subunit. In other embodiments, at least one of the following methods for arranging them in the subunit is used to enhance the immune response.
  • the T cell epitopes are arranged in the order of more antigenic to less antigenic in the direction from the first joint region to the second joint region.
  • the most hydrophobic T cell epitopes may be positioned substantially in the middle of the first antigenic unit and the most hydrophilic T cell epitopes may be positioned towards the joint regions.
  • first antigenic unit comprising an odd number of T cell epitopes
  • substantially in this context refers to first antigenic units comprising an even number of T cell epitopes, wherein the most hydrophobic T cell epitopes are positioned as close to the middle as possible.
  • a first antigenic unit comprises 5 T cell epitopes, which are arranged as follows: l-2-3*-4-5; with 1, 2, 3*, 4 and 5 each being a different T cell epitope and - being a T cell epitope linker and * indicating the most hydrophobic T cell epitope, which is positioned in the middle of the first antigenic unit.
  • a first antigenic unit comprises 6 T cell epitopes, which are arranged as follows: l-2-3*-4-5-6 or, alternatively, as follows: l-2-4-3*-5-6; with 1, 2, 3*, 4, 5 and 6 each being a T cell epitope and - being a T cell epitope linker and * indicating the most hydrophobic T cell epitope, which is positioned substantially in the middle of the first antigenic unit.
  • the T cell epitopes may be arranged by alternating between a hydrophilic and a hydrophobic T cell epitope.
  • GC rich sequences encoding T cell epitopes are arranged in such a way, that GC clusters are avoided.
  • GC rich T cell sequences are arranged such that there is at least one non-GC rich T cell sequence between them.
  • GC rich sequences are sequences with a GC content of 60% or more, such as 65% or more, such as 70% or more, such as 75% or more, such as 80% or more.
  • T cell epitope linkers When more than one T cell epitope is present in the subunit, the T cell epitopes are separated by T cell epitope linkers. This ensures that each T cell epitope is presented in an optimal way to the immune system.
  • the T cell epitope linker is designed to be non- immunogenic.
  • the T cell epitope linker may be a rigid linker, meaning that that it does not allow the two amino acid sequences that it connects to substantially move freely relative to each other.
  • the T cell epitope linker may be a flexible linker, i.e. a linker that allows the two amino acid sequences that it connects to substantially move freely relative to each other.
  • the T cell epitope linker is a flexible linker, which allows for presenting the T cell epitope in an optimal manner to the immune system, even if the first antigenic unit comprises a large number of T cell epitopes.
  • the T cell epitope linker is a peptide consisting of from 4 to 40 amino acids, such as 35, 30, 25 or 20 amino acids, e.g. from 5 to 20 amino acids or 5 to 15 amino acids or 8 to 20 amino acids or 8 to 15 amino acids 10 to 15 amino acids or 8 to 12 amino acids. In some embodiments, the T cell epitope linker consists of 10 amino acids.
  • the T cell epitope linkers comprised in the first antigenic unit are preferably identical. If, however, one or more of the T cell epitopes comprises a sequence similar to that of the linker, it may be an advantage to substitute the neighboring T cell epitope linker with a linker of a different sequence. Also, if a T cell epitope/linker junction is predicted to constitute an epitope in itself, then it is preferred to use a T cell epitope linker of a different sequence.
  • the T cell epitope linker is a flexible linker, preferably a flexible linker which comprises small, non-polar (e.g. glycine, alanine or leucine) or polar (e.g. serine or threonine) amino acids. The small size of these amino acids provides flexibility and allows for mobility of the connected amino acid sequences.
  • the flexible linker is a serine (S) and/or glycine (G) rich linker, i.e. a linker comprising several serine and/or several glycine residues.
  • GGGGS SEQ ID NO: 161
  • GGGSS SEQ ID NO: 8
  • GGGSG SEQ ID NO: 9
  • SGSSGS SEQ ID NO: 10
  • GGGGS GGGGS SEQ ID NO: 11
  • (GGGGS)m SEQ ID NO: 12
  • (GGGSS)m SEQ ID NO: 13
  • the serine and/or glycine rich linker further comprises at least one leucine (L) residue, such as at least 1 or at least 2 or at least 3 leucine residues, e .g. 1, 2, 3 or 4 leucine residues.
  • the T cell epitope linker comprises or consists of LGGGS (SEQ ID NO: 16), GLGGS (SEQ ID NO: 17), GGLGS (SEQ ID NO: 18), GGGLS (SEQ ID NO: 19) or GGGGL (SEQ ID NO: 20).
  • the T cell epitope linker comprises or consists of LGGSG (SEQ ID NO: 21), GLGSG (SEQ ID NO: 22), GGLSG (SEQ ID NO: 23), GGGLG (SEQ ID NO: 24) or GGGSL (SEQ ID NO: 25).
  • the T cell epitope linker comprises or consists of LGGSS (SEQ ID NO: 26), GLGSS (SEQ ID NO: 27) or GGLSS (SEQ ID NO: 28).
  • the T cell epitope linker comprises or consists of LGLGS (SEQ ID NO: 29), GLGLS (SEQ ID NO: 30), GLLGS (SEQ ID NO: 31), LGGLS (SEQ ID NO: 32) or GLGGL (SEQ ID NO: 33).
  • the T cell epitope linker comprises or consists of LGLSG (SEQ ID NO: 34), GLLSG (SEQ ID NO: 35), GGLSL (SEQ ID NO: 36), GGLLG (SEQ ID NO: 37) or GLGSL (SEQ ID NO: 38).
  • the T cell epitope linker comprises or consists of LGLSS (SEQ ID NO: 39), or GGLLS (SEQ ID NO: 40).
  • the T cell epitope linker is a serine-glycine linker that has a length of 10 amino acids and comprises 1 or 2 leucine residues.
  • the T cell epitope linker comprises or consists of LGGGSGGGGS (SEQ ID NO: 41), GLGGSGGGGS (SEQ ID NO: 42), GGLGSGGGGS (SEQ ID NO: 43), GGGLSGGGGS (SEQ ID NO: 44) or
  • the T cell epitope linker comprises or consists of LGGSGGGGSG (SEQ ID NO: 46), GLGSGGGGSG (SEQ ID NO: 47), GGLSGGGGSG (SEQ ID NO: 48), GGGLGGGGS G (SEQ ID NO: 49) or GGGSLGGGSG (SEQ ID NO: 50).
  • the T cell epitope linker comprises or consists of LGGSSGGGSS (SEQ ID NO: 51), GLGSSGGGSS (SEQ ID NO: 52), GGLSSGGGSS (SEQ ID NO: 53), GGGLSGGGSS (SEQ ID NO: 54) or GGGSLGGGSS (SEQ ID NO: 55).
  • the T cell epitope linker comprises or consists of LGGGSLGGGS (SEQ ID NO: 56), GLGGSGLGGS (SEQ ID NO: 57), GGLGSGGLGS (SEQ ID NO: 58), GGGLSGGGLS (SEQ ID NO: 59) or GGGGLGGGGL (SEQ ID NO: 60).
  • the T cell epitope linker comprises or consists of LGGSGLGGSG (SEQ ID NO: 61), GLGSGGLGSG (SEQ ID NO: 62), GGLSGGGLSG (SEQ ID NO: 63), GGGLGGGGLG (SEQ ID NO: 64) or GGGSLGGGSL (SEQ ID NO: 65).
  • the T cell epitope linker comprises or consists of LGGSSLGGSS (SEQ ID NO: 66), GLGSSGLGSS (SEQ ID NO: 67) or GGLSSGGLSS (SEQ ID NO: 68).
  • the T cell linker comprises or consists of GSGGGA (SEQ ID NO: 69), GSGGGAGSGGGA (SEQ ID NO: 70),
  • the T cell linker comprises or consists of SGGGSSGGGS (SEQ ID NO: 74), GGGGSGGGGS (SEQ ID NO: 75), SSGGGSSGGG (SEQ ID NO: 76), GGSGGGGSGG (SEQ ID NO: 77),
  • the T cell epitope linker comprises or consists of the sequence TQKSLSLSPGKGLGGL (SEQ ID NO: 83). In other embodiments, the T cell epitope linker comprises or consists of the sequence SLSLSPGKGLGGL (SEQ ID NO: 84). In other embodiments the T cell epitope linker comprises or consists of AAY or GPGPG (SEQ ID NO: 109)
  • the T cell epitope linker is a GSAT linker, i.e. a linker comprising one or more glycine, serine, alanine and threonine residues, e.g. a linker comprising or consisting of the sequence
  • GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 87) or a SEG linker, i.e. a linker comprising one or more serine, glutamic acid and glycine residues, e.g. a linker comprising or consisting of the sequence GGS GGGSEGGGSEGGGSEGGGSEGGGSEGGGS GGGS (SEQ ID NO: 88), or the T cell epitope linker is ELKTPLGDTTHT (SEQ ID NO: 85; corresponding to amino acids 94-105 of SEQ ID NO: 1).
  • the T cell epitope linker may be a rigid linker, meaning that it does not allow the two amino acid sequences that it connects to substantially move freely relative to each other.
  • it may be a flexible linker, i.e. a linker that allows the two amino acid sequences that it connects to substantially move freely relative to each other.
  • the T cell linker is a rigid linker. Such rigid linkers may be useful to efficiently separate (larger) antigens and prevent their interferences with each other.
  • the T cell linker comprises or consists of KPEPKPAPAPKP (SEQ ID NO: 95), AEAAAKEAAAKA (SEQ ID NO: 96), (EAAAK)m (SEQ ID NO: 97), PSRLEEELRRRLTEP (SEQ ID NO: 98) or SACYCELS (SEQ ID NO: 99).
  • the T cell linker is a cleavable linker, e.g. a linker which includes one or more recognition sites for endopeptidases, e.g. endopeptidases such as furin, caspases, cathepsins and the like.
  • Cleavable linkers may be introduced to release free functional protein domains (e.g. encoded by larger antigens), which may overcome steric hindrance between such domains or other drawbacks due to interference of such domains, like decreased bioactivity, altered biodistribution.
  • T cell epitope linkers are disclosed in paragraphs [0098]-[0099] and in the recited sequences of WO 2020/176797A1 (in particular SEQ ID NOs: 37 to 65 and SEQ ID NOs: 67 to 76), which is incorporated herein by reference and in paragraphs [0135] to [0139] of US 2019/0022202A1, which is incorporated herein by reference.
  • the first antigenic unit does not comprise a T cell epitope linker.
  • the present constructs comprise, as described herein in detail, one or more T cell epitopes.
  • the T cell epitopes are from a pathogen, for example from a virus or a pathogenic bacteria.
  • the T cell epitopes are known to be immunogenic, e.g. their immunogenicity has been confirmed by appropriate methods and the results have been published, e.g. in a scientific publication.
  • T cell epitopes against infection by SARS-CoV2 in humans can be found in Grifoni et al, 2021 (Cell Host Microbe. 2021 Jul 14; 29(7): 1076-1092). Such T cell epitopes may thus be included in the present constructs to provide protection against SARS-CoV-2 in humans.
  • T cell epitopes Another example of such T cell epitopes is the T cell epitope with the sequence CTELKLSDY (SEQ ID NO: 100) of the nucleoprotein from Influenza A virus, which has been studied for immune reactivity in 39 publications, tested in 54 T cell assays and 34 MHC ligand assays.
  • CTELKLSDY SEQ ID NO: 100
  • NLVPMVATV sequence of the 65 kDa phosphoprotein from human herpesvirus 5 (human cytomegalovirus) which has been studied
  • the T cell epitopes are selected based on their predicted ability to bind to HLA class I/II alleles, i.e. selected in silico on the basis of predictive HLA-binding algorithms. After having identified relevant epitopes, the epitopes are ranked according to their ability or predicted ability to bind to HLA class I/II alleles and the epitopes that are predicted to bind best are selected to be included in the antigenic unit.
  • HLA-binding algorithm Any suitable HLA-binding algorithm may be used, such as one of the following: available software analysis of peptide-MHC binding (IEDB, NetMHCpan and NetMHCIIpan) may be downloaded or used online from the following websites: www.iedb.org/ servi ces . healthtech . dtu . dk/servi ce . php?NetMHCpan-4.0 services. healthtech.dtu.dk/service.php?NetMHCIIpan-3.2
  • Commercially available advanced software to predict optimal sequences for vaccine design are found here: www. oncoimmunity . com/ omictools.com/t-cell-epitopes-category github.com/griffithlab/pVAC-Seq crdd . osdd . net/ raghava / cancertope/help . php www.epivax.com/tag/neoantigen/
  • each T cell epitope is ranked with respect to its predicted binding affinity and/or antigenicity and/or immunogenicity, and the predicted most antigenic or most immunogenic epitopes are selected and preferably optimally arranged in the antigenic unit.
  • conserved T cell epitopes for inclusion into the antigenic unit may be identified by the method below which is explained using a betacoronavirus as an example, but which may be used for the identification of conserved T cell epitopes of other viruses or other pathogens.
  • Second antigenic unit comprising one or more antigens or parts or fragments thereof
  • the construct of the disclosure comprises a second antigenic unit comprising one or more antigens or parts thereof, which is separated from the first antigenic unit comprising the one or more T cell epitopes by the second joint region.
  • the second joint region is designed to allow for correct folding of the antigen such that the conformational B cell epitopes are presented to the immune system.
  • Protein modelling may be used to model 3D structures/conformations of the antigen connected to the flexible unit of the second joint region to determine which length and amino acid sequence promotes correct folding, and to suggest sequences of such flexible units that enable conformationally correct folding.
  • the flexible unit of the second joint region consists of from 5 to 60 amino acids, e.g. from 7 to 55 amino acids or 8 to 50 amino acids or 9 to 45 amino acids or 10 to 40 amino acids or 11 to 35 amino acids or 12 to 30 amino acids or 13 to 20 amino acids.
  • the flexible unit of the second joint region is a serine (S) and/or glycine (G) rich linker, which may comprise one or more leucines, e.g. like the T cell epitopes linkers described above.
  • the flexible unit of the second joint region comprises or consists of the sequence TQKSLSLSPGKGLGGL (SEQ ID NO: 83). In other embodiments, the flexible unit comprises or consists of the sequence SLSLSPGKGLGGL (SEQ ID NO: 84).
  • the flexible unit of the second joint region is a GSAT linker, i.e. a linker comprising one or more glycine, serine, alanine and threonine residues, e.g. a linker comprising or consisting of the sequence GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 87) or a SEG linker, i.e. a linker comprising one or more serine, glutamic acid and glycine residues, e.g. a linker comprising or consisting of the sequence GGS GGGSEGGGSEGGGSEGGGSEGGGSEGGGS GGGS (SEQ ID NO: 88), or ELKTPLGDTTHT (SEQ ID NO: 85).
  • GSAT linker i.e. a linker comprising one or more glycine, serine, alanine and threonine residues
  • the flexible unit of the second joint region may be any of the sequences disclosed for the flexible unit of the joint regions.
  • the one or more antigens or parts or fragments thereof are disease-relevant antigens, i.e. they are comprised (or naturally found) in proteins of the pathogen which causes the disease or is involved in causing it, and which is the target of a vaccine comprising the immunogenic construct of the disclosure which includes such antigens.
  • the one or more antigens or parts or fragments thereof comprise conformational B cell epitopes, but may also comprise linear B cell epitopes and/or T cell epitopes. In contrary to the T cell epitopes in the first antigenic unit, these T cell epitopes are not isolated, but are presented to the immune system in their natural environment, i.e. flanked by the amino acid residues which are present in the antigen.
  • the following paragraph describes an embodiment of the second antigenic unit based on an antigen, but the skilled reader will recognize that it applies correspondingly to a part or a fragment of an antigen.
  • the second antigenic unit comprises one or more antigens of a pathogen, i.e. one antigen of a pathogen or more than one antigen of a pathogen, i.e. multiple antigens of a pathogen.
  • the multiple antigens are of the same pathogen, i.e. comprised in the same or different proteins of the pathogen.
  • the multiple antigens are of multiple different pathogens, i.e. comprised (or naturally found) in proteins of different pathogens.
  • a “different pathogen” may, for example be a different virus or bacterium or a different strain of the same virus or bacterium or it may be the same strain, but comprising one or more mutations.
  • the construct of the disclosure may be for use in a pan-vaccine, e.g. a vaccine targeting different (seasonal) viruses.
  • a pan-vaccine e.g. a vaccine targeting different (seasonal) viruses.
  • the pan-vaccine could target betacoronavirus and influenza or target different strains of e.g. betacoronaviruses or different mutations of the same strain.
  • the second antigenic unit comprises one or more antigens derived from surface proteins of pathogens, e.g. viral surface proteins such as the spike protein from SARS-CoV-2, hemagglutinin of the influenza virus or gpl20 of the HIV virus (human immunodeficiency virus).
  • the second antigenic unit comprises or consist of or more antigens or parts or fragments thereof comprising a Hemagglutinin H1N1 sequence, such as the Hemagglutinin H1N1 sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 141.
  • the second antigenic unit comprises or consist of or more antigens or parts or fragments thereof comprising T cell epitopes as given in SEQ ID NO: 158-160.
  • the antigen is a full-length protein of a pathogen, preferably a full-length surface protein, e.g. a full-length viral surface protein or bacterial surface protein or a full-length surface protein of any other pathogen.
  • the antigen is a full-length bacterial protein which is secreted by the bacterium, e.g. secreted into the cytoplasm of infected subjects.
  • the second antigenic unit comprises more than one antigen, i.e. several antigens, each of which being a full-length protein.
  • the second antigenic unit comprises one part of one antigen.
  • the RBD domain of the spike protein of SARS-CoV-2 or the head or stem domain of hemagglutinin of the influenza virus are examples of parts of an antigen. Sequences of the spike protein and of the RBD domain are available in databases. As an example, in the spike protein of the “Wuhan” strain (NCBI accession number YP 009724390), the RBD sequence is positioned at residues 319 to 542.
  • the second antigenic unit comprises several parts of one antigen. This may enhance the cross-binding of B cell receptors which may lead to elevated antibody responses.
  • the second antigenic unit comprises one part of several antigens, e.g. one part of antigen 1 and one part of antigen 2 and 1 part of antigen 3.
  • the second antigenic unit comprises several parts of several antigens, e.g. 2 parts of antigen 1 and 3 parts of antigen 2.
  • the second antigenic unit comprises or consist of or more antigens or parts or fragments thereof comprising a RBD sequence of spike protein of SARS-CoV-2 of the Wuhan strain, such as the RBD sequence set forth in SEQ ID NO: 104.
  • the second antigenic unit comprises or consist of or more antigens or parts or fragments thereof comprising a hemagglutinin sequence from influenza H1N1, such as the hemagglutinin sequence set forth in SEQ ID NO: 141.
  • the one or more antigens or parts or fragments thereof have retained antigen conformation which may enable induction of antibodies with functional activity, such as neutralizing antibodies.
  • the antigens and parts may be separated by a linker.
  • the linker has similar or the same the properties and/or sequences as the T cell epitope linkers or the flexible units described in this application.
  • the targeting unit first joint region, second joint region, T cell epitopes, antigens and parts thereof are as described herein elsewhere.
  • the antigenic unit may be connected to the first and/or second joint region, by a unit linker.
  • the unit linker is preferably non-immunogenic.
  • the unit linker may comprise a restriction site in order to facilitate the construction of the polynucleotide.
  • the unit linker is GLGGL (SEQ ID NO: 33) or GLSGL (SEQ ID NO: 115).
  • the unit linker comprises or consists of GGGGS (SEQ ID NO: 161), GGGGSGGGGS (SEQ ID NO: 75), (GGGGS)m (SEQ ID NO: 12), EAAAK (SEQ ID NO: 162), (EAAAK)m (SEQ ID NO: 97), (EAAAK)mGS (SEQ ID NO: 163), or (EAAK)mGS (SEQ ID NO: 166), where m is an integer greater than or equal to 1,
  • the construct of the disclosure comprises a targeting unit that targets or is capable of targeting antigen-presenting cells (APCs).
  • targeting unit refers to a unit that delivers the construct of the disclosure, more specifically the polypeptide (multimeric or dimeric protein; encoded by the polynucleotides described herein), to an antigen-presenting cell for MHC class II-restricted presentation to CD4+ T cells or for providing cross presentation to CD8+ T cells by MHC class I restriction.
  • APCs include dendritic cells (DCs) and subsets thereof.
  • the construct of the disclosure attracts DCs, neutrophils and other immune cells.
  • the construct will not only target the antigenic unit comprised therein to specific cells, but in addition facilitate a response- amplifying effect (adjuvant effect) by recruiting specific immune cells to the administration site of a vaccine comprising the construct or a polynucleotide encoding it.
  • adjuvant effect a response- amplifying effect
  • This unique mechanism is of great importance in a clinical setting, since the construct can be administered to a subject in the form of a vaccine, which does not need to comprise any adjuvants, since the construct comprised in the vaccine provides the adjuvant effect.
  • the targeting unit is designed to target the construct of the disclosure to surface molecules expressed on the APCs, such as molecules expressed exclusively on subsets ofDCs.
  • Chemokine receptors include C-C motif chemokine receptor 1 (CCR1), C-C motif chemokine receptor 3 (CCR3), C-C motif chemokine receptor 4 (CCR4),C-C motif chemokine receptor 5 (CCR5), C-C motif chemokine receptor 6 (CCR6), C-C motif chemokine receptor 7 (CCR7), C-C motif chemokine receptor 8 (CCR8) and XCR1.
  • Chemokine receptors include C-C motif chemokine receptor 1 (CCR1), C-C motif chemokine receptor 3 (CCR3), C-C motif chemokine receptor 4 (CCR4),C-C motif chemokine receptor 5 (CCR5), C-C motif chemokine receptor 6 (CCR6), C-C motif chemokine receptor 7 (CCR7), C-C motif chemokine receptor 8 (CCR8) and XCR1.
  • Toll-like receptors include TLR-2, TLR-4 and TLR-5
  • the targeting unit is or comprises a moiety that interacts with these surface molecules.
  • the targeting unit may comprise or consist of an antibody-binding region, such as the antibody variable domains (VL and VH), with specificity for HLA, CD 14, CD40, CLEC9A or Toll- like receptors.
  • the targeting unit comprises or consists of a synthetic or natural ligand. Examples include soluble CD40 ligand, natural ligands like chemokines, such as their human form, e.g.
  • chemokine ligand 5 also called C-C motif ligand 5 (CCL5 or RANEES), macrophage inflammatory protein alpha (CCL3 or MIP-la), and human isoforms hCCL3, hCCL3Ll, hCCL3L2 and hCCL3L3, chemokine ligand 4 (CCL4) and its isoform CCL4L, chemokine ligand 19 (CCL19), chemokine ligand 20 (CCL20), chemokine ligand 21 (CCL21), chemokine motif ligand 1 or 2 (XCL1 or XCL2) and bacterial antigens like for example flagellin.
  • CCL5 or RANEES C-C motif ligand 5
  • MIP-la macrophage inflammatory protein alpha
  • CCL4 chemokine ligand 4
  • CCL4L chemokine ligand 19
  • chemokine ligand 20 CL20
  • the targeting unit has affinity for an MHC class II protein.
  • the targeting unit comprises or consists of the antibody variable domains (VL and VH) with specificity for MHC class II proteins selected from the group consisting of anti-HLA-DP, anti-HLA-DR and anti-pan HLA class II.
  • the targeting unit has affinity for a surface molecule selected from the group consisting of CD14, CD40, TLR-2, TLR-4 and TLR-5.
  • the targeting unit comprises or consist of an antibody-binding region such as the antibody variable domains (VL and VH) with specificity for CD 14, CD40, TLR-2, TLR-4 or TLR-5, such as anti-CD 14, anti-CD40, anti-TLR-2, anti- TLR-4 or anti-TLR-5.
  • the targeting unit comprises or consists of flagellin, which has affinity for TLR-5.
  • the targeting unit comprises or consists of an antibody-binding region with specificity for CLEC9A, such as anti-CLEC9A or variants thereof, such as anti-CLEC9A Fv or the targeting unit comprises or consists of a CLEC9 ligand, e.g. a CLEC9 ligand comprising or consisting of the nucleotide sequence with SEQ ID NO: 105 or an amino acid sequence encoded by said nucleotide sequence.
  • the targeting unit has affinity for a chemokine receptor selected from CCR1, CCR3, CCR4, CCR5, CCR6, CCR7 and CCR8, more preferably from CCR1, CCR3 and CCR5.
  • the targeting unit has affinity for the chemokine receptor CCR7.
  • the targeting unit comprises or consists of CCL19 (e.g. comprising or consisting of a nucleotide sequence of SEQ ID NO: 110) or an amino acid sequence encoded by said nucleotide sequence, or CCL21, such as the human forms of CCL19 or CCL21.
  • the targeting comprises or consists of chemokine human macrophage inflammatory protein alpha (human MIP-la (hMIP-la) variant, also called LD78P or CCL3L1) and its isoforms, including mouse (CCL3 or MIP-la), and human isoforms hCCL3, hCCL3Ll, hCCL3L2 and hCCL3L3, chemokine ligand 4 (CCL4) and its isoform CCL4L, chemokine ligand 19 (CCL19), chemokine ligand 20 (CCL20), chemokine ligand 21 (CCL21), which binds to its cognate receptors, CCR1 and CCR5 expressed on the cell surface of APCs.
  • chemokine human macrophage inflammatory protein alpha human MIP-la (hMIP-la) variant, also called LD78P or CCL3L1
  • CCL3L1 human MIP-la (hMIP-la) variant, also called
  • the targeting unit is an antibody-binding region with specificity for a dendritic cell receptor selected from the group CLEC9A, CD1 lc, CD80, CD86, CD141, CD172a, CDllb, CD103, CD83, CD14, CD206, CD303 and CD85g.
  • the targeting unit comprises or is a ligand chosen from the table below of attracting ligands.
  • the targeting unit comprises or is a ligand chosen from the table below.
  • the targeting unit is capable of targeting a receptor selected from the group consisting of P2Y2, TLR2, TLR8, P2X7, TLR1, TLR11, TLR2, TLR3, TLR4, TLR6, TLR6/2, TLR7 and TLR9.
  • a receptor selected from the group consisting of P2Y2, TLR2, TLR8, P2X7, TLR1, TLR11, TLR2, TLR3, TLR4, TLR6, TLR6/2, TLR7 and TLR9.
  • a receptor selected from the group consisting of P2Y2, TLR2, TLR8, P2X7, TLR1, TLR11, TLR2, TLR3, TLR4, TLR6, TLR6/2, TLR7 and TLR9.
  • such receptors could be targeted by antibody-binding regions with specificity for any of the receptors, an activating mAbs or a molecule such as MyD88.
  • the targeting unit is capable of degrading STAT3.
  • antibody mimetics monobodies are activated upon internalization and will trigger the degradation of STAT3 inside the APCs
  • the binding of the targeting unit to its cognate receptors leads to internalization of the construct into the APC and degradation of the protein into small peptides that are loaded onto MHC molecules and presented to CD4+ and CD8+ T cells to induce specific immune responses.
  • Peptides loaded onto MHC class II molecules can be recognized by antigen-specific CD4+ T helper cells, whereas peptides loaded on MHC class I molecules can be recognized by antigen-specific CD8+ T cells, leading to proliferation and activation of cytotoxic function.
  • Presentation of internalized antigens on MHC II molecules is a process termed cross-presentation. Once stimulated, and with help from activated CD4+ T cells, CD8+ T cells will target and kill cells expressing the same antigens.
  • the targeting unit is MIP-la, preferably hMIP-la.
  • MIP-la attract APCs to the construct through its chemotactic ability, it also causes internalization of the construct through both the classical and cross-presentation pathway, whereby the epitopes are processed by enzymes and presented on the cell surface to raise the T cell response, particularly Thl CD4+ responses and CD8+ T cell responses.
  • MIP-la is also capable of supporting the induction of antibody responses, in particular IgG2a, which is important for protection against infection.
  • the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence 24-93 of SEQ ID NO: 1, such as comprising the amino acid sequence 26-93 of SEQ ID NO: 1 or comprising the amino acid sequence 28-93 of SEQ ID NO: 1.
  • the targeting unit comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence 24-93 of SEQ ID NO: 1 or SEQ ID NO: 112, such as at least 86% or at least 87%, 88%, 89%, 90%,
  • the targeting unit has the amino acid sequence 24-93 of SEQ ID NO: 1 or SEQ ID NO: 112.
  • the targeting unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence 24-93 of SEQ ID NO: 1 or SEQ ID NO: 112.
  • the targeting unit consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence 24-93 of SEQ ID NO: 1 or SEQ ID NO: 112, such as at least 86% or at least 87%, 88%, 89%, 90%,
  • the targeting unit consists of the amino acid sequence 24-93 of SEQ ID NO: 1.
  • the targeting unit comprises the amino acid sequence 24-93 of SEQ ID NO: 1 or SEQ ID NO: 112, except that at the most six amino acids have been substituted, deleted or inserted, such as at the most five amino acids, such as at the most four amino acids, such as at the most three amino acids, such as at the most two amino acids or such as at the most one amino acid.
  • the targeting unit consists of the amino acid sequence 24-93 of SEQ ID NO: 1 or SEQ ID NO: 112, except that at the most six amino acids have been substituted, deleted or inserted, such as at the most five amino acids, such as at the most four amino acids, such as at the most three amino acids, such as at the most two amino acids or such as at the most one amino acid.
  • the targeting unit comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence with SEQ ID NO: 106.
  • the targeting unit comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence with SEQ ID NO: 106, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • the targeting unit has the nucleotide sequence of SEQ ID NO: 106.
  • the targeting unit consists of a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence with SEQ ID NO: 106.
  • the targeting unit consists of a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence 70-277 of SEQ ID NO: 106, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • the targeting unit has the nucleotide sequence of SEQ: ID NO: 106.
  • the targeting unit comprises or is anti-pan HLA class II. This targeting unit induces rapid and strong antibody responses with mixed IgGl and IgG2a antibodies. Moreover, it induces a significant cellular response, i.e. CD4+ responses and CD8+ T cell responses.
  • the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence 20-260 of SEQ ID NO: 5.
  • the targeting unit comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence 20-260 of SEQ ID NO: 5, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • the targeting unit has the amino acid sequence 20-260 of SEQ ID NO: 5.
  • the targeting unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence 20-260 of SEQ ID NO: 5.
  • the targeting unit consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence 20-260 of SEQ ID NO: 5, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • the targeting unit consists of the amino acid sequence 20-260 of SEQ ID NO: 5.
  • the targeting unit comprises the amino acid sequence 20-260 of SEQ ID NO: 5, wherein any one of the amino acids has been substituted, deleted or inserted, with the proviso that no more than 22 amino acids have been so altered, such as no more than 21 amino acids, such as no more than 20 amino acids, such as no more than 19 amino acids, such as no more than 18 amino acids, such as no more than 17 amino acids, such as no more than 16 amino acids, such as no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no more than 5 amino acids, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid.
  • no more than 21 amino acids such as no more
  • the targeting unit consists of the amino acid sequence 20-260 of SEQ ID NO: 5, wherein any one of the amino acids has been substituted, deleted or inserted, with the proviso that no more than 22 amino acids have been so substituted, deleted or inserted, such as no more than 21 amino acids, such as no more than 20 amino acids, such as no more than 19 amino acids, such as no more than 18 amino acids, such as no more than 17 amino acids, such as no more than 16 amino acids, such as no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no more than 5 amino acids, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid.
  • first joint region first antigenic unit, second joint region, second antigenic unit, T cell epitopes, antigens and parts thereof are as described herein elsewhere.
  • the construct of the disclosure is a polynucleotide which further comprises a nucleotide sequence encoding a signal peptide.
  • the signal peptide is either located at the N-terminal end of the targeting unit or the C-terminal end of the targeting unit, depending on the orientation of the targeting unit in the polypeptide (Fig. 1).
  • the signal peptide is designed to allow secretion of the polypeptide encoded by the nucleotide comprised in the polynucleotide in the cells transfected with said polynucleotide. Any suitable signal peptide may be used.
  • Suitable peptides are an Ig VH signal peptide, a human TPA signal peptide, such as SEQ ID NO: 6 and a human MIPl-a signal peptide (corresponding to amino acids 24-93 of SEQ ID NO: 1).
  • the polynucleotide comprises a nucleotide sequence encoding a human MIPl-a signal peptide (such as SEQ ID NO: 107) and preferably comprises a nucleotide sequence encoding a human MIPl-a targeting unit (such as SEQ ID NO: 106).
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide that comprises an amino acid sequence having at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99%, sequence identity to the amino acid sequence SEQ ID NO: 111 or 1-23 of SEQ ID NO: 1.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide that comprises the amino acid sequence SEQ ID NO: 111 or 1-23 of SEQ ID NO: 1.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide that consists of an amino acid sequence having at least 80%, preferably at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% to the amino acid sequence SEQ ID NO: 111 or 1-23 of SEQ ID NO: 1.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide that comprises the amino acid sequence SEQ ID NO: 111 or 1-23 of SEQ ID NO: 1, except that at the most four amino acids have been substituted, deleted or inserted, such as at the most three amino acids, such as at the most two amino acids or such as at the most one amino acid.
  • the polynucleotide which comprises a nucleotide sequence encoding a signal peptide with the amino acid sequence SEQ ID NO: 111 or 1-23 of SEQ ID NO: 1.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide, wherein said nucleotide sequence has at least 80% sequence identity to the nucleotide sequence with SEQ ID NO: 107.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide, wherein said nucleotide sequence has at least 85% sequence identity to the nucleotide sequence with SEQ ID NO: 107, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide, wherein said nucleotide sequence is SEQ ID NO: 107.
  • the polynucleotide comprises a nucleotide sequence encoding an Ig VH signal peptide (corresponding to amino acids 1-19 of SEQ ID NO: 5) and preferably comprises a nucleotide sequence encoding an anti-pan HLA class II targeting unit.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide that comprises an amino acid sequence having at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99%, sequence identity to the amino acid sequence 1-19 of SEQ ID NO: 5.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide that comprises the amino acid sequence 1-19 of SEQ ID NO: 5, except that at the most four amino acids have been substituted, deleted or inserted, such as at the most three amino acids, such as at the most two amino acids or such as at the most one amino acid.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide that comprises the amino acid sequence 1-19 of SEQ ID NO: 5.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide that consists of an amino acid sequence having at least 80%, preferably at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% to the amino acid sequence 1-19 of SEQ ID NO: 5.
  • the polynucleotide which comprises a nucleotide sequence encoding a signal peptide with the amino acid sequence 1-19 of SEQ ID NO: 5.
  • Sequence identity may be determined as follows: A high level of sequence identity indicates likelihood that a second sequence is derived from a first sequence. Amino acid sequence identity requires identical amino acid sequences between two aligned sequences. Thus, a candidate sequence sharing 70% amino acid identity with a reference sequence requires that, following alignment, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity may be determined by aid of computer analysis, such as, without limitations, the ClustalW computer alignment program (Higgins D., Thompson I, Gibson T., Thompson J.D., Higgins D.G., Gibson T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res.
  • the ClustalW algorithm may similarly be used to align nucleotide sequences. Sequence identities may be calculated in a similar way as indicated for amino acid sequences.
  • AlignO does not penalize to gaps in the end of the sequences.
  • a BLOSUM50 substitution matrix with gap opening/extension penalties of-12/-2 is preferably used.
  • Amino acid sequence variants may be prepared by introducing appropriate changes into the nucleotide sequence encoding anticancer vaccine, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences.
  • the terms substituted/substitution, deleted/deletions and inserted/insertions as used herein in reference to amino acid sequences and sequence identities are well known and clear to the skilled person in the art. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. For example, deletions, insertions or substitutions of amino acid residues may produce a silent change and result in a functionally equivalent peptide/polypeptide.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • conservative substitutions i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • non conservative substitutions i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine, diaminobutyric acid ornithine, norleucine, ornithine, pyriylalanine, thienylalanine, naphthyl alanine and phenylglycine.
  • Conservative substitutions that may be made are, for example within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), aliphatic amino acids (alanine, aaline, leucine, isoleucine), polar amino acids (glutamine, asparagine, serine, threonine), aromatic amino acids (phenylalanine, tryptophan, tyrosine), hydroxyl amino acids (serine, threonine), large amino acids (phenylalanine, tryptophan) and small amino acids (glycine, alanine).
  • substitutions may also be made by unnatural amino acids and substituting residues include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-CI- phenylalanine*, p-Br-phenylalanine*, p-I- phenylalanine*, L-allyl-glycine*, b- alanine*, L-a-amino butyric acid*, L-y-amino butyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid*, 7-amino heptanoic acid*, L- methionine sulfone*, L- norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L- hydroxyproline*, L- thioproline
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or b-alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or b-alanine residues.
  • a further form of variation involves the presence of one or more amino acid residues in peptoid form.
  • the construct of the disclosure may be in the form of a polynucleotide.
  • a further embodiment of the disclosure is a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof.
  • the polynucleotide may be a DNA or an RNA, including genomic DNA, cDNA, and mRNA, either double-stranded or single-stranded.
  • the construct is a DNA plasmid, i.e. the polynucleotide is a DNA.
  • polynucleotide is optimized for use in the species to which it is administered.
  • polynucleotide sequence is human codon optimized.
  • the construct of the disclosure may be in the form of a polypeptide encoded by the nucleotide sequence comprised in the polynucleotide as described above.
  • a further embodiment of the disclosure is a polypeptide, comprising in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof.
  • the polypeptide may be expressed in vitro for production of e.g. a vaccine, or the polypeptide may be expressed in vivo as a result of the administration of the polynucleotide to a subject, as described above.
  • multimeric proteins or dimeric proteins are formed when the polypeptide is expressed, i.e. by joining multiple polypeptides, such as two polypeptides, via their joint regions.
  • a further embodiment of the disclosure is a multimeric protein consisting of multiple polypeptides, each of which comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof, and wherein the multiple polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions.
  • a further embodiment of the disclosure is a dimeric protein consisting of two polypeptides, each of which comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof, and wherein the two polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions.
  • the dimeric protein may be a homodimer, i.e.
  • the dimeric protein may be a heterodimer comprising two polypeptide chains, wherein polypeptide chain 1 comprises different antigen sequences in its antigenic unit than polypeptide 2.
  • the latter may be relevant if the number of antigens for inclusion into the antigenic unit would exceed an upper size limit for the antigenic unit. It is preferred that the dimeric protein is a homodimeric protein.
  • the multimeric protein or dimeric protein may be prepared by expression of the polypeptide in vitro.
  • a further embodiment of the disclosure is a method for preparing a multimeric protein consisting of multiple polypeptides; each of which comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen- presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof, and wherein the multiple polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions, wherein the method comprises the following steps: a) transfecting or transducing cells with a polynucleotide comprising a nucleotide sequence encoding the polypeptide; b) culturing the cells; c) collecting the multimeric protein from the cells; and d) isolating and optionally purifying the fraction of the multimeric proteins, wherein the multiple polypeptides are linked to each other
  • Isolation of the multimeric protein in step d) and the optional purification can be carried out by methods known in the art, including precipitation, differential solubilization and chromatography.
  • the above-described multimeric protein of the disclosure may be used as the active ingredient in a protein vaccine for the prophylactic or therapeutic treatment of infectious diseases.
  • a further embodiment of the disclosure is a method for preparing a dimeric protein consisting of two polypeptides; each of which comprising, in the specified order, a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof, and wherein the two polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions, wherein the method comprises the following steps: a) transfecting or transducing cells with a polynucleotide comprising a nucleotide sequence encoding the polypeptide; b) culturing the cells; c) collecting the dimeric protein from the cells; and d) isolating and optionally purifying the fraction of the dimeric proteins, wherein the two polypeptides are linked to each other via
  • the polynucleotide may be comprised in a plasmid for transfection or a vector for transduction.
  • Isolation of the dimeric protein in step d) and the optional purification can be carried out by methods known in the art, including precipitation, differential solubilization and chromatography.
  • the above-described dimeric protein of the disclosure may be used as the active ingredient in a protein vaccine for the prophylactic or therapeutic treatment of infectious diseases.
  • Vectors
  • the polynucleotide sequence of the construct may be a DNA polynucleotide comprised in a vector suitable for transfecting or transducing a host cell and expression of a polypeptide or multimeric/dimeric protein encoded by the polynucleotide, i.e. an expression vector.
  • the vector is a DNA plasmid.
  • the vector is a viral vector, such as a retroviral vector.
  • the vector is suitable for transfecting a host cell and expression of a mRNA encoding for the polypeptide/multimeric/dimeric protein.
  • a further embodiment of the disclosure is a vector comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and d) a second antigenic unit comprising one or more antigens or parts or fragments thereof.
  • the vector is a DNA plasmid and the polynucleotide is DNA.
  • the above-described vector is a polycistronic vector that allows the expression of the polypeptide of the disclosure and, in addition, the expression of one or more immunostimulatory compounds as separate molecules.
  • A a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order, a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; and
  • the one or more immunomodulatory compounds may enhance the effect of the construct of the disclosure.
  • the co expression may have marked advantages on the cellular level.
  • the immunostimulatory compound is expressed in and secreted from the same muscle cell, it can stimulate the same antigen-presenting cell and thereby directly affect said antigen-presenting cell, e.g. if the antigen-presenting cell is a dendritic cell, promote the activation and maturation of it.
  • the polycistronic vector of the disclosure may be any suitable vector, e.g. a DNA plasmid or viral vector, such as a retroviral vector.
  • the vector is a polycistronic DNA plasmid.
  • the polycistronic vector of the disclosure will be illustrated discussing a DNA plasmid (i.e. a polycistronic DNA plasmid of the disclosure), but it is understood that the discussion thereof applies also to other vectors, e.g. viral vectors.
  • Polycistronic plasmids are known in the art, hence, the skilled person is able to design and construct the polycistronic plasmid of the disclosure.
  • the polycistronic plasmid of the disclosure comprises one or more co-expression elements, i.e. nucleotide sequences which allow co-expression of the polypeptide and the one or more immunostimulatory compounds from the plasmid as separate molecules.
  • the polycistronic plasmid comprises a co-expression element, which causes that the polypeptide and the one or more immunostimulatory compounds are transcribed on a single transcript but independently translated into the polypeptide and the one or more immunostimulatory compounds. Hence, the presence of the co-expression element results in a final production of separate translation products.
  • such co-expression element is an IRES element (internal ribosome entry site). In other embodiments, such co-expression element is a 2A self- cleaving peptide (2A peptide). Both co-expression elements are known in the art.
  • an immunostimulatory compound is expressed from the polycistronic plasmid of the disclosure, an IRES element and/or 2A peptide needs to be present in plasmid, e.g. upstream of each nucleotide sequence encoding an immunostimulatory compound.
  • the polycistronic plasmid comprises a co-expression element which causes that the polypeptide and the one or more immunostimulatory compounds are transcribed as separate transcripts, which results in separate transcription products and thus separate proteins.
  • such co-expression element is a bidirectional promoter.
  • co-expression elements are various promoters, i.e. the polycistronic plasmid comprises a promoter for each of the nucleotide sequences encoding either the polypeptide or the one or more immunostimulatory compounds. Both co-expression elements are known in the art.
  • co-expression elements can be combined in any manner, i.e. the polycistronic plasmid of the disclosure may comprise one or several of such same or different co-expression elements.
  • Immunostimulatory compounds i.e. the polycistronic plasmid of the disclosure may comprise one or several of such same or different co-expression elements.
  • the polycistronic plasmid of the present disclosure comprises one or more nucleotide sequences encoding one or more immunostimulatory compounds.
  • the immunostimulatory compound is a compound that stimulates antigen-presenting cells and the stimulation results in e.g. attraction, activation, maturation and/or proliferation of APCs.
  • the immunostimulatory compound is one that attracts APCs, preferably one that can interact with the following surface molecules on APCs: CCR1 (C-C motif chemokine receptor 1), CCR3 (C-C motif chemokine receptor 3), CCR4 (C-C motif chemokine receptor 4), CCR5 (C-C motif chemokine receptor 5), CCR6 (C-C motif chemokine receptor 6), CCR7 (C motif chemokine receptor 7), CCR8 (C motif chemokine receptor 8) or XCR1 (X-C motif chemokine receptor 1).
  • CCR1 C-C motif chemokine receptor 1
  • CCR3 C-C motif chemokine receptor 3
  • CCR4 C-C motif chemokine receptor 4
  • CCR5 C-C motif chemokine receptor 5
  • CCR6 C-C motif chemokine receptor 6
  • CCR7 C motif chemokine receptor 7
  • CCR8 C motif chemokine receptor 8
  • the immunostimulatory compound is selected from the list consisting of MIP-la, preferably human MIP-la (h MIP-la or LD78P), RANTES (CCL5), MIP-Ib (CCL4), MIP-3a (CCL20), CCL19, CCL 21, XCL1 or XCL2.
  • MIP-la preferably human MIP-la (h MIP-la or LD78P), RANTES (CCL5), MIP-Ib (CCL4), MIP-3a (CCL20), CCL19, CCL 21, XCL1 or XCL2.
  • the immunostimulatory compound is one that promotes activation and/or maturation of APCs.
  • the immunostimulatory compound can interact with the following surface molecules on APCs: a receptor of the TNF receptor superfamily, including CD40 (cluster of differentiation 40), CD 137 (4- IBB), CD27, ICOSL (CD275) or RANK.
  • Such immunostimulatory compounds may be selected from the list consisting of CD40L (CD40 ligand, CD154), CD137L (4-1BBL, 4-1BB ligand), CD70, ICOS (CD278) or RANKL.
  • the immunostimulatory compound is a cytokine selected from IL-2, IL-10, IL-12, TNFa and IFNy.
  • the immunostimulatory compound can be an immune signaling molecule such as MyD88 and TRIF which activate through TLR receptors.
  • the immunostimulatory compound can be a viral infection sensor such as for example RIG-1 and MDA-5.
  • the immunostimulatory compound can interact with a pattern recognition receptor on APCs, e.g. a Toll-like receptor, including TLR2, TLR4 or TLR5.
  • Such immunostimulatory compounds may be selected from the list consisting of pathogen-associated molecular patterns (PAMPs), such as flagellin, or protein damage-associated molecular patterns (DAMPs), such as HMGB1, HSPs (heat-shock proteins) Calrecticulin and Annexin Al.
  • PAMPs/DAMPs include those can be included as a nucleotide sequence into the DNA plasmid of the disclosure and will be expressed as functional proteins that may comprise functional groups introduced by post-translational modifications.
  • the aforementioned molecules in turn activate the following receptors on APCs: RAGE, TLR4, TLR9 and TIM-3 (for HMGB1), FPR (for Annexin Al), SREC1, LOX1 and CD91 (for HSP).
  • the immunostimulatory compound is one that promotes growth and/or expansion of APCs.
  • the immunostimulatory compound can interact with the following surface molecules on APCs: GM-CSF- receptor (granulocyte-macrophage colony-stimulating factor receptor, CD116), FLT- 3R (fms like tyrosine kinase 3, CD 135), IL-15R or IL-4R.
  • the immunostimulatory compound is a growth factor, such as GM-CSF (granulocyte- macrophage colony- stimulating factor), FLT-3L, IL-15 or IL-4.
  • the polycistronic DNA plasmid comprises nucleotide sequences encoding 2, 3, 4, 5, 6, 7 or 8 immunostimulatory compounds.
  • the DNA plasmid comprises nucleotide sequences encoding 2 to 6 immunostimulatory compounds, i.e. 2 or 3 or 4 or 5 or 6 different immunostimulatory compounds.
  • the immunostimulatory compounds may be the same or different, preferably different.
  • the different immunostimulatory compounds also affect APCs differently, to stimulate the immune system on many different levels and by that maximize the therapeutic or prophylactic effect of the construct of the disclosure.
  • the polycistronic DNA plasmid comprises nucleotides encoding 3 different immunostimulatory compounds, with the first one being an immunostimulatory compound that promotes the attraction of DCs (e.g. XCL1), the second one being an immunostimulatory compound that promotes the growth of DCs (e.g. FLT-3L) and the third one being an immunostimulatory compound that promotes activation of DCs (e.g. CD40L).
  • the selection of the particular immunostimulatory compounds will also depend on the targeting unit, since it targets APCs and may also affect APCs in a similar manner as the immunostimulatory compound, i.e. attract or activate APCs.
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the vector may be the polycistronic vector described herein .
  • suitable host cells include prokaryotes, yeast, insect or higher eukaryotic cells.
  • the host cell is a human cell, preferably a cell of a human individual in need of the vaccine of the disclosure.
  • compositions comprising a construct, polynucleotide, polypeptide, multimeric protein or dimeric protein as disclosed herein, and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, and/or diluents.
  • Suitable pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, such as PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffers, and combinations thereof.
  • the pharmaceutical composition may further comprise an adjuvant.
  • compositions comprising the multimeric protein or dimeric protein
  • pharmaceutically acceptable adjuvants include, but are not limited to poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS 15, BCG, CP-870,893,
  • composition can be administered without additional adjuvant; thus, in some embodiments, the composition does not comprise an adjuvant
  • composition may comprise a pharmaceutically acceptable amphiphilic block co- polymer comprising blocks of poly(ethylene oxide) and polypropylene oxide).
  • an “amphiphilic block co-polymer” as used herein is a linear or branched co polymer comprising or consisting of blocks of poly(ethylene oxide) (“PEO”) and blocks of polypropylene oxide) (“PPO”).
  • PEO poly(ethylene oxide)
  • PPO polypropylene oxide
  • Typical examples of useful PEO-PPO amphiphilic block co-polymers have the general structures PEO-PPO-PEO (poloxamers), PPO PEO PPO, (PEO PPO-)4ED (a poloxamine), and (PPO PEO-)4ED (a reverse poloxamine), where "ED” is a ethylenediaminyl group.
  • a “poloxamer” is a linear amphiphilic block co-polymer constituted by one block of poly(ethylene oxide) coupled to one block of polypropylene oxide) coupled to one block of PEO, i.e. a structure of the formula EOa-POb-EOa, where EO is ethylene oxide, PO is propylene oxide, a is an integer from 2 to 130, and b is an integer from 15 to 67.
  • Poloxamers are conventionally named by using a 3-digit identifier, where the first 2 digits multiplied by 100 provides the approximate molecular mass of the PPO content, and where the last digit multiplied by 10 indicates the approximate percentage of PEO content.
  • Polyxamer 188 refers to a polymer comprising a PPO block of a molecular weight of about 1800 (corresponding to b being about 31 PPO) and approximately 80% (w/w) of PEO (corresponding to a being about 82).
  • the values are known to vary to some degree, and commercial products such as the research grade Lutrol® F68 and the clinical grade Kolliphor®
  • PI 88 which according to the producer's data sheets both are Poloxamer 188, exhibit a large variation in molecular weight (between 7,680 and 9,510) and the values for a and b provided for these particular products are indicated to be approximately 79 and 28, respectively. This reflects the heterogeneous nature of the block co-polymers, meaning that the values of a and b are averages found in a final formulation.
  • a “poloxamine” or “sequential poloxamine” (commercially available under the trade name of Tetronic®) is an X-shaped block co-polymers that bears four PEO-PPO arms connected to a central ethylenediamine moiety via bonds between the free OH groups comprised in the PEO-PPO-arms and the primary amine groups in ethylenediamine moiety.
  • Reverse poloxamines are likewise X- shaped block co polymers that bear four PPO-PEO arms connected to a central ethylenediamine moiety via bonds between the free OH groups comprised in the PPO-PEO arms and the primary amine groups in ethylenediamine.
  • Preferred amphiphilic block co-polymers are poloxamers or poloxamines. Preferred are poloxamer 407 and 188, in particular poloxamer 188. Preferred poloxamines are sequential poloxamines of formula (PEO-PPO)4-ED. Particularly preferred poloxamines are those marketed under the registered trademarks Tetronic® 904, 704, and 304, respectively. The characteristics of these poloxamines are as follows: Tetronic® 904 has a total average molecular weight of 6700, a total average weight of PPO units of 4020, and a PEO percentage of about 40%.
  • Tetronic® 704 has a total average molecular weight of 5500, a total average weight of PPO units of 3300, and a PEO percentage of about 40%; and Tetronic® 304 has a total average molecular weight of 1650, a total average weight of PPO units of 990, and a PEO percentage of about 40%.
  • the composition comprises the amphiphilic block co polymer in an amount of from 0.2% w/v to 20% w/v, such as of from 0.2% w/v to 18% w/v, 0.2% w/v to 16% w/v, 0.2% w/v to 14% w/v, 0.2% w/v to 12% w/v, 0.2% w/v to 10% w/v, 0.2% w/v to 8% w/v, 0.2% w/v to 6% w/v, 0.2% w/v to 4% w/v, 0.4% w/v to 18% w/v, 0.6% w/v to 18% w/v, 0.8% w/v to 18% w/v, 1% w/v to 18% w/v, 2% w/v to 18% w/v, 1% w/v to 5% w/v, or 2% w/v to 4% w/v.
  • the composition comprises the amphiphilic block co- polymer in an amount of from 2% w/v to 5% w/v, such as about 3% w/v.
  • compositions comprising the polynucleotide or vector
  • the pharmaceutical compositions may comprise molecules that ease transfection of cells.
  • the pharmaceutical composition may be formulated in any way suitable for administration to a subject, e.g. such as a liquid formulation for injection, e.g. for intradermal or intramuscular injection.
  • the pharmaceutical composition may be administered in any way suitable for administration to a subject, such as administered by intradermal, intramuscular, or subcutaneous injection, or by mucosal or epithelial application, such as intranasal or oral.
  • the pharmaceutical composition comprises a polynucleotide, e.g. comprised in a vector such as a polycistronic vector, and is administered by intramuscular or intradermal injection.
  • the pharmaceutical composition of the disclosure typically comprises the polynucleotide in a range of from 0.1 to 10 mg, e.g. about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mg or e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg.
  • the pharmaceutical composition of the disclosure typically comprises the polypeptide, dimeric protein and/or multimeric protein in the range of from 5 pg to 5 mg.
  • the amount of polynucleotide, polypeptide, multimeric protein or dimeric protein may vary depending on whether the pharmaceutical composition is administered for prophylactic or therapeutic treatment, the severity of the disease in individuals which are infected, and on parameters like the age, weight, gender, medical history and pre-existing conditions.
  • the construct of the disclosure may be administered to a subject as a vaccine, i.e. a pharmaceutical composition comprising the construct, e.g. the form of a polynucleotide, vector, polypeptide, multimeric protein or dimeric protein and a pharmaceutically acceptable carrier.
  • a vaccine i.e. a pharmaceutical composition comprising the construct, e.g. the form of a polynucleotide, vector, polypeptide, multimeric protein or dimeric protein and a pharmaceutically acceptable carrier.
  • a further embodiment of the disclosure is a vaccine, comprising a pharmaceutically acceptable carrier and
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a multimeric protein such as a dimeric protein, consisting of multiple polypeptides as defined in (ii), such as two polypeptides.
  • a further embodiment of the disclosure is a vaccine, comprising a pharmaceutically acceptable carrier and
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a further embodiment of the disclosure is a vaccine, comprising a pharmaceutically acceptable carrier and
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • Suitable pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, such as PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffers, and combinations thereof.
  • the pharmaceutically acceptable carrier or diluent is an aqueous buffer.
  • the aqueous buffer is Tyrode's buffer, e.g. Tyrode’s buffer comprising 140 mM NaCl, 6 mM KC1, 3 mM CaC12, 2 mM MgC12, 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes) pH 7.4, and 10 mM glucose.
  • the vaccine may further comprise an adjuvant.
  • pharmaceutically acceptable adjuvants include, but are not limited to poly-ICLC, 1018 IS S, aluminum salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFactEVl P321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys
  • the present vaccines can be administered without additional adjuvant; thus, in some embodiments, the vaccine does not comprise an adjuvant, i.e. is adjuvant-free.
  • the vaccine may comprise a pharmaceutically acceptable amphiphilic block co- polymer comprising blocks of poly(ethylene oxide) and polypropylene oxide).
  • an “amphiphilic block co-polymer” as used herein is a linear or branched co polymer comprising or consisting of blocks of poly(ethylene oxide) (“PEO”) and blocks of polypropylene oxide) (“PPO”).
  • PEO poly(ethylene oxide)
  • PPO polypropylene oxide
  • Typical examples of useful PEO-PPO amphiphilic block co-polymers have the general structures PEO-PPO-PEO (poloxamers), PPO PEO PPO, (PEO PPO-)4ED (a poloxamine), and (PPO PEO-)4ED (a reverse poloxamine), where "ED” is a ethylenediaminyl group.
  • a “poloxamer” is a linear amphiphilic block co-polymer constituted by one block of poly(ethylene oxide) coupled to one block of polypropylene oxide) coupled to one block of PEO, i.e. a structure of the formula EOa-POb-EOa, where EO is ethylene oxide, PO is propylene oxide, a is an integer from 2 to 130, and b is an integer from 15 to 67.
  • Poloxamers are conventionally named by using a 3-digit identifier, where the first 2 digits multiplied by 100 provides the approximate molecular mass of the PPO content, and where the last digit multiplied by 10 indicates the approximate percentage of PEO content.
  • Polyxamer 188 refers to a polymer comprising a PPO block of a molecular weight of about 1800 (corresponding to b being about 31 PPO) and approximately 80% (w/w) of PEO (corresponding to a being about 82).
  • the values are known to vary to some degree, and commercial products such as the research grade Lutrol® F68 and the clinical grade Kolliphor®
  • PI 88 which according to the producer's data sheets both are Poloxamer 188, exhibit a large variation in molecular weight (between 7,680 and 9,510) and the values for a and b provided for these particular products are indicated to be approximately 79 and 28, respectively. This reflects the heterogeneous nature of the block co-polymers, meaning that the values of a and b are averages found in a final formulation.
  • a “poloxamine” or “sequential poloxamine” (commercially available under the trade name of Tetronic®) is an X-shaped block co-polymers that bears four PEO-PPO arms connected to a central ethylenediamine moiety via bonds between the free OH groups comprised in the PEO-PPO-arms and the primary amine groups in ethylenediamine moiety.
  • Reverse poloxamines are likewise X- shaped block co polymers that bear four PPO-PEO arms connected to a central ethylenediamine moiety via bonds between the free OH groups comprised in the PPO-PEO arms and the primary amine groups in ethylenediamine.
  • Useful amphiphilic block co-polymers are poloxamers or poloxamines, more particularly poloxamer 407 and 188, and in particular poloxamer 188.
  • Useful poloxamines are sequential poloxamines of formula (PEO-PPO)4-ED; in particular, preferred poloxamines are those marketed under the registered trademarks Tetronic® 904, 704, and 304, respectively.
  • the characteristics of these poloxamines are as follows: Tetronic® 904 has a total average molecular weight of 6700, a total average weight of PPO units of 4020, and a PEO percentage of about 40%.
  • Tetronic® 704 has a total average molecular weight of 5500, a total average weight of PPO units of 3300, and a PEO percentage of about 40%; and Tetronic® 304 has a total average molecular weight of 1650, a total average weight of PPO units of 990, and a PEO percentage of about 40%.
  • the vaccine comprises the amphiphilic block co polymer in an amount of from 0.2% w/v to 20% w/v, such as of from 0.2% w/v to 18% w/v, 0.2% w/v to 16% w/v, 0.2% w/v to 14% w/v, 0.2% w/v to 12% w/v, 0.2% w/v to 10% w/v, 0.2% w/v to 8% w/v, 0.2% w/v to 6% w/v, 0.2% w/v to 4% w/v, 0.4% w/v to 18% w/v, 0.6% w/v to 18% w/v, 0.8% w/v to 18% w/v, 1% w/v to 18% w/v, 2% w/v to 18% w/v, 1% w/v to 5% w/v, or 2% w/v to 4% w/v.
  • the vaccine comprises the amphiphilic block co- polymer in an amount of from 2% w/v to 5% w/v, such as about 3% w/v.
  • the vaccines may further comprise molecules that ease transfection of cells.
  • the vaccine may be formulated in any way suitable for administration to a subject, e.g. a patient suffering or suspected of suffering from an infection caused by a pathogen, for intradermal or intramuscular injection.
  • the vaccine comprising in some embodiments a polynucleotide as described herein, may be administered in any way suitable for administration to a subject, such as administered by intradermal, intramuscular, or subcutaneous injection, or by mucosal or epithelial application, such as intranasal or oral administration.
  • the vaccine comprises a polynucleotide as described herein, e.g. comprised in a vector such as the polycistronic vector, and is administered by intramuscular or intradermal injection.
  • the vaccine of the disclosure typically comprises the polynucleotide in a range of from 0.1 pg to 10 mg, e.g. about 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.75 pg, 1 pg, 5 pg, 10 pg, 25 pg, 50 pg, 75 pg, or more; such as from 0.1 to 10 mg, e.g. about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mg or e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg.
  • the vaccine of the disclosure typically comprises the polypeptide, dimeric protein and/or multimeric protein in the range of from 5 pg to 5 mg.
  • the amount of polynucleotide, polypeptide, dimeric protein and/or multimeric protein may vary depending on whether the vaccine is administered for prophylactic or therapeutic treatment, the severity of the disease in individuals which are infected, and on parameters like the age, weight, gender, medical history and pre-existing conditions.
  • Suitable methods for preparing the vaccine according to the disclosure are disclosed in WO 2004/076489 Al, WO 2011/161244A1, WO 2013/092875A1, WO 2017/118695 A1 and WO 2021/205027 Al, which are incorporated herein by reference, in particular page 15, lines 10-13 and page 17, section “Construction of Vaccibodies” of WO 2004/076489A1; page 10, lines 10-14 and Example 1 of WO 2011/161244A1; page 15 and Example 1 of WO 2013/092875A1; section “Methods for preparing the vaccine” and Example 1 of WO 2017/118695A1 and page 26, line 17 to page 27, line 38, page 30, line 23 to page 31, line 27 and Example 4 of WO 2021/205027 Al, and include preparing the polynucleotide, vector, polycistronic vector, multimeric protein or dimeric protein by the methods described herein and mixing them with a pharmaceutically acceptable carrier.
  • the disclosure relates to a method for preparing a vaccine comprising the multimeric protein, such as the dimeric protein, or the polypeptide as defined above by producing the polypeptides in vitro.
  • the in vitro synthesis of the polypeptides and proteins may be carried out by any suitable method known to the person skilled in the art, such as by peptide synthesis or expression of the polypeptide in a variety of expressions systems followed by purification.
  • the aforementioned compounds are dissolved in said pharmaceutically acceptable carrier.
  • Also disclosed herein is a method for preparing a vaccine which comprises a construct of the disclosure, wherein the method comprises: a) transfecting cells with a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; b) culturing the cells; c) collecting and purifying the polypeptide, dimeric protein or multimeric protein expressed from the cells; and d) mixing the polypeptide, dimeric protein or multimeric protein obtained from step c) with a pharmaceutically acceptable carrier.
  • the polypeptide, dimeric protein or multimeric protein obtained from step c) is dissolved in said pharmaceutically acceptable carrier.
  • the polynucleotide may be prepared by any suitable method known to the skilled person.
  • the polynucleotide may be prepared by chemical synthesis using an oligonucleotide synthesizer.
  • the disclosure relates to the use of the construct, polynucleotide, vector, polypeptide, multimeric protein or dimeric protein described above as a medicament.
  • the present disclosure further relates to the construct, polynucleotide, vector, polypeptide, multimeric protein, dimeric protein, pharmaceutical composition or vaccine for use as a medicament.
  • the construct, pharmaceutical composition and/or vaccine of the disclosure may be used to treat any infectious disease caused by any pathogen, including diseases caused by viruses (such as betacoronaviruses or influenza viruses or HIV), bacteria, fungi or parasites, and treatment may either be for prophylactic or for therapeutic purpose.
  • the construct, pharmaceutical composition and/or vaccine is administered such that it induces an immunoprotective response (for a prophylactic treatment) or an immunotherapeutic response (for a therapeutic treatment) in the individual vaccinated with such vaccine.
  • Such response is induced by either a single vaccination or several vaccinations, e.g. an initial vaccination and one or several booster vaccinations, adequately spaced in time.
  • the disclosure provides a method for treating a subject having an infectious disease or being in need of prevention of an infectious disease, the method comprising administering to the subject a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a method for treating a subject having an infectious disease or being in need of prevention of an infectious disease, the method comprising administering to the subject a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a method for treating a subject having an infectious disease or being in need of prevention of an infectious disease, the method comprising administering to the subject a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a vaccine for use in the prophylactic or therapeutic treatment of an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a vaccine for use in the prophylactic or therapeutic treatment of an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a vaccine for use in the prophylactic or therapeutic treatment of an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a vaccine for use in the prophylactic or therapeutic treatment of an infectious disease in a subject, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a vaccine for use in the prophylactic or therapeutic treatment of an infectious disease in a subject, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a vaccine for use in the prophylactic or therapeutic treatment of an infectious disease in a subject, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides the use of a vaccine for the prophylactic or therapeutic treatment of an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides the use of a vaccine for the prophylactic or therapeutic treatment of an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides the use of a vaccine for the prophylactic or therapeutic treatment of an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides the use of a vaccine for the manufacture of a medicament for the prophylactic or therapeutic treatment of an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides the use of a vaccine for the manufacture of a medicament for the prophylactic or therapeutic treatment of an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides the use of a vaccine for the manufacture of a medicament for the prophylactic or therapeutic treatment of an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides the use of a vaccine for prophylactically or therapeutically treating a subject having an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides the use of a vaccine for prophylactically or therapeutically treating a subject having an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides the use of a vaccine for prophylactically or therapeutically treating a subject having an infectious disease, the vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a medicament for the prophylactic or therapeutic treatment of an infectious disease wherein the medicament comprises a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a medicament for the prophylactic or therapeutic treatment of an infectious disease wherein the medicament comprises a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a medicament for the prophylactic or therapeutic treatment of an infectious disease wherein the medicament comprises a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or (ii) a polypeptide encoded by the nucleotide sequence defined in (i); or
  • a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • the disclosure provides a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a medicament for the treatment of an infectious disease in a subject suffering from an infectious disease by administering to the subject a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a medicament for the treatment of an infectious disease in a subject suffering from an infectious disease by administering to the subject a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a medicament for the treatment of an infectious disease in a subject suffering from an infectious disease by administering to the subject a vaccine comprising a pharmaceutically acceptable carrier and:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen-presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • Example 1 Design and production of immunogenic DNA constructs of the disclosure for use as a vaccine against infection with SARS-CoV-2
  • Table 2 The amino acid sequence of various predicted T cell epitopes encoded by the immunogenic DNA constructs
  • VB1026 (SEQ ID NO: 1) comprises the following units: SEQ ID NOs: 111,
  • Expi293F cells (2x106 cells/ml, 1 ml) were seeded in a 96-well culture plate.
  • the cells were transfected with 0.64 pg/ml plasmid DNA using ExpiFectamine 293 Reagent (100014994 Thermo Fisher Sci.), and the plates were incubated on an orbital shaker (3 mm diameter, 900 rpm) in a humidified C02 cell incubator (8% CO2, 37°C). The plates were incubated for 72 h before the supernatant was harvested.
  • the secreted proteins were characterized in a sandwich ELISA of the supernatant using rabbit anti-SARS-CoV-2 (2019-nCoV) Spike RBD antibody (capture antibody, 100 pl/well, 1:1000 dilution, 40592-T62, Sino Biological) and goat anti-human CCL3/MIP-1 alpha antibody (secondary biotinylated antibody, 100 pl/well, 0.2 pg/ml, AF-270-NA, R&D systems).
  • rabbit anti-SARS-CoV-2 2019-nCoV
  • Spike RBD antibody capture antibody, 100 pl/well, 1:1000 dilution, 40592-T62, Sino Biological
  • goat anti-human CCL3/MIP-1 alpha antibody secondary biotinylated antibody, 100 pl/well, 0.2 pg/ml, AF-270-NA, R&D systems.
  • samples were prepared for SDS-PAGE by mixing 35 pi supernatant from transfected Expi293F cells with 12.5 m ⁇ 4x Laemmli sample buffer (Bio-Rad) with 2.5 m ⁇ DTT (Cayman Chemical) for reducing condition. The samples were heated at 70°C for 10 minutes (min) before adding 40 pL per lane to 4%-20% Criterion TGX Stain-Free precast gels (Bio-Rad). SDS-PAGE was performed in lx Tris/Glycine/SDS running buffer (Bio-Rad) with Precision Plus Protein All Blue Prestained and Unstained protein standards (Bio-Rad).
  • Figure 3 shows that all immunogenic constructs were expressed and secreted from transfected Expi293F cells.
  • the western blot analysis demonstrated that TECH004-IV007, TECH004-IV008, TECH004-IV012, and TECH004-IV013 all are expressed as full-length proteins ( Figure 4).
  • the ELISA and western blot data demonstrate full-length expression of the tested immunogenic constructs comprising varying number of T cell epitopes and/or T cell epitopes of varying lenght together with a constant antigenic protein.
  • Example 3 Assessment of T cell responses induced against T cell epitopes and RBD (aa 319-542) antigen from SARS-COV-2 encoded in immunogenic constructs of the disclosure
  • mice Female, 6-week-old BALB/c mice were obtained from Janvier Labs (France). All animals were housed in the animal facility at the University of Oslo (Oslo, Norway). All animal protocols were approved by the Norwegian Food Safety Authority (Oslo, Norway). 5 mice/group were used for the testing of the constructs comprising an antigenic unit, whereas 3 mice/group were used for the negative control.
  • a final dose of 25 pg DNA plasmid dissolved in sterile PBS was administered by intramuscular needle injection to each tibialis anterior (2 x 25 m ⁇ , 500 pg/ml), followed by electroporation with AgilePulse in vivo electroporation system (BTX, USA).
  • the spleens were collected 13 days after vaccination and mashed in a cell strainer to obtain a single cell suspension.
  • the red blood cells were lysed using ammonium-chloride-potassium (ACK) lysing buffer.
  • ACK ammonium-chloride-potassium
  • the splenocytes were pooled with in respective vaccinated groups and counted using the NucleoCounter NC-202 (ChemoMetec, Denmark) and resuspended to a final concentration of 6x106 cells/ml.
  • the splenocytes were then seeded 6x105 cells/well and re-stimulated with 4 pg/ml of single peptides corresponding to construct encoded T cell epitopes, except pep 08 which was not tested due to unavailbility of stock in the lab at the time of testing (Table 2) and 2 pg/ml RBD peptide pools (Table 3) for 24 hours. No-peptide- stimulation was used as negative control.
  • the stimulated splenocytes were analyzed for IFN-g responses using the IFN-g FluoroSpot kit (Mabtech AB, Sweden).
  • T cell responses against the predicted T cell epitopes from multiple SARS- CoV-2 strains were detected in the splenocytes at day 13 from mice vaccinated once with 25 pg of constructs containing either 3, 5, or 20 predicted T cell epitopes (Figure 5).
  • all constructs were also able to elicit RBD-specific T cell responses ( Figure 6).
  • the combined T cell responses elicited against the RBD (aa319-542) and the additional T cell epitopes encoded in the antigenic unit indicates that combining T cell epitopes with a B cell antigen can broaden the T cell response against SARS-CoV 2 induced by the immunogenic construct.
  • Example 4 Assessment of humoral immune response against SARS-COV-2 RBD (aa319-542. Wuhan variant) induced in mice vaccinated with immunogenic constructs of the disclosure Sera from the mice vaccinated with TECH004-IV007, TECH004-IV008,
  • TECH004-I V 012, TECH004-IV013, and TECH004-IV015 or the VB1026 were collected 12 days after vaccination and tested for anti-RBD IgG antibodies binding the RBD (Wuhan variant) protein. Briefly, blood was collected from the saphenous vein of the vaccinated mice.
  • Coagulated blood was centrifuged twice (1000 g, 15 min) and the serum was collected and transferred to a clean tube.
  • the humoral immune response was evaluated in an ELISA assay detecting total IgG in the sera binding to RBD (aa319-542) from SARS- COV2 (Wuhan variant).
  • ELISA plates (MaxiSorp Nunc-Immuno plates) were coated with 1 pg/ml recombinant RBD-His protein antigen in PBS overnight at 4°C. Plates were blocked with 4% BSA in PBS for 1 hour at RT. Plates were then incubated with serial dilutions of mouse sera and incubated for 2 h at 37°C.
  • Binding antibody endpoint titers were calculated as the reciprocal of the highest dilution resulting in a signal above the cutoff. Binding antigens tested included SARS-COV-2 antigens: RBD (Sino Biological 40592-V08H.
  • the immunogenic constructs of to the disclosure induce similar antibody response against the RBD antigen.
  • Example 5 Design and production of immunogenic DNA constructs of the disclosure for use as a vaccine against infection with Influenza A virus
  • Table 5 The sequence of the various T cell epitopes encoed by the immunogenic DNA constructs
  • Example 6 In vitro assessment of expression and secretion of proteins encoded by immunogenic constructs of the disclosure
  • Expi293F cells (2x106 cells/ml, 1 ml) were seeded in a 96-well culture plate.
  • the cells were transfected with 0.64 pg/ml plasmid DNA using ExpiFectamine 293 Reagent (100014994 Thermo Fisher Sci.), and the plates were incubated on an orbital shaker (3 mm diameter, 900 rpm) in a humidified C02 cell incubator (8% C02, 37°C). The plates were incubated for 72 h before the supernatant was harvested.
  • the secreted proteins were characterized in a sandwich ELISA of the supernatant using mouse anti human IgG CH3 domain antibody (capture antibody, 100 m ⁇ /well, 1 pg/ml, MCA878G, Bio-Rad), rabbit anti-influenza A H1N1 HA domain antibody (detection antibody, 100 pl/well, 0.2 pg/ml, 11684-R107, Sino Biological), and goat anti-rabbit IgG antibody (secondary antibody, 100 m ⁇ /well, 0.16 pg/ml, 31460, Thermo Fisher Scientific).
  • mouse anti human IgG CH3 domain antibody capture antibody, 100 m ⁇ /well, 1 pg/ml, MCA878G, Bio-Rad
  • rabbit anti-influenza A H1N1 HA domain antibody detection antibody, 100 pl/well, 0.2 pg/ml, 11684-R107, Sino Biological
  • goat anti-rabbit IgG antibody secondary antibody, 100
  • Example 7 Assessment of T cell responses induced against H1N1 T cell epitopes and HA antigen encoded by immunogenic constructs of the disclosure
  • mice Female, 6-week-old BALB/c mice were obtained from Janvier Labs (France). All animals were housed in the animal facility at the University of Oslo (Oslo, Norway). All animal protocols were approved by the Norwegian Food Safety Authority (Oslo, Norway). 5 mice/group were used for the testing of the constructs comprising an antigenic unit, whereas 3 mice/group were used for the negative control.
  • a final dose of 25 pg DNA plasmid dissolved in sterile PBS was administered by intramuscular needle injection to each tibialis anterior (2 x 25 m ⁇ , 500 pg/ml), followed by electroporation with AgilePulse in vivo electroporation system (BTX, USA).
  • the spleens were collected 14 days after vaccination and mashed in a cell strainer to obtain a single cell suspension.
  • the red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis buffer.
  • the splenocytes were pooled with in respective vaccinated groups and counted using the NucleoCounter NC-202 (ChemoMetec, Denmark) and resuspended to a final concentration of 6x106 cells/ml.
  • the splenocytes were then seeded 6x105 cells/well and re-stimulating with 4 pg/ml of single peptides corresponding to T cell epitopes (Table 5) and 4 pg/ml of single peptides corresponding to three T cell epitopes within the HA antigen of the second antigenic unit (Table 6) for 24 hours. No-peptide-stimulation was used as negative control.
  • the stimulated splenocytes were analyzed for IFN-g responses using the IFN-g FluoroSpot kit (Mabtech AB, Sweden).
  • T cell epitopes within the HA antigen of the second antigenic unit T cell responses against the predicted T cell epitopes from H1N1 strain were detected in spleens at day 14 from mice vaccinated once with 25 pg of plasmid DNA constructs containing either 3, 5 or 10 predicted T cell epitopes (Figure 9). In addition, all constructs were also able to elicit a HA-specific T cell response ( Figure 10). These results show that the addition of multiple T cell epitopes in combination with a B cell antigen can broaden the total T cell responses elicited against the immunogenic construct.
  • Example 8 Assessment of humoral immune response against H1N1 HA antigen induced in mice vaccinated with immunogenic constructs of the disclosure Sera from the mice vaccinated with TECH004-IV028, TECH004-IV029,
  • TECH004-I V030, TECH004-IV031, and TECH004-IV032 or the VB 1026 control plasmid were collected 14 days after vaccination and tested for total IgG antibodies binding the HA protein. Briefly, blood was collected from the saphenous vein of the vaccinated mice.
  • Coagulated blood was centrifuged twice (1000 g, 15 min) and the serum was collected and transferred to a clean tube.
  • the humoral immune response was evaluated in an ELISA assay detecting total IgG in the sera binding to HA (18-541aa) from Influenza A virus (A/Puerto Rico/8/1934(HlNl).
  • ELISA plates (MaxiSorp Nunc-Immuno plates) were coated with 1 pg/ml recombinant HA-His protein antigen in PBS overnight at 4°C. Plates were blocked with 4% BSA in PBS for 1 hour at RT. Plates were then incubated with serial dilutions of mouse sera and incubated for 2 h at 37°C.
  • Binding antibody endpoint titers were calculated as the reciprocal of the highest dilution resulting in a signal above the cutoff. Binding antigens tested included Influenza A H1N1 (A/Puerto Rico/8/1934) Hemagglutinin/HA Protein (Sino Biological 11684-V08H; (SEQ ID NO: 157)).
  • HA aal8-541
  • HA constant antigenic protein
  • Amino acid sequence of human MIPl-a (amino acids 24-93), signal peptide of human MIPl-a (amino acids 1-23), hinge exon hi from IgG3 (amino acids 94-105), hinge exon h4 from IgG3 (amino acids 106-120), a dimerization unit linker (amino acids 121-130) and the human CH3 domain of IgG3 (amino acids 131-237)
  • amino acids 1-4 amino acids 1-4
  • middle hinge region amino acids 5-15
  • lower hinge region amino acids 16-23
  • Amino acid sequence of anti-pan HLA class II with Ig VH signal peptide (amino acids 1-19), anti-pan HLA class II VL (amino acids 20-127), a linker (amino acids 128-142) and anti-pan HLA class II VH (amino acids 143-260)
  • An immunogenic construct comprising:
  • a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the specified order: a) a targeting unit targeting or capable of targeting antigen- presenting cells, b) a first joint region, c) a first antigenic unit comprising one or more T cell epitopes, d) a second joint region and e) a second antigenic unit comprising one or more antigens or parts or fragments thereof; or
  • a multimeric protein such as a dimeric protein, consisting of multiple polypeptides as defined in (ii), such as two polypeptides.
  • first- and second joint regions comprise a flexible unit and a binding unit.
  • the flexible unit comprises small, non-polar amino acids, such as glycine, alanine or leucine, or polar amino acids, such as serine or threonine.
  • the flexible unit consists of up to 20 amino acids, such as up to 15 amino acids, such as up to 12 amino acids or such as up to 10 amino acids.
  • the flexible unit comprised in the second joint region consists of from 5 to 60 amino acids, such as from 7 to 55 amino acids or 8 to 50 amino acids or 9 to 45 amino acids or 10 to 40 amino acids or 11 to 35 amino acids or 12 to 30 amino acids or 13 to 20 amino acids.
  • the flexible unit comprises or consists of an amino acid sequence having at least 50% sequence identity to the amino acid sequence 94-105 of SEQ ID NO: 1, such as 60%, or such as 70%, or such as 80%, or such as 90% sequence identity.
  • the flexible unit comprises or consists of hinge exon hi of IgG3.
  • the flexible unit comprises or consists of an amino acid sequence having at least 50% sequence identity to the an amino acid sequence 16-23 of SEQ ID NO: 2 or SEQ ID NO: 114, such as 60%, or such as 70%, or such as 80%, or such as 90% sequence identity.
  • the flexible unit comprises or consists of the amino acid sequence 16-23 of SEQ ID NO: 2 or SEQ ID NO: 114, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted , with the proviso that no more than 5 amino acids have been so altered, such as 4 amino acids, no more than 3 amino acids, such as 2 amino acids or no more than 1 amino acid. 0.
  • the flexible unit comprises or consists of the lower hinge region of IgGl. 1.
  • the flexible unit comprises or consists of the amino acid sequence 16-23 of SEQ ID NO: 2 or SEQ ID NO: 114. 2.
  • the binding unit is a covalent binding unit.
  • the covalent binding unit comprises one or more cysteine residues.
  • the covalent binding unit consists of or comprises a cysteine rich sequence. 25.
  • the covalent binding unit comprises at least 2 cysteine residues, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 cysteine residues.
  • the covalent binding unit consists of from 2 to 100 amino acids, such as 3 to 70 amino acids, such as 4 to 50 amino acids or 5 to 30 amino acids.
  • the covalent binding unit is a hinge region derived from an immunoglobulin, such as exon h4 of IgG3 or the middle hinge region of IgGl.
  • hinge region is Ig derived, such as derived from IgG, e.g. IgGl, IgG2 or IgG3.
  • hinge region comprises or consists of the nucleotide sequence with SEQ ID NO: 89 or an amino acid sequence encoded by said nucleotide sequence.
  • the covalent binding unit comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence 106-120 of SEQ ID NO: 1, such as at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity.
  • the covalent binding unit comprises or consists of the amino acid sequence 106-120 of SEQ ID NO: 1, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 6 amino acids have been so altered, such as 5 amino acids, such as 4 amino acids, no more than 3 amino acids, such as 2 amino acids or no more than 1 amino acid.
  • the covalent binding unit comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence 5-15 of SEQ ID NO: 2 or SEQ ID NO: 114, provided that the cysteine residues are retained in their number and position, such as at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity.
  • the covalent binding unit comprises or consists of the amino acid sequence 5-15 of SEQ ID NO: 2 or SEQ ID NO: 114, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 5 amino acids have been so altered, such as 4 amino acids, no more than 3 amino acids, such as 2 amino acids or no more than 1 amino acid.
  • non-covalent binding unit contributes to dimerization through non-covalent interactions, such as hydrophobic interactions.
  • non-covalent binding unit has the ability to form multimeric proteins via non-covalent interactions.
  • the non-covalent binding unit consists of or comprises an immunoglobulin domain, such as an immunoglobulin constant domain (C domain), such as a carboxyterminal constant domain (i.e. a CH3 domain), a CHI domain or a CH2 domain, or a sequence that is substantially identical to the C domain or a variant thereof.
  • C domain immunoglobulin constant domain
  • CH3 domain carboxyterminal constant domain
  • CHI domain CHI domain
  • CH2 domain a sequence that is substantially identical to the C domain or a variant thereof.
  • non-covalent binding unit is a CH3 domain derived from IgG, such as derived from IgG3 or IgGl, preferably derived from IgGl.
  • non-covalent binding unit comprises a CH3 domain derived from IgG3 with an amino acid sequence having at least 80% sequence identity to the amino acid sequence 131-237 of SEQ ID NO: 1 or SEQ ID NO: 113.
  • non-covalent binding unit comprises a carboxyterminal C domain derived from IgG3 with the amino acid sequence 131-237 of SEQ ID NO: l or SEQ ID NO: 113, wherein any one of the amino acids of the non-covalent binding unit has been substituted, deleted or inserted, with the proviso that no more than 22 amino acids have been so altered, such as no more than 21 amino acids , such as no more than 20 amino acids, such as no more than 19 amino acids, such as no more than 18 amino acids, such as no more than 17 amino acids, such as no more than 16 amino acids, such as no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no
  • the non-covalent binding unit comprises a CH3 domain derived from IgG3 with an amino acid sequence having at least 85% sequence identity to the amino acid sequence 131-237 of SEQ ID NO: 1 or SEQ ID NO: 113, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity.
  • non-covalent binding unit comprises or consists of a CH3 domain derived from IgG3 with the amino acid sequence 131-237 of SEQ ID NO: l or SEQ ID NO: 113.
  • non-covalent binding unit comprises or consists of a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence with SEQ ID NO: 90.
  • non-covalent binding unit comprises or consists of a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence with SEQ ID NO: 90, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • non-covalent binding unit comprises or consists of the nucleotide sequence of SEQ ID NO: 90.
  • non-covalent binding unit comprises CH3 domain derived from IgGl with an amino acid sequence having at least 80 % sequence identity to the amino acid sequence according to SEQ ID NO: 3.
  • non-covalent binding unit comprises a CH3 domain derived from IgGl with the amino acid sequence of SEQ ID NO: 3, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 22 amino acids have been so altered, such as no more than 21 amino acids, such as no more than 20 amino acids, such as no more than 19 amino acids, such as no more than 18 amino acids, such as no more than 17 amino acids, such as no more than 16 amino acids, such as no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no more than 5 amino acids, such as no more than 4 amino acids, such as no more than 3 amino
  • non-covalent binding unit comprises a CH3 domain from IgGl with an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 3, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity.
  • the non-covalent binding unit consists of or comprises CH3 of IgGl.
  • non-covalent binding unit consists of or comprises a leucine zipper motif.
  • non-covalent binding unit consists of or comprises a Jun/Fos-based leucine zipper, a ATF6- based leucine zipper, a PAR-based leucine zipper, a C/EBPa-based leucine zipper or an OASIS-based leucine zipper.
  • non-covalent binding unit is or comprises a leucine zipper motif (amino acids 308-336) from the CREB transcription factor (SEQ ID NO: 4).
  • non-covalent binding unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4, such as at least 81% or at least 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • non-covalent binding unit comprises the amino acid sequence of SEQ ID NO: 4, wherein any one of the amino acids of the flexible unit has been substituted, deleted or inserted, with the proviso that no more than 9 amino acids have been so altered, such as no more than 8 amino acids, such as no more than 7 amino acids, such as no more than 6 amino acids, such as no more than 5 amino acids, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, such as no more than 1 amino acid.
  • non-covalent binding unit joins multiple polypeptides, such as two, three, four or more polypeptides, into a multimeric protein, such as a dimeric protein, a trimeric protein or a tetrameric protein.
  • non-covalent binding unit is or comprises a trimerization unit, such as a collagen-derived trimerization unit, such as a human collagen-derived trimerization domain, such as human collagen derived XVIII trimerization domain or human collagen XV trimerization domain.
  • the non-covalent binding unit is a trimerization unit that comprises or consists of the nucleotide sequence with SEQ ID NO: 91, or an amino acid sequence encoded by said nucleotide sequence.
  • the trimerization unit is the C-terminal domain of T4 fibritin.
  • the non-covalent binding unit is a trimerization unit that comprises or consists of the amino acid sequence with SEQ ID NO: 92 , or a nucleotide sequence encoding said amino acid sequence.
  • non-covalent binding unit is or comprises a tetramerization unit, such as a domain derived from p53.
  • non-covalent binding unit is a tetramerization unit that comprises or consists of the nucleotide sequence with SEQ ID NO: 93 , or an amino acid sequence encoded by said nucleotide sequence.
  • first- and/or second joint regions comprise or consist of a naturally occurring sequence. 80.
  • the construct of any of the preceding embodiments, wherein the first- and/or second joint regions comprise or consist of an artificial sequence.
  • the second antigenic unit comprises one or more antigens which are full-length proteins of a pathogen, preferably full-length surface proteins of a pathogen.
  • the one or more antigens or parts or fragments thereof is derived from viral surface proteins, such as the spike protein from SARS-CoV-2 or hemagglutinin of the influenza virus.
  • the one or more antigens or parts or fragments thereof comprises a RBD sequence of spike protein of SARS-CoV-2 of the Wuhan strain, such as the RBD sequence set forth in SEQ ID NO: 104.
  • the first antigenic unit comprises 1 to 10 T cell epitopes such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 or 10 T cell epitopes, alternatively 11 to 20 T cell epitopes, such as 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 T cell epitopes, alternatively 21 to 30 T cell epitopes, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 T cell epitopes, alternatively 31 to 40 T cell epitopes, such as 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 T cell epitopes, alternatively 41 to 50 T cell epitopes, such as 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 T cell epitopes.
  • T cell epitopes are known to be immunogenic or selected for inclusion into said first antigenic unit based on the predicted ability to bind to HLA class Eli alleles.
  • T cell epitopes are comprised in structural proteins, such as surface proteins, and/or non- structural proteins.
  • structural proteins such as surface proteins, and/or non- structural proteins.
  • the one or more T cell epitopes are from a conserved region of the pathogen.
  • the first antigenic unit comprises T cell epitopes with a length of from 7 to 150 amino acids, preferably of from 7 to 100 amino acids, e.g. from about 10 to about 100 amino acids or from about 15 to about 100 amino acids or from about 20 to about 75 amino acids or from about 25 to about 50 amino acids.
  • the first antigenic unit comprises up to 3500 amino acids, such as from 60 to 3500 amino acids, e.g. from about 80 or about 100 or about 150 amino acids to about a 3000 amino acids, such as from about 200 to about 2500 amino acids, such as from about 300 to about 2000 amino acids or from about 400 to about 1500 amino acids or from about 500 to about 1000 amino acids.
  • up to 3500 amino acids such as from 60 to 3500 amino acids, e.g. from about 80 or about 100 or about 150 amino acids to about a 3000 amino acids, such as from about 200 to about 2500 amino acids, such as from about 300 to about 2000 amino acids or from about 400 to about 1500 amino acids or from about 500 to about 1000 amino acids.
  • T cell epitopes are from a conserved region of a betacoronavirus.
  • T cell epitopes are from a region conserved between SARS- CoV-2 and SARS-CoV.
  • T cell epitopes are from a region of a human Influenza virus, such as human Influenza virus A, human Influenza virus B, human Influenza virus C and human Influenza virus D.
  • the construct according to any of the preceding embodiments, wherein the one or more T cell epitopes are from a human Influenza virus of a specific hemagglutinin (HA) subtype, such as HI, H2, and H3, and/or a specific neuraminidase (NA) subtype, such as N1 or N5. 112.
  • HA hemagglutinin
  • NA neuraminidase
  • T cell epitopes are from a region of a human papilloma virus (HPV), such as from HPV16 or HPV18.
  • HPV human papilloma virus
  • T cell epitopes are arranged in the order of more antigenic to less antigenic in the direction from the first joint region to the second joint region.
  • T cell epitopes are arranged by alternating between a hydrophilic and a hydrophobic T cell epitope.
  • said targeting unit is or comprises a moiety that interacts with one or more surface molecules or receptors on antigen presenting cells (APCs) selected from the group consisting of HLA, cluster of differentiation 14 (CD 14), cluster of differentiation 40 (CD40), a chemokine receptor and a Toll-like receptor (TLR).
  • APCs antigen presenting cells
  • CD 14 cluster of differentiation 14
  • CD40 cluster of differentiation 40
  • TLR Toll-like receptor
  • said chemokine receptor is selected from the group consisting of CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 and XCR1.
  • TLR Toll-like receptor
  • TLR-5 Toll-like receptor
  • said targeting unit is selected from an antibody-binding region, a synthetic ligand and a natural ligand.
  • said targeting unit comprises or consist of a moiety selected from the group consisting of an antibody-binding region, a synthetic ligand and a natural ligand.
  • said targeting unit comprises antibody binding regions with specificity for surface molecules or receptors on antigen presenting cells (APCs), preferably specificity for CD14, CD40, Toll- like receptor, CCR1, CCR3, CCR5, MHC class I proteins or MHC class II proteins.
  • APCs antigen presenting cells
  • the targeting unit has affinity for a chemokine receptor selected from CCR1, CCR3 and CCR5.
  • said targeting unit has affinity for MHC class II proteins, preferably MHC class II proteins selected from the group consisting of anti-HLA-DP, anti-HLA-DR and anti-pan HLA class II.
  • the targeting unit is selected from pan-HLAII and MIP-la and preferably selected from pan-HLAII and human MIP-la.
  • the targeting unit comprises or consists of a compound selected from the group consisting of soluble CD40 ligands, natural ligands, such as chemokines, and bacterial antigens, such as flagellin.
  • the targeting unit comprises or consists of a compound selected from the group consisting chemokine ligand 5, also called C-C motif ligand 5 (CCL5 or RANTES), macrophage inflammatory protein alpha (CCL3 or MIP-la), chemokine ligand 4 (CCL4), chemokine ligand 19 (CCL19), chemokine ligand 20 (CCL20), chemokine ligand 21 (CCL21), chemokine motif ligand 1 (XCL1) or chemokine motif ligand 2 (XCL2).
  • chemokine ligand 5 also called C-C motif ligand 5
  • RANTES macrophage inflammatory protein alpha
  • CCL4 chemokine ligand 4
  • chemokine ligand 19 CL19
  • chemokine ligand 20 CCL20
  • chemokine ligand 21 CCL21
  • chemokine motif ligand 1 XCL1
  • XCL2 chemokine motif lig
  • the targeting unit is MIP-la, preferably human MIP-la.
  • the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence 24-93 of SEQ ID NO:
  • the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence 24-93 of SEQ ID NO:
  • the targeting unit comprises or consists of the amino acid sequence 24-93 of SEQ ID NO: 1 or SEQ ID NO: 112.
  • the targeting unit consists of the amino acid sequence 24-93 of SEQ ID NO: 1 or SEQ ID NO: 112, except that at the most six amino acids have been substituted, deleted or inserted, such as at the most five amino acids, such as at the most four amino acids, such as at the most three amino acids, such as at the most two amino acids or such as at the most one amino acid.
  • the targeting unit comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence with SEQ ID NO: 106.
  • the targeting unit comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence with SEQ ID NO: 106, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • the targeting unit has the nucleotide sequence of SEQ ID NO: 106.
  • the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence 20-260 of SEQ ID NO: 5.
  • the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence 20-260 of SEQ ID NO: 5, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.
  • the targeting unit comprises or consists of the amino acid sequence 20-260 of SEQ ID NO: 5.
  • constructs wherein the construct is the polynucleotide and wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.
  • a polynucleotide as defined in any of the preceding embodiments. A vector comprising the polynucleotide according to embodiment 145. .
  • a host cell comprising the polynucleotide according to embodiment 145 and/or the vector according to embodiment 146. .
  • a pharmaceutical composition comprising the construct according to any of embodiments 1-144 and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising the polynucleotide as defined in any of embodiments 1-145, the vector as defined in embodiment 146, the polypeptide as defined in embodiment 148, the dimeric protein as defined in embodiment 149 or the multimeric protein as defined in embodiment 150 and a pharmaceutically acceptable carrier.
  • composition according to any of embodiments 153- 154 further comprising one or more pharmaceutically acceptable excipients and/or diluents.
  • composition according to any of embodiments 153- 155, wherein the pharmaceutically acceptable carrier is selected from the group consisting of saline, buffered saline, PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffers, and combinations thereof.
  • a vaccine comprising the construct according to any of embodiments 1- 144 and a pharmaceutically acceptable carrier.
  • a vaccine comprising the polynucleotide as defined in any of embodiments 1-145, the vector as defined in embodiment 146, the polypeptide as defined in embodiment 148, the dimeric protein as defined in embodiment 149 or the multimeric protein as defined in embodiment 150 and a pharmaceutically acceptable carrier.
  • the vaccine according to any of embodiments 157-158 further comprising one or more pharmaceutically acceptable excipients and/or diluents.
  • the pharmaceutically acceptable carrier is selected from the group consisting of saline, buffered saline, PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffers, and combinations thereof.
  • a method of preparing a polypeptide or dimeric protein according to any of embodiments 1-144 comprising: a) transducing or transfecting cells with the vector as defined in embodiment 146 or the polynucleotide as defined in embodiment 145; b) culturing the cells, whereby the cells express a polypeptide encoded by said polynucleotide; and c) obtaining and optionally purifying the dimeric protein and/or the polypeptide expressed by the cells.
  • step c) comprises the step of isolating and purifying the fraction containing the dimeric protein.
  • a method for preparing a multimeric protein according to any of embodiments 1-144, wherein the multiple polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions comprises the following steps: a) transfecting or transducing cells with a polynucleotide as defined in embodiment 145; b) culturing the cells; c) collecting the multimeric protein from the cells; and d) isolating and optionally purifying the fraction of the multimeric proteins, wherein the multiple polypeptides are linked to each other via their respective first joint regions and via their respective second joint regions.
  • a method for preparing a vaccine which comprises a construct according to any of embodiments 1-144, wherein the method comprises the following steps: a) transfecting cells with a polynucleotide as defined in embodiment 145; b) culturing the cells; c) collecting and purifying the polypeptide, dimeric protein or multimeric protein expressed from the cells; and d) mixing the polypeptide, dimeric protein or multimeric protein obtained from step c) with a pharmaceutically acceptable carrier.
  • composition according to any of embodiments 153- 156 or the vaccine according to any of embodiments 157-160 for use in the treatment of an infectious disease, such as in the prophylactic or therapeutic treatment of an infectious disease.
  • a method for treating an infectious disease comprises administering to a subject in need thereof the pharmaceutical composition according to any of embodiments 153-156 or the vaccine according to any of embodiments 157-160.
  • a medicament comprising the construct according to any of embodiments 1-144 for treatment of an infectious disease, such as in the prophylactic or therapeutic treatment of an infectious disease.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the construct according to any of embodiments 1-144 for the manufacture of a medicament for the treatment of a subject having an infectious disease, such as prophylactic or therapeutic treatment of a subject having an infectious disease.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the construct according to any of embodiments 1-144 for the treatment of a subject having an infectious disease, such as in the prophylactic or therapeutic treatment of a subject having an infectious disease.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the construct according to any of embodiments 1-144 when used in the treatment of an infectious disease, such as in the prophylactic or therapeutic treatment of an infectious disease.

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Abstract

La présente invention concerne des constructions immunogènes, tels que des polynucléotides, des polypeptides, des protéines multimères et des protéines dimériques, et des vaccins comprenant de telles constructions immunogènes, qui sont utiles pour le traitement prophylactique et thérapeutique de maladies infectieuses, ainsi que des procédés de production et d'utilisation des constructions immunogènes et des vaccins.
PCT/EP2022/062556 2021-05-10 2022-05-10 Constructions immunogènes et vaccins destinés à être utilisés dans le traitement prophylactique et thérapeutique de maladies infectieuses WO2022238363A1 (fr)

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